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Doctoral Thesis
Activity and controlled delivery of epigallocatechin 3-gallate inthe treatment of degenerative disc disease
Author(s): Krupkova, Olga
Publication Date: 2016
Permanent Link: https://doi.org/10.3929/ethz-a-010738702
Rights / License: In Copyright - Non-Commercial Use Permitted
This page was generated automatically upon download from the ETH Zurich Research Collection. For moreinformation please consult the Terms of use.
ETH Library
Diss. ETH. No. 23749
Activity and controlled delivery of
epigallocatechin 3-gallate in the treatment
of degenerative disc disease
A thesis submitted to attain the degree of
DOCTOR OF SCIENCES of ETH ZURICH
(Dr. sc. ETH Zurich)
presented by
Olga Krupkova
MSc in Molecular Biology and Genetics, Masaryk University
Born on 01.09.1986
Citizen of Czech Republic
Accepted on the recommendation of
Prof. Dr. Stephen Ferguson
Prof. Dr. Karin Wuertz-Kozak
Prof. Dr. Benjamin Gantenbein
2016
3
Contents
Zusammenfassung ...................................................................................................................................... 6
Abstract ......................................................................................................................................................... 8
1. Introduction .............................................................................................................................................. 11
1.1 Motivation ................................................................................................................................... 13
1.2 Goal, Aims and Hypotheses ......................................................................................................... 15
1.3 Thesis outline............................................................................................................................... 18
2. Background ............................................................................................................................................. 19
2.1 Anatomy of the spine .................................................................................................................. 21
2.2 Intervertebral disc ....................................................................................................................... 21
2.2.1 Functional anatomy .............................................................................................................. 21
2.2.2 Nutrition and Innervation ..................................................................................................... 23
2.3 Intervertebral disc degeneration ................................................................................................ 24
2.3.1 Extracellular matrix decline .................................................................................................. 25
2.3.2 Reduced nutrition ................................................................................................................. 26
2.3.3 Cell reactions ........................................................................................................................ 26
2.3.4 Gene polymorphisms ........................................................................................................... 28
2.4 Pathologies of the intervertebral disc ......................................................................................... 29
2.4.1 Degenerative disc disease .................................................................................................... 30
2.4.2 Disc herniations .................................................................................................................... 31
2.4.3 Diagnostics of disc pathologies ............................................................................................ 32
2.5 Treatment of low back pain in clinical practice ........................................................................... 33
2.5.1 Physiotherapy and exercise .................................................................................................. 33
2.5.2 Pharmacotherapy ................................................................................................................. 34
2.5.3 Surgical treatments .............................................................................................................. 34
2.6 Therapeutic strategies in preclinical development ..................................................................... 35
2.6.1 Molecular therapy ................................................................................................................ 36
2.6.2 Cell-based regeneration ....................................................................................................... 38
2.6.3 Gene therapy ........................................................................................................................ 39
2.6.4 Bio-enhanced materials ........................................................................................................ 40
2.6.5 Future perspectives .............................................................................................................. 41
4
3. Epigallocatechin 3-gallate suppresses interleukin-1β-induced inflammatory responses in
intervertebral disc cells in vitro and reduces radiculopathic pain in rats ........................................ 43
3.1 Abstract ....................................................................................................................................... 45
3.2 Introduction ................................................................................................................................. 46
3.3 Methods ...................................................................................................................................... 48
3.4 Results ......................................................................................................................................... 54
3.5 Discussion .................................................................................................................................... 59
3.6 Conclusion ................................................................................................................................... 63
3.7 Author’s contributions ................................................................................................................ 63
3.8 Acknowledgement ....................................................................................................................... 63
4. The natural polyphenol epigallocatechin gallate protects intervertebral disc cells from
oxidative stress .......................................................................................................................................... 65
4.1 Abstract ....................................................................................................................................... 67
4.2 Introduction ................................................................................................................................. 68
4.3 Methods ...................................................................................................................................... 70
4.4 Results ......................................................................................................................................... 77
4.5 Discussion .................................................................................................................................... 85
4.6 Conclusions .................................................................................................................................. 88
4.7 Authors’ contributions ................................................................................................................ 88
4.8 Acknowledgement ....................................................................................................................... 89
4.9 Supplement ................................................................................................................................. 89
5. An inflammatory nucleus pulposus tissue culture model to test molecular regenerative
therapies: Validation with epigallocatechin 3-gallate ........................................................................ 93
5.1 Abstract ....................................................................................................................................... 95
5.2 Introduction ................................................................................................................................. 96
5.3 Methods ...................................................................................................................................... 98
5.4 Results ....................................................................................................................................... 103
5.5 Discussion .................................................................................................................................. 108
5.6 Conclusions ................................................................................................................................ 112
5.7 Author’s contributions .............................................................................................................. 112
5.8 Acknowledgement ..................................................................................................................... 112
5.9 Supplement ............................................................................................................................... 113
6. Stability of (–)-epigallocatechin gallate and its activity in liquid formulations and delivery
systems ....................................................................................................................................................... 115
6.1 Abstract ..................................................................................................................................... 117
6.2 Biological effects of (–)-epigallocatechin gallate ....................................................................... 118
6.3 Degradation of (–)-epigallocatechin gallate .............................................................................. 122
6.4 Stability of (–)-epigallocatechin gallate in solution ................................................................... 124
6.5 Stabilization of (–)-epigallocatechin gallate for clinical application .......................................... 127
5
6.6 Conclusions ................................................................................................................................ 134
6.7 Acknowledgement ..................................................................................................................... 134
6.8 Supplement ............................................................................................................................... 135
7. Towards controlled delivery of epigallocatechin 3-gallate as an anti-inflammatory treatment
for degenerative disc disease................................................................................................................. 139
7.1 Abstract ..................................................................................................................................... 141
7.2 Introduction ............................................................................................................................... 142
7.3 Methods .................................................................................................................................... 144
7.4 Results ....................................................................................................................................... 145
7.5 Discussion .................................................................................................................................. 149
8. Discussion .............................................................................................................................................. 153
8.1 Summary.................................................................................................................................... 155
8.1.1 Clinical relevance ................................................................................................................ 155
8.1.2 Clinical applicability ............................................................................................................ 158
8.2 Limitations ................................................................................................................................. 160
8.3 Future steps ............................................................................................................................... 161
8.4 Conclusion ................................................................................................................................. 162
8.6 References ................................................................................................................................. 163
Appendix .................................................................................................................................................... 191
List of abbreviations ........................................................................................................................ 193
Acknowledgement ........................................................................................................................... 196
Curriculum Vitae .............................................................................................................................. 197
6
Zusammenfassung
Bei der degenerativen Bandscheibenerkrankung (im Englischen degenerative disc
disease = DDD) handelt es sich um ein progressives, multifaktorielles Krankheitsbild, das eine
entscheidende Rolle bei der Entstehung von Rückenschmerzen spielt. Ein typisches Merkmal
der schmerzhaften DDD ist die gesteigerte Expression von Zytokinen und Schmerzmediatoren,
die zur Reizung von nozizeptiven Nerven sowie zur neuronale Schädigung beitragen können.
Die klinische Behandlung von DDD zielt zwar auf eine Bekämpfung der Schmerzen ab, diese ist
jedoch rein symptomatisch. Da die angewendeten Methoden nicht die verursachenden
biologischen Vorgänge, wie etwa den Abbau der extrazellulären Matrix oder das Vorkommen
von oxidativem Stress, Entzündungen, Seneszenz und Zelltod bekämpfen, kommt es meist zu
einer Verschlimmerung der DDD. Neue therapeutische Ansätze sind also von Nöten, um die
Behandlung zu verbessern. Vielsprechend als Therapeutika sind jene Moleküle, die die während
der DDD abnormal aktivierten Signalwege regulieren können. Präklinische Studien der
vergangenen Jahre haben vor allem Wirkstoffe mit anaboler Wirkung untersucht, während
anti-entzündliche und anti-oxidative Stoffe erst seit kurzem im Forschungs-Fokus sind, so dass
es bisher kaum Daten zu deren Effektivität gibt. Deswegen hatte die hier vorliegende
Doktorarbeit das Ziel, neue molekulare Therapeutika zu identifizieren und zu testen. Im Detail
sollen solche Therapeutika untersucht werden, die die während der schmerzhaften DDD
vorliegenden molekularpathologischen Veränderungen (Entzündung, Katabolismus, oxidativer
Stress) positiv beeinflussen können. Die durchgeführten Versuche zielten darauf ab, die
Wirksamkeit des natürlichen Polyphenols Epigallocatechin 3-gallat (EGCG) bei DDD zu testen.
Der therapeutische Einfluss von EGCG auf Entzündung, Katabolismus und oxidativen Stress
wurde an primären Bandscheibenzellen untersucht. Die Zellantworten wurden mittels Gen-
und Proteinexpression (RT-qPCR, WB, ELISA) und mittels spezifischen Assay (NF-kB Bindungs-
Assay, Seneszenz-assoziierte β-Galactosidase Färbung, Zellviabilität, mitochondriales
Membranpotential) ermittelt. Um den Einfluss von EGCG auf komplettes Bandscheibengewebe
untersuchen zu können, wurde ein degeneratives Organkulturmodel entwickelt, das aus
bovinem Nucleus Pulposus Gewebe in einem artifiziellen Annulus Fibrosus besteht. Um die
Induktion degenerativer Prozesse und deren Kontrolle durch EGCG nachweisen zu können,
wurde die Gewebezusammensetzung (Proteoglykane, Kollagene, Wasser und DNA) sowie die
Genexpression analysiert. Des Weiteren wurden die analgetischen Eigenschaften von EGCG in
einem Radikulopathie-Rattenmodel mittels von Frey Test überprüft. Um die Wirksamkeit von
EGCG in der klinischen Anwendung sicher zu stellen, wird zurzeit eine Methode zur
intradiskalen Applikation mit retardierter Freisetzung entwickelt. Das Freisetzungssystem
besteht aus EGCG-Gelatine-Mikropartikeln, die mit einem injizierbarem HA-pNIPAM Hydrogel
kombiniert werden. Die Mikropartikel werden durch Electrospraying hergestellt und mittels
7
Rasterelektronen-Mikroskopie analysiert. Zur Ermittlung der Konzentration und Stabilität des
freigesetzten EGCG werden der Ferrous Tartarate Assay und der DPPH Assay verwendet. Nach
Abschluss der Entwicklungsarbeiten kann das Freisetzungssystem an Bandscheibenzellen und
im NP Organkulturmodel getestet werden.
In primären Bandscheibenzellen konnte EGCG die durch IL-1β-induzierte
Entzündungskaskade therapeutisch beeinflussen. Die bei Entzündungen relevanten Pathways
NF-kB, JNK and p38 wurden gehemmt und die Gen- und Proteinexpression von entzündlichen
und katabolen Markern reduziert. Bei Ratten mit künstlich induziertem Bandscheibenvorfall
konnte durch lokale Applikation der radikulopathische Schmerz verringert werden, wodurch die
klinische Relevanz von EGCG weiter bestätigt wird. Zusätzlich zu einer anti-entzündlichen
Wirkung vermag EGCG des Weiteren, den durch oxidativen Stress ausgelösten Zelltod durch
eine Hochregulierung des PI3K/Akt Pathway zu verringern. Im NP Organkulturmodel konnte
EGCG ebenfalls die Expression entzündlicher und kataboler Gene hemmen. Da EGCG also auf
viele der dysregulierten, molekularpathologischen Prozesse bei DDD einen positiven Einfluss
nimmt, hat es hohes therapeutisches Potential für die Behandlung von degenerativen
Bandscheibenerkrankungen. Nachdem die vorhandene Literatur über die Stabilität von EGCG in
Flüssigformulierungen und Freisetzungssystemen umfassend untersucht wurde, konnte eine
geeignete intradiskale Applikationsmethode mit retardierter Freisetzung von EGCG entworfen
werden. Die Herstellung biokompatibler EGCG-Gelatine-Mikropartikel mittels Elektrospraying
konnte bereits erfolgreich umgesetzt werden.
In dieser Doktorarbeit konnte gezeigt werden, dass es sich bei EGCG um einen
vielversprechenden therapeutischen Kandidaten für die Behandlung von DDD handelt. EGCG
konnte nicht nur die Zellen der Bandscheibe vor Entzündungen und oxidativem Stress
schützen, sondern konnte zusätzlich auch das Schmerzempfinden in vivo reduzieren. Um die
klinische Anwendbarkeit von EGCG zu erhöhen, wird aktuell ein minimal-invasiv applizierbares
Freisetzungssystem für EGCG entwickelt. Nach weiteren präklinischen und möglicherweise
klinischen Tests wird sich zeigen, ob die in dieser Doktorarbeit entwickelte Strategie erfolgreich
umsetzbar ist.
8
Abstract
Degenerative disc disease (DDD) is progressive multifactorial disorder of the
intervertebral disc (IVD) and one of the major factors in the development of low back pain.
Painful DDD is typically accompanied by elevated levels of cytokines and pain-mediating
chemicals that irritate nociceptive nerves and promote neuronal damage. Clinical treatments
of DDD are aimed at resolution of pain and do not target the biological hallmarks of disc
degeneration, such as extracellular matrix decline, oxidative stress, inflammation, senescence,
and cell death. As a result, DDD can advance. Novel molecules targeting signaling pathways
involved in DDD are promising therapeutic candidates. Although progress in the use of ECM
anabolics has been made in preclinical research, anti-inflammatory and antioxidant
approaches have not yet been thoroughly explored. Therefore, the overall goal of the thesis was
to identify and test novel therapeutic candidates that can counteract inflammation,
catabolism, and oxidative stress involved in painful DDD.
The experiments conducted in this thesis were designed to test the effect of the
compound epigallocatechin 3-gallate (EGCG) on processes involved in DDD. The effects of EGCG
on inflammation, catabolism, and oxidative stress were investigated in primary IVD cell
cultures. Cellular responses were measured by gene and protein expression (RT-qPCR, WB,
ELISA) and by specific assays (NF-kB binding, senescence associated β-galactosidase, cell
viability, mitochondrial membrane potential). To evaluate the effects of EGCG on the tissue, a
degenerative organ culture model of bovine nucleus pulposus (NP) in an artificial annulus
fibrosus was developed and tissue composition (proteoglycan, collagen, water, DNA) and gene
expression were analyzed. Furthermore, the analgesic properties of EGCG were determined in a
rat model of radicular pain, using von Frey test. To promote clinical translation of EGCG-based
therapy, a system with the ability to provide sustained delivery in the IVD has been designed.
The delivery system consists of EGCG-gelatin microparticles, which are dispersed in injectable
HA-pNIPAM. The EGCG microparticles were produced by electrospraying and analyzed by
scanning electron microscopy. The concentration of EGCG released from the HA-pNIPAM was
measured by ferrous tartarate assay.
In primary IVD cell cultures, EGCG interfered with the IL-1β-induced inflammatory
cascade, inhibiting the key inflammatory mediators NF-kB, JNK and p38 and reducing the
expression of inflammatory and catabolic genes/proteins. Furthermore, local application of
EGCG in a rat model of IVD herniation attenuated radiculopathy, hence supporting the clinical
relevance of this compound. In addition to its anti-inflammatory effects, EGCG also possessed
9
the ability to reduce oxidative stress-induced cell death of IVD cells, via up-regulation of the
PI3K/Akt pathway. This suggests that EGCG constitutes a promising therapeutic option as it
targets multiple deregulated processes in the degenerative IVD. In the NP culture model, EGCG
reduced RNA expression of inflammatory and catabolic genes. After reviewing the stability of
EGCG in liquid formulations and delivery systems, an intradiscal sustained release system for
EGCG, composed of gelatin microparticles and HA-pNIPAM, was designed. Electrospraying of
biocompatible gelatin-based microparticles for the encapsulation of EGCG has been optimized.
In this thesis, evidence was provided that EGCG is a promising therapeutic candidate for
the treatment of DDD, as it can reduce inflammation and oxidative stress in vitro as well as
pain intensity in vivo. To increase the clinical applicability of our findings, a minimally invasive
delivery system for EGCG is currently under development. The question whether our strategy is
optimal for the clinical use will hence be answered in the future, after additional preclinical and
possibly also clinical testing.
.
10
11
Chapter 1
Introduction
12
13
1.1 Motivation
Degenerative disc disease (DDD) is a common progressive disorder with both genetic
and environmental components. DDD has been defined as accelerated ageing of intervertebral
disc (IVD), accompanied by inflammation, cell senescence and cell death [1-3], resulting in a
structural failure [4]. It is now established that DDD causes nerve irritation and compression,
largely contributing to the development of low back pain (LBP) [5-7]. Up to 80 % of the
population suffers from LBP at least once in their life, most frequently between the ages of 35
to 55. As LBP constitutes the 2nd leading cause for doctors’ visit and 3rd most common cause for
surgery in the economically active population, LBP plays a crucial role in work absence [8].
Moreover, 5% of all low back pain cases lead to chronic disability [9].
Despite increasing prevalence of discogenic back pain and the consequently high
socioeconomic burden [9], only symptomatic therapies are currently available [10, 11].
Conservative treatment is comprised of analgesics, with side effects such as gastric and
cardiovascular damage or drug dependence [12, 13]. Surgical treatment entails complications
arising from implant displacement, reduced spinal flexibility, and accelerated degeneration of
adjacent IVDs [14]. As current therapies have only limited success, the need for advancement in
long-term therapeutic strategies is apparent.
Therefore, research focus has shifted from symptom-driven medicine towards
regenerative medicine and tissue engineering of the IVD. Nowadays, the objective is to restore
IVD homeostasis and function via inhibiting inflammation, preventing premature aging, and
delaying cell death, ideally in a minimally invasive manner. This can be achieved by intradiscal
injection of biologics (cells, molecules) embedded in biocompatible materials. Although
injected cells can support the existing cell population and “rejuvenate” the aging disc [15, 16],
they are at risk of apoptosis due to the surrounding degenerative and inflammatory
environment [15, 17]. Injectable molecular therapeutics such as anti-inflammatory compounds
[18, 19] or growth factors [20, 21], can reduce inflammation and catabolism, thus enhancing
survival and function of the resident disc cells. Therefore, identifying and testing of novel
compounds that are able to interfere with degenerative processes within the IVD is crucial for
therapeutic advancement. Phytochemicals are good therapeutic candidates for the treatment
of DDD, as they exhibit multiple effects via modulation of several signal transduction and
apoptotic cascades, often with minimal adverse effects [22]. In the future, novel compounds
could be used as single treatments or in combination with conservative therapies or cells.
Drug development process consists of several consecutive phases (Figure 1). An initial
phase of preclinical testing takes place in in vitro models, where biological activities of
candidate compounds are evaluated. Due to the multifactorial nature of DDD, it is challenging
to develop a clinically relevant and reliable in vitro model. It requires thorough understanding
14
of the pathophysiological processes in the IVD tissue and the ability to perfectly represent these
processes in vitro. Despite the existing challenges, a number of cell and organ culture models
have been developed over the past years [23-29]. Although whole-disc organ cultures in
bioreactors appropriately mimic the in vivo status, they do not allow for high throughput
experiments, due to space-related demands. On the other hand, space-saving cell cultures lack
native extracellular matrix (ECM). Recently, a new organ culture model, comprised of a bovine
nucleus pulposus in an artificial annulus fibrosus, has been developed [30]. This model
overcomes space-related constrains, allowing for long-term high throughput experiments.
However, a degenerative phenotype has not yet been induced in this model. Activation of
appropriate degenerative changes in this model will be crucial for advancement in preclinical
testing of compounds that interfere with DDD. Compounds and formulations with successful
outcomes in cell and organ culture studies can further progress towards animal studies and
clinical trials (Figure 1).
Understanding compounds’ degradation properties is essential for successful clinical
translation. The disadvantage of some pharmacological compounds is their low stability,
diminishing their therapeutic effects in vivo and resulting in unsuccessful drug development
process. Encapsulation into protective biomaterials can overcome this drawback by improving a
compounds’ chemical stability and prolonging its activity. In fact, appropriately designed drug
delivery systems are able to improve the clinical outcome and provide long-term therapeutic
effects [31].
Figure 1. Drug development process. Candidate compounds are identified and tested in appropriate cell and organ culture tests. Following successful in vitro tests, the activity of compounds is verified in a suitable animal model before clinical trials are initiated. Then, clinical trials follow. The clinical trials comprise of sequential evaluation of toleration (Phase I), efficacy and dose range (Phase II), followed by trials in a large number of patients to develop a broad database of efficacy and safety (Phase III). After successful clinical trials, the candidate compound can receive regulatory approval, e.g. through the FDA. The approved compound formulation can be applied in the treatment of the disease for which it was designed [32].
15
1.2 Goal, Aims and Hypotheses
The overall goal of the thesis was to identify and test novel molecular therapeutic(s)
with multiple beneficial effects against DDD. Upon identification of a promising candidate
(epigallocatechin 3-gallate, EGCG), four specific aims were formulated to investigate its
properties (Figure 2). Each of these aims constitutes an independent study with objectives and
hypotheses outlined below and in.
Figure 2. Aims of the thesis. Biological effects of epigallocatechin gallate (EGCG) were analyzed in vitro (Aim 1 and 2). A degenerative nucleus pulposus model was developed and biological effects of EGCG were evaluated in this organ culture model (Aim 3). Further, a drug delivery system for intradiscal delivery of EGCG was designed (Aim 4).
Aim 1
The first study aimed to identify the effects of EGCG on the inflammatory and catabolic
responses of IVD cells. This study, published in 2014 in European cells & Materials, forms
Chapter 3: Epigallocatechin 3-gallate suppresses interleukin-1β-induced inflammatory
responses in intervertebral disc cells in vitro and reduces radiculopathic pain in rats.
Inflammatory cytokines IL-1β and TNF-α play a major role in the pathogenesis of DDD
and discogenic back pain [2]. Therefore, compounds interfering with cytokine-induced signaling
pathways can inhibit degenerative process in the IVD. Recently, EGCG showed anti-
inflammatory effects in osteoarthritic (OA) chondrocytes [33], which bear similarities to IVD
16
cells. EGCG reduced an IL-1β-induced response in chondrocytes by interacting with c-Jun N-
terminal protein kinase (JNK), nuclear factor kappa B (NF-κB) and p38 MAPK [34-36]. In another
study, EGCG inhibited the gene expression of cyclooxygenase 2 (COX-2) and prostaglandin E
synthase (PGES), molecules involved in the transduction and sensation of pain [37]. Based on
the promising effects of EGCG on OA chondrocytes, we hypothesized that EGCG can possess
anti-inflammatory and anti-catabolic effects in IVD cells and thus have a potential for the
treatment of inflammation and pain in DDD. To test this hypothesis, we examined the effects
of EGCG on gene and protein expression in IVD cells with simulated inflammation and the
effects of EGCG on IVD-related radiculopathy in rats.
Aim 2
The second study aimed to identify the effects of EGCG on IVD cells exposed to
oxidative stress in vitro. This study, published in 2016 in Oxidative Medicine and Cellular
Longevity, forms Chapter 4: The natural polyphenol epigallocatechin gallate protects
intervertebral disc cells from oxidative stress.
Oxidative stress in tissues is caused by extensive formation of reactive oxygen species
(ROS) and insufficient cellular anti-oxidant capacity [38]. Oxidative stress in the IVD contributes
to local inflammation, enhances expression of matrix degrading enzymes [39], causes cellular
damage and senescence, and activates cell death [40]. Moreover, oxidative damage positively
correlates with age and with the degree of disc degeneration [3, 40-47]. EGCG was shown to
directly interact with ROS or operate in an indirect manner by inhibiting ROS-generating
enzymes, by chelating pro-oxidant metal ions [31], or by enhancing the activity of pro-survival
pathways [48]. Therefore, we hypothesized that EGCG can protect IVD cells against oxidative
stress. To test this hypothesis, we examined the effects of EGCG on senescence and cell death
in ROS-treated IVD cells in vitro and we investigated the involved molecular mechanisms.
Aim 3
Organ culture models are valid alternatives for screening and feasibility testing of novel
therapeutic strategies for the treatment of DDD [23]. However, currently available organ
culture models have several limitations such as size, cost, and durability [23, 27]. The main
objective of the third study was to develop a cost-efficient, degenerative organ culture model
for preclinical testing of molecular therapeutics. An additional aim was to validate this model
with EGCG. This study, under review in the International Journal of Molecular Sciences, forms
Chapter 5: A novel tissue culture model simulating inflammatory degenerative disc disease to
test molecular regenerative therapies: Validation with epigallocatechin 3-gallate.
We hypothesized that a previously described bovine artificial annulus fibrosus system
may constitute a suitable base to develop a degeneration model, either by (1) inducing ECM
17
degradation through injection of a biologically relevant enzyme [49] or by (2) activating
degenerative responses through diffusion of cytokines from the culture media [50]. To test this
hypothesis, ECM composition and the expression of inflammatory and catabolic genes was
examined upon the above mentioned treatments.
Aim 4
The fourth aim focused on understanding and improving the stability of EGCG in order
to enhance its clinical applicability [51]. The fourth aim comprises two studies.
A first study, published in The Journal of Nutritional Biochemistry in 2016, forms
Chapter 6: Stability of (–)-epigallocatechin gallate and its activity in liquid formulations and
delivery systems. The objective of this review was to provide detailed information on prior
findings about EGCG stability in solution and biological systems as well as to evaluate available
encapsulation methods to improve the stability of EGCG.
It has been reported that the molecular structure of EGCG allows fast degradation [52,
53] and limits the efficiency of common encapsulation techniques [31, 51]. Therefore, clinical
translation of EGCG-based therapies is challenging. The second study, presented as a report of
work in progress, forms Chapter 7: Towards controlled delivery of epigallocatechin 3-gallate as
an anti-inflammatory treatment for degenerative disc disease. In this study the intradiscal
delivery system for EGCG was designed. We hypothesized that EGCG can be efficiently
encapsulated using a gentle electrospraying process [54], ultimately prolonging stability and
providing sustained release upon injection. To test this hypothesis, release and activity tests are
being performed.
18
1.3 Thesis outline
Chapter 2 provides relevant background information on the anatomy and function of
the IVD and describes the main biological processes involved in IVD degeneration and DDD.
Further, current clinical treatments for DDD are summarized. The chapter ends with the
discussion of novel therapeutic strategies in preclinical research. Chapters 3 to 7 comprise the
main body of work of the thesis. Chapter 3 to 6 represents unmodified manuscripts, published
or under review in peer-reviewed journals. Chapter 7 includes report on the currently ongoing
study, providing a summary of preliminary data and perspectives for the future research. In
Chapter 8, the findings and limitations are discussed in a general context and the thesis closes
with conclusion.
19
Chapter 2
Background
20
21
2.1 Anatomy of the spine
The spine has three primary functions: to support the upper body, to allow movement
of the trunk, and to protect the spinal cord and spinal nerve roots. The bony vertebrae and the
surrounding soft tissues including intervertebral discs, muscles and ligaments, perform these
functions [55]. A human spine is composed of 34 vertebrae: 7 cervical, 12 thoracic, 5 lumbar, 5
sacral, and 4 coccygeal vertebrae. In the cervical, thoracic and lumbar spine region, the
vertebrae are connected with 24 intervertebral discs, forming a flexible column that supports
the weight of the trunk and head. The sacral and coccygeal vertebrae are fused. 31 pairs of
spinal nerves exit the spine to transmit motor, sensory, and autonomic signals between the
spinal cord and the body. 8 nerve pairs exit at the cervical region, 12 pairs at the thoracic region,
5 pairs at the lumbar region, 5 pairs at the sacrum region, and 1 pair exits at the coccygeal
region [55]. Muscles and ligaments provide further support to the spinal integrity, and prevent
damages to the spinal column by restricting excessive movement.
2.2 Intervertebral disc
The intervertebral discs (IVD) are cartilaginous joints connecting two adjacent
vertebrae, from the second segment of cervical spine to the first sacral segment. The major
functions of the IVD are to distribute the applied loads to the vertebrae, and to provide spinal
flexibility within the limited range [55].
2.2.1 Functional anatomy
The IVD is anatomically divided into 3 distinct parts with substantially different
structural and mechanical properties: outer annulus fibrosus (AF), inner nucleus pulposus (NP),
and two cartilaginous endplates (CEPs) [1, 56]. The AF is composed of circumferential layers of
lamellae formed by closely arranged fibers of collagen type I. The AF has significant load-
bearing function, provides tensile resistance to the applied loads and aids the maintenance of
adequate NP pressure. The NP, located inside the AF, is predominantly comprised of a loose
network of collagen type II and highly hydrated proteoglycans. This gel-like structure equally
distributes the compressional and torsional stresses applied to the spinal column [57]. The
main differences between the composition of AF and NP are shown in Table 1. The CEPs, that
connect the IVDs with the vertebrae, are composed of hyaline cartilage located against the
vertebral body, and the fibrocartilage adjacent to the IVD. The CEPs are porous and allow fluid
exchange between the vertebrae and the IVD [55, 58].
22
Table 1. Composition of nucleus pulposus (NP) and annulus fibrosus (AF) in the lumbar spine [3, 55, 59]. †Percentage of wet weight of IVD; ‡Percentage of dry weight of IVD.
Water Collagens Proteoglycans Elastin Other proteins
Cells Enzymes
NP 70-90%† 15-20%‡ mainly type II
65%‡ 20-45%‡ Minor
AF 60-70%† 50-60‡ mainly type I
20%‡ 10%‡ Minor
Function Hydrostatic pressure
Tensile strength
Osmotic pressure
Support lamellae and cells
Homeostasis
IVD contains ~1% of cells, responsible for the maintenance of IVD homeostasis [60]. The
cells of the NP are chondrocyte-like with oval shape, whereas the cells of the AF, particularly in
the outer region, are elongated and aligned parallel to the collagen fibers [59, 61]. As the
cellular activities govern the organization and properties of the extracellular matrix (ECM),
healthy IVD cells are essential for normal IVD function. To ensure proper turnover of the ECM,
cells balance the synthesis of macromolecules by the expression of proteinases such as matrix
metalloproteinases (MMPs), aggrecanases, and their inhibitors such as tissue inhibitors of
matrix metalloproteinases (TIMPs) [58, 59]. The main macromolecules synthesized by the disc
cells are outlined below.
Aggrecan
The most abundant proteoglycan synthesized by IVD cells is aggrecan (>2,500 kDa).
Aggrecan is formed by a core protein with ~100 covalently attached glycaosaminoglycan (GAG)
chains of highly sulfated chondroitin sulfate and keratan sulfate. The core protein allows
aggrecan to aggregate with hyaluronic acid (HA). Resulting large-sized complexes have limited
diffusion, hence maintaining their location within the disc. The GAG chains provide aggrecan
with its osmotic properties, and therefore, the IVDs possess the ability to swell and resist
against the compressive loads. The high concentration of aggrecan also protects the IVDs from
neovascularization and innervation [62]. The turnover time for proteoglycans in the disc is
between 3 to 5 years [55].
Collagens
To date, 19 different types of collagens have been identified (collagen I – XIX). Types I to
V are the most abundant, whereas types VI to XIX occur only in small quantities. The IVD is
mainly comprised of the fibers of collagen I and II, with minor amounts of other collagen types
(III, VI, IX, X, XI, XII, and XIV) [55, 61]. The collagen synthesis starts with formation of triple helix
pro-collagen molecules inside a disc cell. Pro-collagen is then secreted into the ECM, where final
collagen assembly takes place. Flexible collagen fibers are spontaneously formed from a bundle
23
of threadlike pro-collagen fibrils and are cross-linked by lysyl oxidases to gain additional
strength [55, 63]. The principal amino acids of the collagen are glycine (35%), proline (12%),
hydroxyproline (10%), and alanine (11%) [55]. Type I collagen fibers form lamellae of the AF and
are found to have a diameter between 1 to 12 μm. Type II collagen fibers in the NP are smaller,
with average diameter of 20 nm [55]. Under the physiological conditions, collagens are stable
throughout the life [64].
2.2.2 Nutrition and Innervation
An adult IVD is an avascular structure, except for the most peripheral region of the AF.
The IVD cells receive nutrients mainly by diffusion from vertebrae, as vertebral capillaries
penetrate through the bone marrow spaces and terminate in the vicinity of the CEPs (Figure 1)
[58, 59]. The majority of fluids received by the IVD are absorbed by the NP, due to the presence
of negatively charged proteoglycans. Their fixed charges are electrically balanced by positive
cations (mainly potassium and sodium) in the interstitial fluid. Upon application of a load, the
NP loses water but not the ions, while removal of the applied load causes rapid rehydration due
to the osmotic gradient in the NP [55, 59, 60]. Therefore, spinal loading, proteoglycan content
and structure of the CEPs play major roles in the nutrition of the IVD cells.
Lumbar IVDs are innervated by a branching network of afferent and efferent fibers that
arise from the spinal nerves. While the NP is aneural, the outer third of a healthy AF is
innervated by sensory fibers with nociceptive and proprioceptive functions (Figure 2) [65, 66].
The AF also contains vasomotor nerve fibers associated with the small blood vessels on the
surface of the AF [55]. The CEPs are also innervated, especially at their center [67].
24
Figure 1. Nutrition (left) and innervation (right) of the IVD. Blood vessels terminate in the vicinity of CEP and nutrients are delivered to the NP via diffusion. Outer part of the AF is innervated via nerve branches arising from dorsal root ganglion (DRG) and spinal nerves (cauda equina). AF = annulus fibrosus, NP = nucleus pulposus, DRG = dorsal root ganglion, CE = cauda equina. Reprinted from [58] with permission from Macmillan Publishers Ltd. Reprinted from [55] with permission from Elsevier. http://www.sciencedirect.com/science/book/9780323079549 http://www.nature.com/nrrheum/journal/v10/n9/full/nrrheum.2014.91.html
2.3 Intervertebral disc degeneration
The lumbar IVDs are the most susceptible to degeneration, as they are too large for
efficient diffusion and carry the highest body load. The ability of human IVDs to withstand the
applied compressive loads gradually decreases throughout lifetime, with the onset of
degeneration observed from the second life decade. IVD degeneration is caused by a
combination of decreased tissue cellularity and molecular, structural, and mechanical
alterations of the ECM (Figure 2) [3]. Age-related disc degeneration starts in the NP and
progressively changes the load distribution and reduces the spinal flexibility [60]. A structural
disorganization of the CEPs affects the transport of nutrients, oxygen and lactic acid across the
disc [1, 61]. As a consequence, activity of the IVD cells is altered, leading to an impairment of IVD
repair [3]. It is difficult to distinguish between the normal IVD ageing and the pathologies of
the IVD due to the similarities in the mechanisms that are involved [60]. Clinically relevant
features associated with lumbar IVD degeneration are described below.
25
Figure 2. Early signs of the IVD degeneration appear during the second life decade (left). Tears in the AF start to emerge and begin to spread. The CEP shows early signs of cracking and plugging. Advanced signs of degeneration can be found later in life (right). The IVD dehydrates and bulges in several locations. The border between the AF and the NP becomes indistinctive and multiple circumferential and radial tears form in the AF, allowing the NP to move through the AF. The CEPs become thinner, dry and brittle [55]. AF = annulus fibrosus, NP = nucleus pulposus, CEP = cartilaginous endplate. Reprinted from [55] with permission from Elsevier. http://www.sciencedirect.com/science/book/9780323079549
2.3.1 Extracellular matrix decline
The most significant biochemical change of a degenerating NP is the age-related loss of
proteoglycans. For normal IVD function it is essential that the aggrecan content, charge and
size remain as large as possible. Aggrecan can be enzymatically cleaved by proteinases such as
the MMPs and aggrecanases [68]. The proteinases are produced by the IVD cells to regulate
the ECM turnover, however, in a degenerated IVD, the expression of these proteinases
increases [69]. Although IVD proteinases degrade collagens (MMP1, 2, 3, 7, 8, 9, 12, 13), they also
actively degrade aggrecan, especially MMP3, 7 and 12. The most active aggrecanases (a
disintegrin and metalloproteinase with thrombospondin motifs, ADAMTS) are ADAMTS4 and 5
[68-70]. HA can be degraded by hyaluronidases (especially HYAL2) and non-enzymatically by
free radicals [71]. In addition, aggrecan and collagens are negatively affected by irreversible
cross-linking, formed by non-enzymatic glycation due to the high blood glucose. The resulting
advanced glycation end products (AGEs) accumulate in long-lived proteins of the IVD,
preventing their repair and turnover [72, 73].
ECM turnover can be influenced by mechanical loads. Physiological loading can restore
proteoglycans in the IVD [74]. On the other hand, excessive dynamic and static compressive
loading activates MMP synthesis, leading to decreased aggrecan content and cell viability [75,
76]. Similarly, excessive torsional and multi-axial loads promote lamellar disorganization of the
AF, leading to the formation of fissures [55, 61]. The formation of fissures alters the stress
distribution in the AF and result in the loss of disc height, increasing the risk of annular injuries
26
and load-induced structural failure (herniation) [60, 77]. The loss of aggrecan also promotes the
neural and vascular ingrowth into the IVD [62].
2.3.2 Reduced nutrition
The IVD is the largest avascular organ in human body. As mentioned before, the
majority of the IVD cells receive their nutrients by diffusion through the CEPs from vertebral
capillaries. Throughout the lifetime, the CEPs calcify, become thinner, brittle and lose their
permeability for nutrients [55, 78]. The malnutrition of IVD cells can also occur with
pathological changes of the vertebrae such as trabecular fractures, inflammation, or
obstruction of capillaries [58]. Once nutrient supply is restricted, oxygen and glucose levels in
the center of the NP drop and at the same time lactic acid, which is produced by the IVD cells at
high rates, accumulates and reduces tissue pH [78]. Inefficient transport of solutes have
multiple negative consequences such as accelerated cell death, reduced ECM production, and
increased ECM degradation [78].
Cells in degenerative discs are able to activate the production of vascular endothelial
growth factor (VEGF) [79]. This suggests that attenuated nutrient supply can promote vascular
ingrowth, possibly as an attempt to enhance the availability of nutrients. However, an
abnormal blood supply in an originally avascular tissue can increase oxidative stress and
promote cytokine release. Moreover, in severely degenerated discs, newly formed blood vessels
are frequently accompanied by nociceptive nerves, mediating pain sensation [1].
2.3.3 Cell reactions
Inflammatory phenotype
Inflammatory cytokines are key molecules involved in IVD degeneration (Figure 3) [80].
The event that promotes cytokine production by IVD cells, especially in the absence of trauma
or herniation, is usually chronic stress such as smoking, IVD overload or spinal infection [2, 81].
Increased levels of interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) are
frequently found in the degenerative IVDs. The presence of IL-1β and TNF-α correlates with the
degradation of ECM [28, 82-84], as cytokines can induce the expression of collagenases (MMPs)
and aggrecanases (ADAMTSs) [85-87]. Mechanistically, IL-1β binds to a cell surface receptor IL-
1R1, activating nuclear factor κB (NF-κB), c-jun N-terminal protein kinase (JNK), and p38
mitogen-activated protein kinase (MAPK). The activation of these signaling molecules results in
the transcription of inflammatory and catabolic genes such as interleukins (IL-1α, IL-1β, IL-6, IL-
8), matrix metalloproteinases (MMP1, MMP3 and MMP13), and molecules that promote pain
(COX-2, NGF, iNOS) [2, 86]. Similarly, the interaction of TNF-α with its receptor TNFR1 leads to an
27
activation of NF-κB and MAPK pathways through tumor necrosis factor receptor-associated
factors (TRAFs) [2, 88]. In addition to the catabolic and pro-inflammatory effects, IL-1β and TNF-α
can influence cell senescence, autophagy and expression of genes involved in proliferation [80].
Inflammatory cytokines play important role in the neuronal sensitization.
Figure 3. Signaling pathways activated in IVD cells by inflammatory cytokines IL-1β (left) and TNF-α (right). After the interaction with their specific receptors, cytokines activate key inflammatory mediators (p38, JNK, NF-kB), which results in the expression of inflammatory and catabolic genes. This further promotes tissue inflammation and pain [2, 18]. IL-1R = interleukin 1 receptor, IRAK-1 = interleukin 1 receptor-associated kinase 1, TNRR1 = TNF receptor, TRAF = TNF receptor-associated factor.
Cell senescence and cell death
Although cells form only ~1% of the IVD, they are extremely important for IVD
maintenance [61]. Environmental factors such as non-physiological loading, oxidative stress, or
acidic environment can alter the cellular function and enhance premature senescence and cell
death in the IVD (Figure 4). Cell senescence is defined as permanent, irreversible replication
arrest, which occurs after a certain number of cell divisions [89], or when the cell encounters
sub-lethal stress. Senescent cells with reduced functionality accumulate in aged tissues, secrete
pro-inflammatory cytokines and promote tissue fibrosis [90, 91]. Stress-induced death of the
IVD cells, executed either through apoptosis or necrosis, is frequently found during IVD
degeneration [39, 61, 92-96]. The senescence and death of IVD cells do not only positively
correlate with age, but also with the degree of disc degeneration [3, 40-47].
Oxidative damage of macromolecules, mediated by reactive oxygen species (ROS)
including superoxide anion radical (O2−), hydroxyl radicals (OH·) or singlet oxygen (1O2), is one of
28
the most common causes of senescence and cell death. Cellular antioxidants such as
superoxide dismutase, catalase and glutathione peroxidase counteract the formation of ROS,
but their activity decreases with age [97]. Additional radical scavengers namely vitamins C and
E, carotenoids, or flavonoids, can be obtained through dietary sources, but the delivery of the
antioxidants into the disc is dependent on healthy blood supply [98].
Figure 4. Signaling pathways activated in IVD cells by oxidative stress. Sub-lethal damage (left) leads to the activation of p53-p21 and/or p16 pathways, causing irreversible cell cycle arrest via the inhibition of protein RB. Lethal stress (right) commonly induces intrinsic apoptotic pathway. Depolarized mitochondria release cytochrome C, which is implicated in the activation of caspase 3 or 7. The activation of caspase 3 or 7 executes apoptosis. Anti-apoptotic Bcl proteins (such as Bcl-xL or Bcl-2) and pro-apoptotic BH3 proteins (such as PUMA or NOXA) play major roles in the regulation of this pathway [40, 44, 99]. 2.3.4 Gene polymorphisms
Although the pathogenesis of IVD degeneration is not completely understood, it was
found to be associated with several genetic polymorphisms. Polymorphic genes are
represented by more than one allele within a population, and this allele commonly causes
abnormal gene expression and protein production. A polymorphism can alter promoter activity,
influence transcription factor binding, may result in shorter transcript, or faster product decay.
Investigating the polymorphisms associated with IVD degeneration could provide an insight
into the disease mechanism, reveal therapeutic targets, and provide useful information for the
identification of patients at risk [100, 101]. The important gene polymorphisms are outlined in
Table 2.
29
Table 2. The effects of selected polymorphisms on gene and protein level, and their participation in the
degenerative process of IVD [100]. TF = transcription factor, GDF = Growth Differentiation Factor 5, VDR
= vitamin D receptor, COL = collagen, MMP = matrix metalloproteinase, IL = interleukin, COX =
cyclooxygenase, PGE = prostaglandin E, uk = unknown.
Gene Function Functional change GENE
Functional change PROTEIN
GDF5 Growth and repair ↓ transcription ↓ growth and repair
VDR Sulfate concentration ↓TF binding , ↑ decay ↓ amount of sulfate in proteoglycans
Aggrecan Structural Shorter allele ↓ functional proteoglycan
COL1 Structural ↑ TF binding Impaired COL1 protein
COL9 Structural Addition of Tryptophan ↓ interactions with matrix molecules
Fibronectin Structural Fragments ↓ aggrecan, ↑ MMP
MMP2 Catabolic ↑ TF binding ↑ MMP
MMP3 Catabolic ↑ promoter activity ↑ MMP
IL1 Inflammation ↑ transcription ↑ IL1 and inflammation
IL6 Inflammation uk ↑ IL6 and inflammation
COX-2 Pain sensation uk ↑ COX2, PGE, pain and inflammation
2.4 Pathologies of the intervertebral disc
A pathological IVD degeneration is defined as accelerated ageing process of a disc,
accompanied with pain and structural failure [4]. Two types of pain are commonly associated
with IVD degeneration: a nociceptive pain, caused by a neuronal sensitization due to a tissue
damage, and a radicular pain, resulting from neuronal damage and inflammation (Table 3) [55].
Therefore, a degenerated IVD invaded with newly formed nerve fibers is the primary source of
nociceptive back pain [61], while IVD-related compression of nerve roots and spinal nerves
causes radiculopathy and muscle weakness [55, 66]. Both sensitization and damage of the
nerves can occur simultaneously. Although there can be various reasons for low back pain, an
IVD-related problem is generally accepted to be the most common cause. Two of the major
problems associated with the IVD are the DDD and the IVD herniation. These painful disc
pathologies have a strong impact on the quality of life [9].
30
Table 3. Classification and causes of IVD-related back pain. Pain arising from the IVD can be caused by internal disc disruption, injury or herniation. IVD-related pain have both nociceptive and neuropathic component (radicular pain). AF = annulus fibrosus, CEP = cartilaginous endplate.
IVD-related pain
Nociceptive Radicular
Internal disc disruption Internal disc disruption
Torsion AF injuries Herniations
Compression CEP injuries Loss of disc height
↓ ↓
Nerve sensitization Nerve damage
2.4.1 Degenerative disc disease
Both, the outer part of the AF and the central part of CEP are innervated by sensory
nerves. These nerves can be sensitized by a variety of chemicals directly released from disc cells,
among which cytokines are the most prominent. Both TNF-α and IL-1β have been implicated in
nerve sensitization and nerve ingrowth in a non-herniated degenerative IVD [66]. They act as
nociceptive triggers and also induce the expression of other potentially nociceptive cytokines
(e.g. IL-6, IL-8, IL-12, IL-17) and direct pain mediators (nitric oxide, prostaglandin E) [66, 80, 102-
104]. Neuropeptides such as substance P are located in the sensory nerve endings [105] and also
produced by the IVD cells [106]. By-products of disc cell metabolism such as lactic acid can also
be noxious [58, 107]. Additionally, IVD cells express neurotrophins such as nerve growth factor
(NGF) and brain-derived neurotrophic factor (BDNF), which contribute to neo-innervation of the
disc [106, 108]. Molecules implicated in nociception are listed in Table 4.
Early degenerative disc disease
It has been reported that the molecules larger than 400 Da have difficulties to
penetrate across a healthy IVD [66, 109]. Early degenerative IVDs still contain a dense
proteoglycan network, which does not allow for free diffusion of large cytokines such as TNF-α
and IL-1β, reducing their ability to reach the nerves in the AF and CEP. However, cytokines
released in the center of NP disturb local homeostasis and enhance degradation of the ECM,
hence facilitating a progression of the inflammatory process from the NP towards the AF [110].
On the other hand, smaller pain-mediating molecules that are able to diffuse freely can be
implicated in the pain sensation at earlier stages of DDD.
Advanced degenerative disc disease
A moderate or advanced IVD disruption leads to a direct chemical stimulation of
nociceptors by cytokines. Pain develops after torsional injuries in the AF (collagen disruption),
with neural ingrowth into the annular tears worsening the outcome, or after compression
31
injuries, causing nerve irritation [55]. Compressive loading was shown to cause CEP fractures
and internal disc disruption [26, 111], with invading leukocytes possibly worsening the process
[112]. An exposure of NP to the blood circulation breaks the immune privilege, further
promoting infiltration of nerves and blood vessels and resulting in immune reactions within
the IVD. It has been suggested that not only the proteoglycans, but also the IVD cells contribute
to the immune privilege of the IVD. Fas ligand (Fas-L) presence in the membranes of healthy
human NP cells can prevent angiogenesis in the IVD by inducing Fas-mediated apoptosis of
vascular endothelial cells and white blood cells [113]. However, Fas-L expression and the ability
of NP cells to induce Fas-mediated apoptosis of leukocytes decrease with advancement of IVD
degeneration [114]. Compression and torsion injuries in some cases progress to IVD herniations
and spinal stenosis [55].
Table 4. Molecules released from IVD cells and implicated in nociception. NGF = nerve growth factor, BDNF = brain-derive neurotrophic factor, CGRP = calcitonin gene-related peptide. M = membrane, S = soluble. Molecule Size Role References
TNF-α 26 kDa (M) 17 kDa (S)
Pro-inflammatory cytokine Neuronal sensitization
[80, 88]
IL-1β 17 kDa Pro-inflammatory cytokine Neuronal sensitization
[2, 88]
Nitric oxide 30 Da Inflammatory mediator Neuronal sensitization
[102, 115]
Lactic acid 90 Da Glycolysis end product Neuronal sensitization
[116, 117]
COX-2 69 kDa Inflammatory mediator Converts arachidonic acid to prostaglandins
[18, 39]
Prostaglandin E 352 Da Inflammatory mediator Transmits pain signal within the disc
[118, 119]
Substance P 1.35 kDa Pro-inflammatory Transmits pain signal within the disc
[105, 106]
CGRP 15 kDa Transmits pain signal in neurons [108, 120]
NGF 26 kDa Nerve growth Neuronal sensitization
[106, 121]
BDNF 14 kDa Nerve growth Neuronal sensitization
[106, 121]
2.4.2 Disc herniations
The lumbar IVDs are in close contact with dorsal nerve roots. Therefore, lumbar IVD
herniations are considered to be a major cause of radicular pain (Figure 5). Prolapsed IVD can
apply pressure on the spinal cord, dorsal root or DRG, inducing nerve damage and pain. A
32
radicular pain occurs along the nerves branching from the affected dorsal root. Tissues
surrounding the affected nerves are sensitized to motions and pressures that normally do not
cause pain [55]. In addition, pain-mediating chemicals released from the herniated NP activate
inflammation of the DRG and contribute to functional and structural nerve damage [122]. Apart
from the herniations, loss of disc height also causes mechanical deformation of DRGs by
compression, which is associated with radiculopathy [123].
Figure 5. Two major types of IVD herniation. In IVD protrusion (left), the NP has herniated through several lamellae of the AF and pushes on the spinal nerves, but has not penetrated through the most external lamella. In IVD extrusion (right), the NP has passed through the most external lamella of the AF and comes in contact with the spinal nerves or DRG. NP = nucleus pulposus, AF = annulus fibrosus, DRG = dorsal root ganglion, CE = cauda equina. Reprinted from [1] with permission from Elsevier. http://www.sciencedirect.com/science/book/9780323079549
2.4.3 Diagnostics of disc pathologies
Most spinal structures are innervated by nerves from multiple vertebral levels, which
poses difficulties in the diagnostics of back pain [55]. Nevertheless, axial back pain is often
diagnosed as DDD. Diagnostic methods used in patients with back pain include lumbar
radiographs, computed tomography (CT) scans, magnetic resonance imaging (MRI) and
provocative discography [124]. The lumbar radiographs are commonly used to assess the
anatomy of vertebrae and exclude other diagnoses such as displacement (spondylolisthesis),
fractures or scoliosis of the spine. The 3D CT scans are an alternative diagnostic method to
better assess the spinal canal, the anatomy of vertebrae, the appearance of osteophytes, and
endplate sclerosis. Although CT scan does not directly visualize the soft tissues, it allows
diagnostics of disc herniations [124, 125]. With MRI images, soft tissues are visualized, hence
permitting direct assessment of the nerves and discs. A darker MRI signal in the center of the
disc is associated with a loss of proteoglycans, whereas brighter irregular signals suggest the
presence of inflammation or infection [124]. Other common structural changes visualized using
33
MRI include radial fissures, radial bulging of the annulus, reduced disc height, osteophyte
formation and endplate defects. The major problem of MRI as a diagnostic method for DDD is
the fact that scans of symptomatic patients sometimes do not show any morphological signs
of IVD degeneration and vice versa, the degenerated discs are not always painful. It has been
reported that IVDs of up to 30% of asymptomatic volunteers contained protrusions and
herniations as well as reduced proteoglycan content [124]. Issues in diagnostics cause
difficulties to determine the clinically relevant structural hallmarks of DDD [100]. Nowadays it
is becoming widely accepted that persistent inflammation and innervation, rather than other
morphological changes, are the key factors distinguishing symptomatic and asymptomatic
discs [126].
In symptomatic patients, lumbar discography can help determining which disc is
painful. An injection of radiopaque dye followed by a CT scan enables the evaluation of disc
morphology and its defects, as the dye can leak through the disrupted layers of the AF. The
discography also reproduces the patient’s symptoms, since flux caused by injection of the dye
irritates the nerve endings. The discography is the only currently available diagnostic method to
evaluate the actual pain response of a patient [124]. However, it has been reported that needle
puncture of the AF can accelerate degeneration, especially in healthy IVDs that are used as
controls for reliable evaluation of painful disc [127].
2.5 Treatment of low back pain in clinical practice
Low back pain can be treated conservatively by physiotherapy and pharmacotherapy.
However, the success of these methods is not always guaranteed. Especially in patients with
chronic pain, complete eradication of pain can almost never be achieved. The long-term goals,
when treating the patients with chronic pain, are moderation of pain and decreased healthcare
utilization [128]. Upon failure of conservative treatment, a disc surgery is commonly performed.
The treatment strategies in the clinical practice are described below.
2.5.1 Physiotherapy and exercise
It is accepted that rehabilitation and exercise can reduce chronic low back pain and
disability [129, 130]. Physical therapies possibly promote healing of the disc by stimulating cells,
boosting metabolite transport, and preventing re-injury by strengthening back muscles [131].
However, excessive loading can have deleterious effects on both, tissues and cells, and further
promote the degenerative cascade [132]. Recently it has been suggested that the use of isolated
lumbar extension (ILEX) may be the most effective exercise to reduce LBP, thus potentially
improving the disc condition [130]. ILEX trains isolated muscles that extend the lower back and
34
represents a relatively high loading on the IVD at a low frequency [129]. This can promote fluid
flow across the disc, which in turn also enhances transport of larger molecules into the NP
[109]. However, in many cases, physiotherapy and exercise do not lead to major improvement
[133] and it is still unknown whether specific loading modes applied through exercise can heal
or regenerate the IVDs.
2.5.2 Pharmacotherapy
The most common analgesics for discogenic pain are paracetamol, non-steroid anti-
inflammatory drugs (NSAIDs) and opioids. Paracetamol is generally safe, but does not have
significant anti-inflammatory effects and likely does not ameliorate the neuropathic
component of pain [134].
Traditional NSAIDs (e.g. ibuprofen, aspirin) inhibit both COX-1 and COX-2, enzymes
involved in the synthesis of prostaglandins. More advanced COX-2 NSAIDs (e.g. celecoxib)
selectively inhibit COX-2-mediated synthesis of pro-inflammatory prostaglandins. NSAIDs are
effective for short-term symptomatic relief in patients with acute low back pain, but are
ineffective in the management of chronic pain and pain arising from neural damage [12, 135,
136]. Traditional NSAIDs can cause gastrointestinal complications in elderly, while specific COX-
2 inhibitors have less such side effects [137]. In addition, the long-term use of NSAIDs is
associated with serious cardiovascular events [12, 138, 139].
Opioids (e.g. morphine, oxycodone) are effective in treating chronic back pain with a
neuropathic component [13, 140]. They interact with neuronal opioid receptors in the spinal
cord and brain, alleviating pain signals. However, their use poses the risk for analgesic tolerance
over time [12, 141]. Complications of opioid use further include addiction and overdose-related
mortality, which have risen in parallel with the prescription rates [142]. Despite these
limitations, opioids are considered both effective and safe if used appropriately [143].
The majority of patients can be successfully treated by conservative therapies. However,
especially in the case of chronic back pain, these therapies can fail. None of the available dugs
can be used on the long term basis without side effects. Therefore, a surgical treatment is
required for those patients suffering from prolonged, life-limiting pain.
2.5.3 Surgical treatments
Injection therapy is usually the first line of more invasive treatment offered to the
patient. A needle is used to deliver drugs (local anesthetics, NSAIDs, opioids) into and around
the IVD and the nerve roots. This allows local delivery of higher doses, but inflammation and
pain is suppressed only temporarily. As the degenerative process progresses, disc surgery
usually follows [128].
35
Spinal fusion is the gold standard surgical treatment for disc removal. It alleviates pain
by removing a painful disc and preventing movement between affected spinal segments.
Overall, lumbar fusion mostly results in significant reduction in back and leg pain intensities
[128]. However, fusion of a spinal segment may have detrimental effects on the normal
physiological and biomechanical function of the rest of the spine, causing adjacent segment
disease [128].
Total disc replacement (TDR) may be able to prevent adjacent segment disease, as it
allows motion to be preserved at the “degenerated” level. Pain relief is thought to be due to a
combination of removal of the painful disc and improvement of load transfer. Short-term trials
of TDR indicate quicker recovery and better clinical outcome compared to fusion surgery.
However, 5-years follow ups show less significant differences between these 2 groups and the
long term effectiveness and safety of TDR is unclear. Controlled trials with relevant control
group and long-term follow-up are needed to evaluate the efficacy and safety of TDR [128, 144].
Pain arising from herniated lumbar discs can be treated with conservative therapy and
normally improves in 6 weeks. Surgical removal of herniated tissue (discectomy) is performed
when the nerve root is compressed and at the same time no improvement was observed after 6
weeks of conservative therapy. Discectomy have high success rates, but disturbed nerves may
become a source of chronic pain later in the life [145]. Discectomy is also one of the most
common risk factors for recurrent disc herniation [146].
2.6 Therapeutic strategies in preclinical development
Biological hallmarks of DDD, including inflammation, catabolism, senescence, and ECM
decline, can be specifically targeted. In the future, strategies such as in vivo molecular and cell
therapies may achieve inhibition of catabolism and activation of ECM production in the disc. In
more advanced stages of the DDD, degenerated discs could be replaced with tissue-engineered
implants.
Currently, molecular mechanisms of novel therapeutics are being evaluated in cell and
organ cultures, derived from human or animal discs, as well as in animal models. Organ
cultures provide valuable information about the ECM, but they possess limitations related to
explant size and culture durability. Although there are large differences between the tissue
composition, anatomy or physiology of animal species and human, large animal models are the
last and most important part of the complex preclinical testing. Biological treatments with
promising results in research and preclinical development are outlined in the following
subchapters.
36
2.6.1 Molecular therapy
Molecules promoting ECM regeneration
Growth factors are polypeptides involved in the maintenance of the disc ECM integrity
[147]. Their application as ECM stimulating agents is promising, as growth factors, namely bone
morphogenetic proteins (BMP-2, BMP-7), insulin growth factor (IGF-1), tumor growth factor
(TGF-β3), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and growth
differentiation factor-5 (GDF-5), induce the formation of new disc ECM via interaction with
their surface receptors on the disc cells [147]. The drawbacks of exogenous growth factors are
their costs and their short half-life (hours to days), resulting in only transient effects and the
need for repeated injections. Another complication is their large size, which can impair their
diffusion through the NP [109]. Moreover, optimal doses of growth factors for clinical use still
remain to be elucidated.
Link protein is found in the disc ECM, stabilizing the interaction between
aggrecan/hyaluronan aggregates and blocking the access for hyaluronidases. The N-Terminus
of Link Protein (LinkN) is a peptide cleaved by stromelysin from the Link protein, acting similarly
as growth factors hence stimulating ECM synthesis [21, 147].
Notochordal cells are present in young NP tissue. They play an important role in the
maintenance of a healthy IVD by secreting growth and pro-survival factors. However, in
humans these cells disappear during the first life decade, which is thought to contribute to the
early onset of disc degeneration. Notochordal cell-conditioned medium (NCCM), obtained from
either isolated notochordal cells encapsulated in alginate beads, or directly from the
notochordal cell-rich NP tissue, is capable of stimulating the ECM production in the disc organ
cultures [148]. The drawback of the use of NCCM is the uncertainty in the composition of
NCCM. Therefore, the identification of bioactive factors and their active concentrations in
NCCM is crucial for further development of this technique [149].
Similarly, blood plasma that has been enriched with platelets (platelet rich plasma, PRP)
can be used to stimulate healing of bone and soft tissues. Activated platelets release various
growth factors (e.g. PDGF, CTGF, bFGF, EGF, IGF1) and healing-promoting cytokines. This
mixture maintains the intervertebral disc homeostasis by shifting the cellular catabolism
towards the anabolic state, as shown in the cell and organ culture studies [20, 150]. However,
the composition of PRP is variable and requires standardization for the clinical use.
Other molecules, such as anti-catabolic enzymes (e.g. TIMPs), intracellular regulators
(e.g. Sox-2) [147] and inorganic polyphosphates [151] also have promising anabolic effects on the
ECM.
37
Molecular antagonists of degenerative pathways
Activation of inflammatory pathways is caused by the imbalance between the
inflammatory cytokines and their antagonists in the disc tissue, commonly observed during
human disc degeneration [152]. Interleukin-1 receptor antagonist (IL-1Ra) was shown to inhibit
ECM degradation in an intact degenerative human IVD [153]. Similarly, TNF-α receptor
antagonist inhibits inflammatory responses in the human IVD cells [154]. TNF-α receptor
antagonist (etanercept) or antibodies (adalimumab) that attenuate TNF-α-mediated effects
and reduce inflammation and pain are currently under clinical investigation [155].
As mitogen-activated protein kinases (MAPKs) are directly affected by pro-inflammatory
cytokines and regulate progression of inflammation, they are promising targets for the
treatment of DDD. Several specific small molecule MAPK inhibitors of p38, JNK and ERK are
available (e.g. SB202580, SP600125, PD0325901). The role of p38 in the expression and activity of
COX-2 is well established [156] and therefore p38 inhibition reduces cytokine-activated
prostaglandin E2 (PGE2) accumulation in the IVD [157]. However, gene expression of MMPs and
cytokines in the disc cells is more complex, commonly regulated by p38, JNK and ERK together
[18, 158, 159]. Therefore, a simultaneous inhibition of multiple MAPKs can synergistically block
the inflammatory and pain reactions arising from the IVD.
NF-κB is another direct target of pro-inflammatory cytokines. Therefore, NF-κB
inhibition can become a valuable therapeutic strategy for DDD. In healthy cells, an inactive NF-
κB complex binds to an IκB inhibitor located in the cell cytoplasm. In contrast, IκB degradation
allows nuclear translocation of NF-κB subunit p65 during inflammation. Its transcription
activity correlates with the degenerative changes of the IVD [160]. IL-1β-activated NF-κB
increases the expression of matrix degrading enzymes, promoting ECM degradation in the IVD
[160]. In addition, the expression of VEGF in IVD cells is regulated by TNF-α-activated NF-κB
[161]. Small molecule inhibitors of NF-κB such as MG132, BAY11-7082 and IMD0354 are available.
Although these inhibitors can be used as therapeutic agents, they have limitations. MG132 is a
proteasome inhibitor, preventing IκB phosphorylation, ubiquitination and degradation.
However, MG132 is not NF-κB-specific and can thus interfere with normal protein turnover and
activate JNK-mediated apoptosis [162]. BAY11-7082 and IMD0354 block specifically IκB
phosphorylation [163], however, their overall effects on the cells are not yet well understood
[164]. Another therapeutic target associated with NF-κB is receptor activator of NF-κB (RANK), a
member of the TNFR molecular sub-family, as the expression of its ligand (RANKL) is elevated in
DRG during disc-related pain. RANKL antibodies suppress the expression of TNF-α and IL-6 in
injured lumbar IVDs as well as the expression of CGRP in DRG neurons. This indicates the
possible use of RANKL as a pain control agent in the patients with lumbar IVD degeneration
[165].
38
Oxidative stress in the IVD is caused by the imbalance between the production of ROS
and the activity of the antioxidant protection systems. As cellular capacity to repair oxidative
damage decreases with age, antioxidants can constitute a therapeutic strategy to counteract
IVD degeneration [98]. It has been shown that antioxidants such as N-acetyl cytosine (NAC) or
ferulic acid alleviate the detrimental effects of oxidative stress in the IVD cells [166, 167].
Oxidative stress is also induced by high glucose (e.g. in diabetic patients), causing accumulation
of AGEs [168] and premature senescence of IVD cells in vitro and in vivo [19]. A combination of
pyridoxamine (AGE inhibitor) and pentosan polysulphate (anti-inflammatory) was shown to
protect diabetic mice from high glucose-induced IVD degeneration [19].
In addition to the inflammatory and nociceptive factors, degenerative and painful
human IVDs release NGF and BDNF, promoting pathological nerve ingrowth [108]. An
intradiscal administration of anti-NGF antibody reduces nerve sprouting and suppresses the
expression of CGRP in the rat DRG, located next to the injured IVD [169].
Due to the deregulation of diverse signaling pathways in a degenerated IVD,
compounds with multiple beneficial effects are the most interesting option. Compounds
derived from traditional medicinal plants (phytochemicals) exhibit antioxidant, anti-
inflammatory and pro-survival activities in the treatment of various age-related diseases.
Phytochemicals act on several signal transduction and apoptotic cascades with minimal
adverse effects [22]. Naturals compounds such as resveratrol [170], curcumin [171], osthole [172],
ferulic acid [167], or triptolide [173] demonstrated beneficial activity in reducing inflammation,
catabolism and oxidative stress in IVD-related studies. Although further research is required to
fully understand the effects of phytochemicals on the integrity of the IVD, it has been
suggested that they can delay or revert the degeneration of the NP. Polyphenols can also
enhance the disc nutrition by improving vascular health [174], and relieve pain [170].
2.6.2 Cell-based regeneration
Transplantation of autologous NP cells, which is currently under evaluation in the
clinical trials, restores the proteoglycan content in the IVD [175]. However, poor expansion rates
and the loss of native phenotypic features of the NP cells during the expansion in monolayer
cultures are major drawbacks [176]. Autologous NP cells for transplantation can be obtained
from surgical specimens, removed during discectomy. Nevertheless, the numbers of obtained
cells are low, and the cells are likely to be affected by the degeneration [17].
Stem cells possess the capacity of self-renewal, maintaining their undifferentiated
phenotype in multiple subcultures. Upon application of appropriate stimuli, stem cells undergo
differentiation. Various types of adult stem cells were tested for IVD regeneration [176, 177] such
as bone marrow mesenchymal stromal/stem cells (MSCs) [178, 179], adipose tissue-derived
stem cells [180] and muscle-derived stem cells [181]. For example MSCs cultured under
39
chondrogenic conditions are capable of differentiating towards the NP cells [182]. Stem cells
can be injected into the disc as free undifferentiated cells or encapsulated in hydrogels. In
animal models of IVD degeneration, implantation of MSCs has resulted in restoration of the
IVD height, IVD-like phenotype expression, ECM synthesis, and improvement in MRI signals [17,
56, 177]. Although promising outcomes were found in pilot clinical applications (phase I/III
clinical trials [155]), no fully conclusive data exist to date [16, 183].
The major drawback of cell therapies is the poor survival rate of implanted cells due to
the low oxygen content, low glucose, acidic pH, and complex mechanical loading [16, 58]. In
addition, cells can undergo apoptosis as a result of limited nutrition caused by the calcification
of CEPs or age-related vascular changes in the vertebrae [1]. Implanted cells can also escape the
disc and induce the formation of osteophytes [184]. Nevertheless, the disc microenvironment
may be able to aid the differentiation into NP phenotype in some cases [185]. As the
microenvironment plays likely a crucial role in the clinical outcome of stem cell therapies,
careful patient selection is required [186]. In addition, future therapeutic strategies can use
genetically modified cells, with unfavorable polymorphisms being corrected, using an ex vivo
gene therapy strategy [107].
2.6.3 Gene therapy
A gene therapy is a technique used to deliver exogenous genetic material (RNA or DNA)
into cells in order to provide prolonged protein synthesis. Gene therapy using TGF-β, BMP-2,
SOX-2 or BMP-7 was tested in several in vitro and in vivo IVD studies [187, 188]. These showed
improvements in disc height and ECM synthesis and demonstrated the ability to modulate the
biological processes of IVD cells. However, severe drawbacks of in vivo gene delivery still exist.
Viral vectors can be immunogenic and non-viral vectors exhibit low transfection efficiency. In
addition, a misdirected injection may lead to the expression of transgenic growth factors
outside the IVD, leading to toxicity with detrimental consequences [189]. For this reason,
inducible gene expression systems were developed to reduce these problems [176]. Ex vivo gene
delivery has been tested as well. IVD cells were infected with adenoviral IL-1Ra in vitro and
injected into degenerative IVD tissue explants, which resulted in increased IL-1Ra production
[190]. However, the NP cells are not the ideal target for genetic alterations, due to their scarcity
in the adult IVD, inaccessibility, and slow proliferation rate with dedifferentiation in vitro. On
the other hand, stem cells (such as MSCs or induced pluripotent stem cells) modified by
targeting genome editing systems (such as CRISPR/Cas9) could provide better results in the
future. Gene silencing using RNA interference in the treatment of DDD is currently also being
investigated in vitro and ex vivo [191].
40
2.6.4 Bio-enhanced materials
Biocompatible materials mixed with biologics are commonly used for tissue
engineering (biomaterials enhanced with cells) and drug delivery (biomaterials enhanced with
drugs). Natural materials such as polysaccharides (e.g. chitosan) or proteins (e.g. collagen)
mimic the in vivo environment, as they bear similarities with the native ECM [150]. Widely used
synthetic biodegradable polymers such as poly(lactide) (PLA), poly(glycolide) (PGA), poly(ε-
caprolactone) (PCL) and their copolymers have tunable properties and low immunogenicity.
Biomaterials from both natural sources and synthetic sources were tested and are used in
clinical applications [192, 193].
Biomaterial-based scaffolds are ideal for AF tissue engineering. Scaffolds can be created
by electrospinning, which allows fiber alignment resembling the AF structure. Electrospinning
generates solid structures in a one-step process, without the need of employing high
temperatures or toxic solvents. The scaffolds generated by electrospinning can retain cells at a
desired location, provide optimal mechanical properties, and facilitate tissue ingrowth. The
fiber diameter and the stiffness of a scaffold influence the cell function and proliferation [194].
Growth factors and small molecules can be incorporated into electro-spun scaffolds to further
enhance their regenerative function [195].
Hydrogels are extremely useful biomaterials for the regeneration of the NP. They
consist of highly-hydrated networks of hydrophilic polymers, capable of absorbing a large
amount of water. Due to their similar composition, hydrogels are used to deliver drugs and cells
into the NP. Upon the injection of environmentally-responsive hydrogels into the NP, the
hydrogels polymerize in situ to form a depot for drug release or cell encapsulation [155].
Thermo-reversible hydrogels are one of the most promising in situ polymerizing hydrogels.
Poly(N-isopropylacrylamide) (pNIPAM) is a frequently investigated thermo-reversible
polymer[196], which undergoes sol-gel transition upon change in temperature via intra- and
inter-molecular hydrophobic interactions between its isopropyl groups (gel point ~32°C).
pNIPAM’s ability to solidify at human body temperature is extremely attractive for in vivo
applications (e.g. injections) [155]. A modified pNIPAM was recently used as a vehicle for siRNA
and celecoxib in experimental IVD degeneration studies [191, 197]. Hyaluronan-grafted pNIPAM
(HA-pNIPAM) has been used to deliver growth factors and cells into degenerated IVDs [198-
200]. Even though hydrogels have mechanical properties similar to the native NP tissue, they
are generally weak and their extrusion through an injection site can occur [155]. For this reason
simultaneous regeneration or repair of the AF is preferred.
It is well documented that the efficiency of pharmaceutical agents can be improved by
controlled delivery using biomaterial carriers. For example, PLGA microparticles, used to
encapsulate IL1-Ra or TFG-β, exhibit superior effects on the formation of ECM compared to the
injection of free agents [155]. Another example is a sustained local delivery of encapsulated
41
COX-2 inhibitor, which showed better results in the reduction of inflammation than its free
form [201]. Biologics-loaded particles can be created by modification of electrospinning called
electrospraying. During electrospraying, the solution jet breaks down into fine droplets which
acquire spherical shapes due to the surface tension, subsequently producing solid
microparticles upon solvent evaporation [54]. These biologics-loaded microparticles can be
administered into the disc by injection, which is less invasive than the total disc replacement
surgery.
2.6.5 Future perspectives
DDD is a multifactorial disorder accompanied by nerve compressions and irritations,
which strongly contributes to life-limiting low back pain. Traditional clinical treatment
strategies are mainly focused on the resolution of pain, rather than targeting the biological
causes of IVD degeneration. Nowadays, the main focus in research has shifted towards
regenerative therapies and tissue engineering of the IVD. The aim of these novel approaches is
to decelerate or reverse IVD degeneration and to restore the IVD for long-term prevention of
pain. Several molecular therapeutics and cell-based strategies have been tested in preclinical
and clinical settings and showed promising results. However, at present, there is no consensus
on the optimal method to resolve DDD using these strategies. It seems clear that the best
results will be achieved by the combination of multiple approaches in a patient-specific
manner. A suitable biomaterial will facilitate the molecular and cellular delivery into the disc,
improve the IVD microenvironment, release substances, reduce pain and enhance the
mechanical properties of the degenerative disc.
42
43
Chapter 3
Epigallocatechin 3-gallate suppresses
interleukin-1β-induced inflammatory responses
in intervertebral disc cells in vitro and reduces
radiculopathic pain in rats
Olga Krupkova, Miho Sekiguchi, Juergen Klasen, Oliver Hausmann,
Shinischi Konno, Stephen John Ferguson, Karin Wuertz-Kozak
European cells & Materials, 2014, 28: 372-86.
44
45
3.1 Abstract
Intervertebral disc (IVD) disease, which is characterized by are-related changes in the
adult disc, is the most common cause of disc failure and low back pain. The purpose of this
study was to analyze the potential of the biologically active polyphenol Epigallocatechin 3-
gallate (EGCG) for the treatment of painful IVD disease by identifying and explaining its anti-
inflammatory and anti-catabolic activity. Human IVD cells were isolated from patients
undergoing surgery due to degenerative disc disease (n=34) and cultured in 2D or 3D. An
inflammatory response was activated by IL-1β, EGCG was added, and the expression/activity of
inflammatory mediators and pathways was measured by qPCR (mRNA), Western blotting,
ELISA, Immunofluorescence and Transcription Factor assay. The small molecule inhibitor
SB203580 was used to investigate the involvement of the p38 pathway in the observed effects.
The analgesic properties of EGCG were analyzed by von Frey filament test in Sprague-Dawley
rats (n=60). EGCG significantly inhibited the expression of pro-inflammatory mediators and
matrix metalloproteinases in vitro, as well as radiculpathic pain in vivo, most probably via
modulation of the activity of IRAK-1 and its downstream effectors p38, JNK and NF-κB.
Key words
Epigallocatechin 3-gallate; intervertebral disc degeneration; low back pain; IRAK-1; p38; NF-κB;
inflammation, radiculopathy
46
3.2 Introduction
Intervertebral disc (IVD) degeneration is a normal part of the aging process that starts in
the late second or early third decade [202]. Although often asymptomatic, specific histological,
biochemical and functional changes have been observed during IVD degeneration. These
changes are consistent with pain generation and disc degeneration has been suggested as the
most common cause of low back pain in adults [202]. Costs associated with health services for
spinal problems have been estimated at 85.9 billion USD in 2005 in the United States [203, 204]
and many studies have shown that indirect costs related to reduced productivity and
subsequent impact on national economy are also significant [202].
Degeneration of the IVD occurs due to an imbalance between anabolic and catabolic
processes, leading to a loss in collagen and proteoglycan and a concomitant reduction in water
content by enhanced exposure to matrix metalloproteinases (MMPs) and a disintegrin and
metalloproteinases with thrombospondin motifs (ADAMTs) [3, 86, 205-207]. These
degenerative processes lead to mechanical dysfunction and altered stress distribution in the
tissue, hence increasing the risk for load-induced structural failure in the annulus fibrosus (AF),
so-called clefts, tears or fissures. Under these circumstances, pain sensation can be provoked by
leakage of nucleus pulposus (NP) material through the AF (i.e. disc herniation) and consequent
irritation of spinal nerves (radiculopathy) and/or nerve infiltration into the compromised disc
(nociception).
However, even without a disc prolapse, the degenerated IVD can be painful, especially if
high levels of pro-inflammatory mediators are secreted. Cytokines, e.g. from the interleukin-1
(IL-1) superfamily, not only irritate nerve endings in the AF [56], but also stimulate the
production of matrix-degrading enzymes, hence further worsening the degenerative processes
[82, 110, 208]. The healthy IVD is excluded from the development of immunologic tolerance as
an immune-privileged organ with no access to systemic circulation [209]. In contrast, diseased
human discs are heavily invaded by blood vessels and nociceptive nerve fibers [210], so that
anti-angiogenic and anti-neurogenic factors are of therapeutic interest in terms of nociceptive
pain [209]. Once NP extrudes from the IVD to the systemic circulation, the immune system
recognizes it as a foreign body, leading to autoimmune reactions conducted by activated B and
cytotoxic T lymphocytes, which may further enhance the destruction of NP tissue inside the
disc [211]. On the other hand, some immune cells (macrophages, mast cells) might be beneficial
to the regrowth of the IVD wounds and immune privilege maintenance at early stage of the
degeneration [212] as well as in the absorption of herniated NP [209, 213, 214]. When
conservative treatment (physical therapy, pharmacological treatment) fails, patients with disc
herniation or painful degenerative disc disease are likely to undergo surgical interventions,
discectomy in case of herniation, spinal fusion or disc replacement in case of disc disease.
47
Imaging techniques such as MRI are a valuable tool to identify the source of pain in disc
herniation patients, however in patients with degenerative disc disease, identification of the
painful disc is error-prone and removal of the degenerated tissue has a negative impact on disc
height or load-bearing capacity [194]. In case of spinal fusion, a consequent risk for adjacent
segment degeneration, with the need of additional surgeries, is also significant [14, 56, 194].
Currently used procedures for the treatment of degenerative disc disease are therefore
not optimal and new, less invasive but more targeted strategies are being developed [56]: NP
replacement, e.g. by the injection of biocompatible hydrogels with or without cells may have
the potential to restore normal disc height and load distribution, as well as limit degenerative
changes in adjacent discs [56]. Recently, tissue-engineered IVDs that formed a functional
motion segment were used to replace the degenerated discs in rodent caudal spine [215] and
chemical crosslinking was applied to stabilise the AF tissue in vitro [216]. Anti-inflammatory and
anti-catabolic substances that target the metabolism and inflammatory signaling within the
IVD also represent a new, interesting treatment option [170, 171, 173, 217].
Epigallocatechin 3-gallate (EGCG) is a biologically active polyphenolic catechin present
in green tea. EGCG forms 40-60 % of all green tea catechins and is reported to be responsible
for the major health benefits of green tea due to its anti-oxidant, anti-aging and anti-
inflammatory properties, which are exhibited by direct or indirect interaction with many
molecules and signaling transduction pathways in cells [218, 219]. EGCG was shown to have
beneficial effects for a number of clinical conditions including cancer, obesity, atherosclerosis,
diabetes, liver and neurodegenerative diseases [220-222], with cell-type specific effects and
modes of action [218, 221]. Although EGCG has chemopreventive and anticancer effects and acts
synergistically with several anticancer drugs [223], it has also been shown that EGCG can block
the function of boronic acid proteasome inhibitors [224] and reduce the bioavailability of the
novel oral multi-target tyrosine kinase inhibitor sunitinib [225]. Moreover, the consumption of
EGCG by pregnant women could increase a risk of innate acute myeloid leukemia in children,
because EGCG inhibits topoisomerase II activity not only in cancer cells, but also in the foetus.
This can lead to the translocation at chromosome 11q23 involving the mixed-lineage leukemia
(MLL) gene [226, 227]. Therefore, EGCG is possibly contraindicated in particular cases.
Beneficial effects of EGCG and green tea extract were described in osteoarthritic (OA)
chondrocytes [33], which bear similarities to IVD cells. It has recently been demonstrated that
EGCG inhibits the general IL-1β-induced response, but does not have significant anabolic effects
in chondrocytes. The anti-inflammatory effect of EGCG in human chondrocytes is mainly
mediated by inhibition of c-Jun N-terminal protein kinase (JNK) and nuclear factor kappa-light-
chain-enhancer of activated B cells (NF-κB) activity [34, 35]. EGCG was shown to inhibit NF-κB
activation by suppressing the degradation of its inhibitory protein in the cytoplasm [36]. EGCG
also reduces the activity of activator protein 1 (AP-1) transcription factor [34], p38 mitogen-
48
activated protein kinase (MAPK) [36] and blocks interleukin-1 receptor-associated kinase 1 (IRAK-
1) degradation [35] in OA chondrocytes. EGCG is able to inhibit the expression of cyclooxygenase
2 (COX-2) and prostaglandin E synthase (PGES) [37], which are both involved in the development
and transduction of inflammation and pain. EGCG also potently blocks the toll-like receptor 4
(TLR4) signaling pathway, which may play an important role in the occurrence and
development of neuropathic pain in rats [228]. Based on the promising findings of EGCG effects
in cartilage, we hypothesize that EGCG may also possess anti-inflammatory and anti-catabolic
effects in the IVD and thus have a potential for the treatment of pain and inflammation in
degenerative disc disease.
3.3 Methods
Human IVD cell culture preparation
The study was approved by ethic committees Kantonale Ethikkommission Zürich EK-
16/2005 and Ethikkommission des Kantons Luzern 1007IVD. Human NP tissue was removed
from patients undergoing spinal surgery for degenerative disc disease or disc herniation after
informed consent was granted. The details about the donors used in this study are listed in
Table 1. Tissue was enzymatically digested using a mixture of 0.2% collagenase NB4 (17454,
Serva) and 0.3% dispase II (04942078001, Roche) for 4–8 h at 37°C and isolated primary cells
were seeded in Dulbecco’s Modified Eagle’s Medium (DMEM/F12, D8437, Sigma) supplemented
with 10% fetal calf serum (F7524, Sigma), penicillin (50 units/ml), streptomycin (50 μg/ml) and
ampicillin (125 ng/ml, 15240-062, Gibco) and sub-cultured up to passage 3 using 1.5% trypsin
(15090-046, Gibco). Cells in passage 1-3 that were cultured either adherently (2D) or in alginate
beads (3D) were used for experiments.
Alginate beads preparation
Alginate was prepared as a solution of 1.2 % alginic acid sodium salt (180947, Sigma-
Aldrich) in 0.9% sodium chloride, stirred overnight and sterile filtered. Human IVD cells in
passage 1-3 were harvested with trypsin and gently mixed with alginate (4 x 106 cells per 1 ml of
alginate). Alginate-cell suspension was slowly pushed through a sterile syringe with a 21G
needle and dropped into a 102 mM calcium chloride solution with constant speed. Beads were
formed after 5 minutes of stirring in calcium chloride solution and then washed with 0.9%
NaCl and PBS. Beads were spread into 6-well plates with cell culture medium containing 50
µg/ml Vitamin C (A4403, Sigma) and cultured for 7 days with one media exchange.
49
Table 1. Demographic data of all donors used for the experiments. STEN = discogenic stenosis, DH = disc herniation, DDD = degenerative disc disease, SP = spondylolisthesis. E = ELISA, G = gene expression, W = Western blotting, V = Viability, TF = transcription factor assay, IC = immunocytochemistry, uk = unknown.
Donor Sex Age Level Pathology Grade Experiment
1 F 43 L4/5 DH IV E(3D), G(3D), W
2 F 62 L4/5 SP V E(3D), G(3D), W
3 F 49 L5/S1 protrusion IV E(3D), G(3D)
4 M 42 C6/7 DH III E(3D), G(3D)
5 M 47 L2/3 DH IV E(3D), G(3D)
6 F 67 L4/5 DH IV E(3D), G(3D)
7 uk uk uk uk uk E(2D,3D), G(2D,3D)
8 M 50 L5/S1 SP III E(3D), G(3D)
9 uk uk uk uk uk E(3D), G(3D)
10 M 41 L4/5 DH IV E(2D), G(2D), G(SB), W
11 M 52 L1/2 DH V E(2D), G(2D), TF
12 F 53 C6/7 DH IV E(2D), G(2D), IS
13 F 46 L5/S1 DH V E(2D), G(2D), IS
14 M 42 L4/5 DH IV G(2D),W
15 M 39 L4/5 DH IV G(2D,3D), G(SB)
16 F 54 L3/4 SP V G(2D), TF
17 F 38 L5/S1 DH IV G(2D), G(SB), TF
18 M 59 L4/5 DH IV G(3D)
19 uk uk uk uk uk G(3D), W
20 M 59 L3/4 DH V G(2D), W
21 M 57 L5/S1 DH V G(2D), W
22 uk uk uk uk uk G(2D), G(SB)
23 M 48 L5/S1 DH IV G(2D), G(SB), W
24 uk uk uk uk uk G(3D), W
25 M 54 L4/5 DH III G(2D),W
26 F 49 C4/5 DH II V, G(2D)
27 M 62 C5/6-C6/7 STEN III V, G(2D)
28 uk uk uk uk uk V, G(2D)
29 uk uk uk uk uk V, G(2D)
30 F 57 L3/4 SP III V, G(2D)
31 M 56 L5/S1 DH III V, G(2D)
32 M 63 L2/3 DH IV V, G(2D)
33 uk uk uk uk uk V, G(2D)
34 F 25 L5/S1 DDD III G(2D)
Viability measurement
Non-toxic concentrations of EGCG (E4243, Sigma) were defined using the MTT assay in
2D cell culture. Cells were seeded in 12-well plates (1 x 105 cells/well) and treated with different
concentrations of EGCG (0.1 - 50 μM) in medium with 10% FCS or without FCS. After 24 and 48
hours, fresh MTT solution (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide,
M5655, Sigma) in media (0.5 mg/ml) was added and kept for 4 hours in 37°C. MTT was
50
discarded, cells were lysed in DMSO (D8418, Sigma) and absorbance was measured at 565 nm
relative to untreated controls. The non-toxic concentration 10 μM was chosen for subsequent
experiments. Data not shown.
Gene expression analysis
The effect of EGCG on the expression of inflammatory mediators and matrix degrading
enzymes was studied in 2D and 3D cell culture models that were pre-treated with IL-1β (211-11,
Peprotech) in order to induce the expression of pro-inflammatory and catabolic genes and
hence simulate the changes seen during inflammation-related disc degeneration [82, 229]. IVD
cells were seeded in 6-well plates (3 x 105 cells/well) (2D) or alginate beads (3D), serum-starved
for 2 hours and then exposed to 10 ng/ml IL-1β for 2 hours before treatment with 10 μM EGCG.
The involvement of the p38 pathway was studied using the small molecule inhibitor of p38
MAPK SB203580 (SB, S8307, Sigma), which was added 2 hours after IL-1β pre-stimulation in the
concentration of 10 µM to the cells seeded in 2D (n = 3 for MMPs and 4 for iNOS and COX-2).
After 18 hours, RNA was extracted with the TRIzol/chloroform method according to the
manufacturer instructions (15596-018, Invitrogen AG) and 1 µg was reverse transcribed to
complementary DNA (cDNA) using a reverse transcription kit (4374966, Applied Biosystems).
cDNA was then mixed with primers and master mix (4352042, Applied Biosystems) and gene
expression was measured using real-time PCR. Data was analysed by the comparative Cq
method (2ΔΔCq method, housekeeping gene Tata Box binding protein = TBP). Primer details are
given in Table 2. Results are presented as a gene expression relative to IL-1β pre-stimulation (100
%).
Table 2. Primers used for real-time RT-PCR (TaqMan Gene Expression Assays, Applied Biosystems).
Gene Primer Sequence Number Base Pairs TATA box binding protein (TBP) Hs00427620_m1 91 Interleukin-6 (IL-6) Hs00174131_m1 95 Interleukin-8 (IL-8) Hs00174103_m1 101 Matrix metalloproteinase-1 (MMP1) Hs00233958_m1 133 Matrix metalloproteinase-3 (MMP3) Hs00968308_m1 98 Matrixmetalloproteinase-13 (MMP13) Hs00233992_m1 91 Toll-like receptor 2 (TLR2) Hs00152932_m1 80 Cyclooxygenase-2 (COX-2) Hs00153133_m1 75 Inducible nitric oxide synthase (iNOS) Hs01075521_m1 82 Nerve growth factor (NGF) Hs00171458_m1 102
Western blotting
IVD cells seeded in 12-well plates (1 x 105 cells/well) were serum starved for 2 hours and
then exposed to 10 ng/ml IL-1β for 2 hours before treatment with 10 μM EGCG or 10 μM SB, 15
minutes for p-p38 (n = 9), p-JNK (n = 3) and IRAK-1 (n = 5), 6 hours for COX-2 (n = 1). For total
protein analysis, cells were lysed in 0.1% SDS buffer, mixed with Laemmli buffer (S3401, Sigma),
51
heated up (99°C, 5 minutes) and lysates were loaded onto 10% SDS-polyacrylamide gels.
Separated proteins were transferred to PVDF membranes (RPN303F, GE Healthcare) and
membranes were blocked in 5% non-fat milk in TBS-T for 1 hour/RT. Primary antibodies were
applied overnight at 4 °C. After washing in 1% non-fat milk in TBS-T (3×10 minutes), membranes
were incubated with secondary antibody conjugated to horseradish peroxidase (HRP) for 1
hour/RT. Visualization was performed on medical X-ray film (28906836, GE Healthcare) using a
chemiluminescence kit West Dura (34076, Thermo Scientific), films were scanned and scans
were processed by GIMP2. Tubulin was used as a loading control. Antibodies and dilutions used:
p38 MAPK, 1:1000 (9212, Cell Signaling); phospho-SAPK/JNK, 1:1000 (9251, Cell Signaling);
phospho-p38MAPK, 1:1000 (9211, Cell Signaling); α-Tubulin, 1:1000 (2144, Cell Signaling); IRAK-1,
1:1000 (4359, Cell Signaling); COX-2, 1:1000 (4842, Cell Signaling) and mouse anti-rabbit IgG
HRP, 1:5000 (211-032-171, Jackson Immuno Research).
Immunocytochemistry
IVD cells (n = 2) seeded onto cover slips in Petri dishes were serum-starved for 2 hours
and then exposed to 10 ng/ml IL-1β alone or in combination with 10 μM EGCG. After 1 hour of
simultaneous treatment, cells were washed with PBS (3×5 minutes), fixed with ice-cold
methanol (10 minutes on ice), washed with PBS again (3×5 minutes) and blocked with 2% BSA
(A4503, Sigma) with the addition of 5% goat serum (G9023, Sigma) (1 hour/RT). Primary
antibody p65 (RelA, the large subunit of NF-κB) was applied overnight at 4 °C. The next day,
cells were washed with PBS (3×5 minutes) and secondary antibody (CY2 goat anti-rabbit IgG)
was applied for 1 hour at RT. After washing in PBS (3×5 minutes), DAPI (D9542, Sigma) was
applied (5 minutes), cells were washed and embedded in Mowiol 4-88 (81381, Sigma). Analysis
of p65 translocation was performed with a fluorescent microscope (Olympus IX51) and
micrographs were processed in ImageJ. Cells without primary antibody were used as a non-
specific binding control, untreated cells were used as a negative control and cells treated with
10 ng/ml IL-1β were used as a positive control for p65 translocation. Antibodies and dilutions
used: NF-κB p65, 1:200 (Santa Cruz, sc-372) and CY2 goat anti-rabbit IgG, 1:400 (Jackson Immuno
Research, 111-165-144).
Nuclear extraction and Transcription factor assay
Nuclear extracts were prepared using a Nuclear Extraction kit (10009277, Cayman). Cells
(n = 3) were seeded in T75 flasks in the density 5 x 106 cells/flask and pre-cultured 24 hours. Cells
were serum-starved for 2 hours and then exposed to 10 ng/ml IL-1β alone or in combination
with 10 μM EGCG. After 1 hour of simultaneous treatment, nuclear extraction was performed
according to the producer’s protocol. Briefly, cells were collected into 15 ml pre-chilled tubes,
centrifuged 2 times with kit PBS/Phosphatase inhibitor solution and then re-suspended in ice-
52
cold kit hypotonic buffer. After 15 minutes of incubation, 10% Nonidet P-40 was added, cells
were centrifuged for 1 minute/140×g and supernatants containing the cytoplasmic fraction
were collected. Pellets were further re-suspended in kit complete nuclear extraction buffer by
vortexing (15 seconds), shaking (15 minutes on ice) and vortexing (15 seconds). Supernatants
containing the nuclear fraction were collected into new pre-chilled tubes after 10 minutes of
centrifugation (16000×g/4 °C). Protein concentration was determined using BCA assay kit
(23227, Thermo Scientific). NF-κB (p65) Transcription Factor Assay Kit (10007889, Cayman) was
used for the assessment of NF-κB (p65) DNA-binding activity in nuclear extracts according to
the producer’s protocol on a 96-well plate. An equal amount of protein (15 μg/well) was loaded
in duplicates for each sample. Provided non-specific binding control, competitor dsDNA control
and positive control were used to monitor appropriate assay function. The 96-well plate was
incubated 2 hours at RT, washed with kit wash buffer and primary antibody was added. After
one hour, the plate was washed, incubated with secondary antibody, washed again and
incubated with kit developing solution for 30 minutes. Finally, kit stop solution was applied and
absorbance at 450 nm was measured immediately. The result is presented as NF-κB (p65) DNA-
binding activity relative to IL-1β pre-stimulation (100 %).
Enzyme-linked immunosorbent assay (ELISA)
To detect secreted proteins, IVD cells in passage 1-3 were seeded on 6-well plates (3 x 105
cells/well) (n = 5) or alginate beads (n = 9), serum starved for 2 hours and then exposed to 10
ng/ml IL-1β for 2 hours before treatment with 10 μM EGCG. Cell culture media was collected
after 18 hours and the level of IL-6 protein expression was analysed by ELISA according to the
producer’s protocol (Human IL-6 ELISA set, 555220 with Reagent set B, 550534, BD Biosciences).
Briefly, 96-well plates were coated by kit capture antibody at 4 °C overnight, washed and
blocked in kit assay diluent. After washing, samples with controls and standards were
incubated on the plates for 2 hours at RT. Then plates were washed, kit detection antibody,
streptavidin-HRP and substrate solution were applied according to the producer’s protocol.
Absorbance was measured within 30 minutes at 450 nm with 570 nm correction after the
addition of a kit stop solution. Results are presented as protein expression relative to IL-1β pre-
stimulation (100 %).
In vivo study on pain behaviour
All animal experiments were carried out under the control of the Animal Care and Use
Committee in accordance with local guidelines for animal experiments and government law
concerning the protection and control of animals. Female Sprague-Dawley rats (n = 60, 200–
250 g) (Japan SLC, Shizuoka, Japan) were used in this study. A combination of 0.3 ml
medetomidine hydrochloride (1.0 mg/ml), 0.8 ml midazolam (5.0 mg/ml), and 1.0 ml
53
butophanol tartrate (5.0 mg/ml) was prepared as an anesthetic. Animals were anesthetized by
intraperitoneal injection of 0.1 ml/100g of BW of the above described mixed anesthetic.
Animals were placed in a prone position and the surgical intervention was performed with a
stereo operating microscope and microsurgical instruments. An incision was made to the
spinal midline, then fascia and multifidus muscle were resected, and the left L5 nerve root and
dorsal root ganglion (DRG) were exposed to L5-L6 facetectomy on the left side, with great care
taken to avoid trauma to the tissue. In the NP application group (NP group, n=48), autologous
NP was harvested from the tail and applied to the DRG [230-232]. In the sham-operated group
(Sham group, n = 12), the left L5 nerve root and DRG were exposed to L5-L6 facetectomy, but no
other procedures were performed. Animals from the NP application group were divided into
four groups (n=12 in each): NP + EGCG 10 µM (0.1 ml of 10 µM EGCG), NP + EGCG 100 µM (0.1 ml
of 100 µM EGCG), NP + water (0.1 ml of water as a vehicle), and NP (non-treatment) group.
Animals in the treatment groups were injected with 0.1 ml of the designed treatment solution
into the underlayer of epineurium, just distal to the NP, before closing the incisions [170, 232].
Sensitivity to non-noxious mechanical stimuli was tested using the von Frey Filament test.
Baseline testing was performed before the start of the experiment to accommodate animals
with normal responses. Hind paw withdrawal response to von Frey hair (North Coast Medical,
Morgan Hill, CA, USA) stimulation of the plantar surface of the footpads was determined at day
2, 7, 14, 21, and 28 after surgery. Individual rats were placed in an acrylic cage with a mesh floor
and allowed to acclimate for 15 minutes or until cage exploration and major grooming activities
ceased. The lateral plantar surface of the operated hind paw, innervated by the L5 nerve, was
stimulated with nine von Frey filaments (1.02, 1.4, 2.0, 4.1, 6.1, 8.0, 10.6, 15.4, and 26.0 g) threaded
under the mesh floor. The gram ratings for von Frey hairs were based on the ratings supplied by
the manufacturer. The filaments were sequentially applied to the paw surface just until the
filament bent and was held for 3 seconds. The response was considered positive if the rat lifted
the foot in combination with either licking or shaking of the foot as an escape response [170,
232].
Statistical Analysis
Statistical significance between IL-1β and IL-1β+EGCG groups (Figure 1, 2) as well as
between IL-1β and IL-1β+SB groups (Figure 3) was analyzed using Student’s T-test. Asterisks
represent a significance level P ˂ 0.05. For the animal study, behavioural data between the
groups over the experimental period (28 days) was statistically evaluated by ANOVA with
Bonferroni post-hoc testing to correct for multiple comparisons. Asterisks represent a
significance level P ˂ 0.01.
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3.4 Results
EGCG causes a significant decrease in the expression of inflammatory mediators and matrix
metalloproteinases in IVD cells cultured in vitro
The effect of EGCG on the expression of inflammatory mediators and matrix
metalloproteinases was determined in adherent 2D cell culture and in 3D alginate beads on the
mRNA level after 18 hours. A significant inhibition of mRNA expression of interleukins (IL-6, IL-
8), matrix metalloproteinases (MMP1, MMP3, MMP13), toll-like receptor 2 (TLR2), nerve growth
factor (NGF), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) was detected
upon EGCG treatment in both, 2D and 3D (Figure 1a, 1b). The exact values for each gene,
including p-values, are listed in Table 3. To confirm these observations on the protein level, IL-6
was chosen to be measured in cell culture media by ELISA, due to its strong regulation on the
mRNA level and pathological relevance. EGCG treatment significantly decreased IL-6 secretion
to the media, compared to the IL-1β pre-stimulated cells both in 2D (31.26 %, p = 0.0007) and 3D
(64.45 %, p = 0.0012) (Fig. 1c). Displaying the data relative to the IL-1β pre-stimulation (set as
100%) minimizes the inter-donor variation and shows clearly the effect of studied compound
on the acute inflammatory response.
Figure 1. EGCG exhibits anti-inflammatory and anti-catabolic effects in 2D and 3D IVD cell culture. Cells were pre-stimulated with IL-1β for 2 hours before treatment with EGCG and compared to cells treated with IL-1β and EGCG separately. After 18 hours, EGCG significantly reduced mRNA expression of IL-6, IL-8, MMP1, MMP3, MMP13, TLR2, NGF, iNOS and COX-2, both in 2D adherent cell culture (a) and in 3D alginate beads (b). IL-6 protein secretion in cell culture media was significantly decreased as well (c). Data is
55
presented as mRNA or protein expression relative to IL-1β pre-stimulation. Asterisks indicate statistical significance (p ˂ 0.05, Student´s T-test). Table 3. Percentage of mRNA expression in samples treated with IL-1β + EGCG, relative to IL-1β pre-stimulation, showed in Figure 1.
Gene 2D 3D Expression, P-Value Expression, P-Value
IL-6 22.72 % P ˂ 0.0001 32.79 % P = 0.0002 IL-8 4.99 % P ˂ 0.0001 25.69 % P = 0.0002 MMP1 30.40 % P ˂ 0.0001 41.40 % P = 0.0013 MMP3 19.73 % P ˂ 0.0001 32.55 % P = 0.0020 MMP13 28.93 % P ˂ 0.0001 54.08 % P = 0.0096 TLR2 10.43 % P ˂ 0.0001 3.25 % P = 0.0001 NGF 10.67 % P = 0.0002 46.79 % P = 0.0003 iNOS 5.91 % P ˂ 0.0001 45.44 % P = 0.0096 COX-2 20.68 % P ˂ 0.0001 39.62 % P = 0.0067
EGCG inhibits IL-1β-induced IRAK-1 degradation and partially blocks NF-κB, p38 and JNK activity
IRAK-1 was reported to be important for the transduction of inflammatory signals (IL-1β,
LPS), which cause its phosphorylation and degradation, hence enabling the activation of stress-
related signaling molecules NF-κB, p38 and JNK [233]. IL-1β pre-stimulated IVD cells were treated
with EGCG and the level of IRAK-1, together with the activity of its downstream effectors p38
and JNK, were determined by Western blotting. IRAK-1 was degraded in the cells stimulated
with IL-1β, but the level of IRAK-1 degradation was reduced in cells treated with IL-1β + EGCG
(Figure 2a). Phosphorylation of p38 and JNK was also decreased in IL-1β + EGCG-treated cells,
compared to IL-1β stimulated cells, i.e. EGCG reduced p38 and JNK activity (Figure 2b). The effect
of EGCG on the activity of the transcription factor NF-κB was studied by immunofluorescence,
visualising the translocation of NF-κB subunit p65 (RelA) to the nucleus and by NF-κB (p65)
Transcription Factor Assay. The effect of EGCG + IL-1β studied by immunocytochemistry
prevented p65 translocation in approximately 50% of cells. Cells without primary antibody,
untreated cells and cells treated with IL-1β were used as controls. As nuclear NF- κB was still
detectable in the EGCG + IL-1β-treated cells (Figure 2c), a Transcription Factor Assay was
performed to quantify the nuclear localization of NF-κB in EGCG + IL-1β-treated cells via DNA
binding activity. Results of the Transcription Factor Assay showed 50.83 % (p = 0.0327) decrease
in NF-κB DNA-binding activity upon EGCG treatment (Figure 2d). Results are presented as NF-κB
DNA binding activity relative to IL-1β stimulated cells (100%). Suggested mechanism of EGCG
action in IVD cells is shown in Figure 5.
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Figure 2. EGCG inhibits IRAK-1 - NF-κB/p38 /JNK signaling in IVD cells. Cells were pre-stimulated with IL-1β for 2 hours before EGCG was added. After 15 minutes, cells were lysed and Western blotting was performed. IRAK-1 degradation (a), p38 and JNK phosphorylation (b) were inhibited upon EGCG treatment of IL-1β stimulated cells. Combination of IL-1β with EGCG partially blocks NF-κB (p65) translocation to the nucleus (c) and NF-κB (p65) DNA-binding activity (d) after 1 hour. Asterisk indicates statistical significance (p ˂ 0.05, Student´s T-test).
EGCG inhibits p38 MAPK-dependent expression of COX-2, iNOS, MMP1 and MMP13
As shown in Fig 2, EGCG modulates the IRAK-1 - p38 signaling pathway in IVD cells. In
order to reveal which inflammation-related molecules are regulated by p38 MAPK on the gene
expression level, a small molecule inhibitor of p38 MAPK SB203580 (SB) was used. IL-1β pre-
stimulated cells were treated with either EGCG or SB and mRNA expression was measured after
18 hours. Both, EGCG and SB treatment significantly inhibits COX-2 (SB: 26.79 %, p = 0.0151),
iNOS (SB: 19.39 %, p = 0.0042), MMP1 (SB: 44.54 %, p = 0.0494) and MMP13 (SB: 6.66 %, p =
0.0042) expression compared to IL-1β pre-stimulated cells (100 %) (p ˂ 0.05) (Figure 3a). The
ability of SB to inhibit p38 activity was confirmed by Western blotting (Figure 3b). The
regulation of COX-2 expression by EGCG and SB was proved also on protein level by Western
blotting (Figure 3c). The expression of other studied genes (IL-6, IL-8, MMP3, TLR2, NGF) is only
partially p38 dependent or p38 independent (data not shown).
57
Figure 3. EGCG supresses p38 MAPK-dependent MMP1, MMP13, iNOS and COX-2 expression in IL-1β pre-stimulated IVD cells. Cells were pre-stimulated with IL-1β for 2 hours before treatments with EGCG and SB203580 (SB). Control cells were either untreated or treated with IL-1β, EGCG and SB separately. After 18 hours, EGCG and SB203580 both reduced mRNA expression of MMP1, MMP13, iNOS and COX-2. Data are presented as mRNA expression relative to IL-1β pre-stimulation. Asterisks indicate statistical significance (p ˂ 0.05, Student´s T-test) (a). The function of p38 MAPK inhibitor SB203580 was confirmed by Western blotting (b). The addition of EGCG or SB to IL-1β-stimulated cells inhibits p38-dependent COX-2 protein expression (c).
EGCG inhibits pain behaviour in vivo
Animal behaviour in response to mechanical stimulation by von Frey filaments was
compared between the NP + EGCG treatment groups (NP + EGCG 10 µM or NP + EGCG 100 µM),
the NP group (NP + no treatment), the water group (NP + water) and the sham group (only
facetectomy). Furthermore, the NP + EGCG treatment groups were compared amongst each
other. In the NP groups without EGCG treatment, the mechanical withdrawal thresholds were
significantly decreased for 28 days compared with the sham group, indicating that pain was
evoked by application of NP tissue to the DRG. Over the experimental period, mechanical
withdrawal thresholds upon EGCG treatment were significantly higher than in the NP group
and NP + water group and reached levels measured in the sham group. The mean hind paw
withdrawal thresholds for each group at each time point are listed in Table 4. The exact P-
Values for each comparison are listed in Table 5. Results indicate that EGCG treatment reduced
pain perception and this effect was independent of the applied concentration of EGCG (NP +
EGCG 10 µM vs NP + EGCG 100 µM = n.s.). Decrease in the mechanical withdrawal threshold
observed in the sham group arises from facetectomy-related distress (Figure 4).
58
Table 4. The mean hind paw withdrawal thresholds for each group and time point.
Baseline day 2 day 7 day 14 day 21 sham 26±0 23.5±8.0 22.8±7.2 15.6±9.5 16.6±9.2 NP 26±0 10.2±6.3 9.5±6.3 5.7±4.7 9.1±6.2 NP + water 26±0 10.9±5.9 7±1.8 7.8±3.9 9.9±6.2 NP + EGCG 10 µM 26±0 15±7.3 11.5±7.5 15.4±8.5 17.8±8.9 NP + EGCG 100 µM 26±0 16.9±8.7 11.9±7.0 17.5±10.6 19.6±8.4 Table 5. Statistical analysis of von Frey filament test between the groups. ns = non-significant. sham NP NP
+ water NP + EGCG 10
NP + EGCG 100
sham x P = 0.0001 P < 0.0001 ns ns NP P = 0.0001 x ns P = 0.0098 P = 0.0004 NP + water P < 0.0001 ns x P = 0.0026 P < 0.0001 NP + EGCG 10 µM ns P = 0.0098 P = 0.0026 x ns NP + EGCG 100 µM ns P = 0.0004 P < 0.0001 ns x
Figure 4. EGCG inhibits pain behaviour in vivo. Pain sensitivity was measured by von Frey filament test for 28 days. When NP tissue was placed on the DRG without therapeutic treatment (NP group and NP + water group), the mechanical withdrawal thresholds were significantly decreased. EGCG treatment (NP + EGCG 10 µM, NP + EGCG 100 µM) prevented the decrease in the mechanical withdrawal threshold observed in the NP group and NP + water group and restored the threshold level observed in the sham group.
59
Figure 5. Suggested mechanism of EGCG action in IVD cells. Upon IL-1β stimulation, IRAK-1 is phosphorylated and degraded, which enables the formation of multiprotein complex (TRAF6/TAB1/TAKs) and activation of key stress-related effectors NF-κB, p38 and JNK, which are further involved in the activation of inflammation, matrix breakdown and pain (A). EGCG blocks IRAK-1 degradation, formation of multiprotein complex and hence subsequent activation of NF-κB, p38 and JNK (B). TRAF6: TNF receptor-associated factor 6, TAK: TGF-beta activated kinase 1, TABs - TGF-beta activated kinase 1 binding proteins (schematic).
3.5 Discussion
The herein presented study clearly demonstrates the anti-inflammatory and anti-
catabolic properties of EGCG in human IVD cells in vitro, hence highlights its potential for the
treatment of IVD-related back pain. The development of back pain involves both nociceptive
and neuropathic pathophysiological mechanisms [12, 234] with IVD degeneration being one of
the major underlying causes. IVD degeneration is induced by phenotypic changes in the NP
cells, leading to tissue degradation [86], as well as to promotion of neoinnervation of the disc
and hence the transmission of pain in vivo [235, 236]. The evidence that IVD degeneration and
low back pain are correlated with increased levels of pro-inflammatory cytokines is increasing
[110]. Inflammatory processes are regulated, for example, by toll-like receptors (TLRs), which are
primarily expressed on the immune cells as a first line of host defence, but have also been
detected in the other cell types, including IVD [237, 238]. Recently, our group confirmed basal
expression of TLRs in IVD cells and showed that expression of TLR2, 4 and 6 is increased during
60
IVD degeneration [239] thus further underlying the clinical relevance of inhibiting IVD
inflammation. In this study, we demonstrated that 10 μM EGCG can significantly inhibit the
mRNA expression of inflammatory and pain-related mediators IL-6, IL-8, TLR2, NGF, iNOS, COX-2
and matrix metalloproteinases MMP1, MMP3, MMP13 in 2D and 3D IL-1β- stimulated IVD cell
culture models. All of these markers have been described to be of relevance during IVD
degeneration and inflammation and hence pain development [240, 241]. Although all cells used
in this study were isolated from the degenerated discs, the difference in the expression of
studied inflammation-related genes between untreated control group and EGCG-treated group
is not significant, because basal expression of these genes decreases when cells are transferred
to in vitro conditions (data not shown).
While this is the first IVD-related study, protective effects of EGCG and green tea extract
have been reported in inflammatory arthritis [33], where EGCG was shown to block IL-1β-
induced expression of IL-6, IL-7, IL-8, IL-1β, TNF-α [35], MMP1 and MMP13 [34], iNOS [242], COX-2
[243] and these effects were linked mostly to NF-κB and JNK signalling pathways [35-37, 242,
244]. In order to determine the underlying mechanisms in the IVD, potential pathways were
identified from the literature [35-37, 242, 244] and the effects of EGCG on IRAK-1 degradation as
well as NF-kB and MAPK activity in IVD cells were analyzed. We propose that EGCG acts as an IL-
1 receptor antagonist in IL-1β stimulated IVD cells. Our suggested mechanism of action, which is
illustrated in Figure 5, is through the inhibition of IRAK-1 degradation. As IL-1 receptors do not
possess intrinsic kinase activity, they rely on the recruitment and activation of intrinsic kinases
(IRAKs) [233]. Upon phosphorylation, IRAK-1 is ubiquitinated and degraded, which initiates a
formation of cytosolic multi-protein complex. The IRAK-1-activated protein complex is involved
in the phosphorylation of the inhibitor of κB kinase (IKK) as well as of MAPK kinases 3/4/6,
leading to the activation of NF-κB and JNK/p38 MAPKs, respectively [233, 245]. In this study, we
demonstrate inhibition of IRAK-1 degradation in IVD cells, similar to IL-1β-stimulated OA
chondrocytes [35]. As a result, activity of NF-κB was decreased in IL-1β-stimulated EGCG-treated
IVD cells. This effect was observed after 1 hour, since NF-κB is a protein complex that functions
as a “rapid-acting” transcription factor and as such regulates the transcription of more than 150
genes related to stress responses, including MMPs [110, 229, 246, 247].
The inhibition of IRAK-1 degradation is accompanied by a decrease in the activity of its
downstream effectors p38 and JNK. p38 kinase is a MAPK family member that is activated in
various stress conditions like UV irradiation, hypoxia, oxidative stress and inflammation and
regulates the expression of many stress-related genes, leading either to restoration of
homeostasis or to alteration of cellular functions and apoptosis. Recently it was published that
blockage of p38 MAPK activity in rabbit [248] and human [156] NP cells reduces IL-1β-induced
NO and prostaglandin E-2 accumulation [248, 249] and partially restores proteoglycan
synthesis [248]. Using the small-molecule inhibitor SB203580, we confirmed that not only iNOS
61
and COX-2, but also MMP1 and MMP13 are p38 MAPK target genes in human IVD cells (similar
to bovine IVD cells [250]), hence indicating that the p38 pathway may play an essential role in
ECM degradation and nociception in the disc. Importantly, the concomitant regulation of MMP1
and MMP13 suggests that EGCG may slow down MMP-induced collagen loss in all stages of IVD
degeneration as MMP1 has been shown to be significantly increased in severely degenerated
NPs, whereas MMP13 is increased in earlier stages of NP degeneration [69, 86]. The expression
of other studied genes (IL-6, IL-8, MMP3, TLR2, NGF) is regulated differently in IVD cells,
although p38 may co-operate too. In the context of nociception, the observed decrease in the
prostaglandin H synthase COX-2 can be specifically relevant as it catalyses the initial step in the
conversion of arachidonic acid to prostaglandins, which have a variety of physiological effects
including sensitisation of spinal neurons to pain [248]. Similarly, NOS plays an important role in
the processing of pain [251] so that the reduction of iNOS expression by EGCG should be
highlighted. Interestingly, it was recently reported that intrathecal administration of EGCG
could produce an antiallodynic effect against spinal nerve ligation-induced neuropathic pain,
mediated by blockade of neuronal NOS protein expression and inhibition of nitric oxide (NO)
[252]. As described above, our in vitro results suggest that EGCG-driven p38-mediated inhibition
of COX-2 and iNOS expression may be helpful for nociceptive as well as neuropathic pain
reduction. In order to monitor changes in pain behaviour upon EGCG treatment in vivo, we used
an animal model in which NP-mediated pain is simulated by the application of NP tissue to the
DRG, thus representing typical radiculopathic pain [253-255]. EGCG treatment was able to
prevent the threshold reduction and thus the pain-related behavior. The full recovery to the
sham threshold level was observed on day 14 in both EGCG treatment groups. Our results
correspond to recent findings using the rat model of neuropathic pain (sciatic nerve
constriction) where intrathecal injection of EGCG markedly improved pain behaviour in rats
and decreased the expression of TLR4, NF-κB, high-mobility group protein B1 (HMGB1), tumor
necrosis factor alpha (TNF-α) and IL-1β in the spinal cord [228]. In humans, back pain is however
caused by complex interactions of biological, psychological and social factors and can be
considered as a syndrome with both nociceptive and neuropathic components (mixed pain)
[256]. Nociceptive nerves are a major transmitter of discogenic back pain [257], therefore
alteration of the nerve ingrowth into the disc by EGCG-mediated inhibition of neurotrophins
can modulate the development of acute and chronic nociceptive pain. On the other hand,
discogenic radiculopathy occurs when functional, vascular and morphological changes of the
nerve root are activated, e.g. by disc-related inflammation, which leads to the intraradicular
fibrosis and nerve fiber atrophy [257]. From this point of view and according to our in vivo
results, EGCG-mediated inhibition of the inflammatory response of NP tissue may be beneficial
also against the development of disc-related radiculopathy. Based on these results, we
hypothesize that EGCG may reduce pain behaviour in vivo via inhibiting the expression of
62
cytokines and pain-related inflammatory mediators, similar to the mechanism observed in our
in vitro cell culture study.
This study however has certain limitations; firstly, the mechanism of EGCG action is not
entirely clear. During disc degeneration, the regulation and function of IL-1-mediated pathway
is altered: IL-1α and IL-1β isoforms as well as IL-1 receptor are synthesized in bigger quantities
compared to healthy tissue, whereas the expression of IL-1 receptor antagonist is unchanged
[84]. This imbalance activates the IL-1 pathway in NP tissue, which leads to the induction of the
expression of MMPs, ADAMTs, as well as to the enhanced angiogenesis and neurogenesis [257].
Although our study does not address the exact mechanism of how EGCG influences the IL-
mediated pathway, it provides evidence that EGCG can act as an IL-1 pathway antagonist,
therefore can possibly help to restore the tissue homeostasis in vivo. The second limitation is
that NP cells were isolated from donors with different medical conditions due to the limited
number of available biopsies. In addition, exact separation of NP and AF tissue (which was
performed in the surgical as well as the laboratory setting) can be challenging in degenerated
material so that certain impurities are possible. Nevertheless, all donors respond very similar.
Finally, the effect of EGCG on the tissue level (matrix degradation) was not analyzed in this
study.
Considering the anti-inflammatory, anti-catabolic and analgesic properties of EGCG
presented in this study, we suggest that EGCG could in the future supplement or even
substitute drugs which are used currently for the treatment of low back pain and disc
degeneration. However, the best mode of application is still a matter of debate. While oral
application has been considered for diseases such as obesity or diabetes, large in vitro/in vivo
discrepancy was observed [258] due to the low bioavailability of EGCG, with an elimination
half-life in plasma of around 3.5 hours [259]. For the treatment of back pain, oral application of
EGCG is certainly not a promising approach because the inflammatory processes within the IVD
need to be targeted directly. Therefore, local application (e.g. intradiscal or epidural injection)
will be required, which could be done in conjunction with other surgical or regenerative
interventions or independently, i.e. as an early, non-invasive treatment. However, it is likely that
the effects of non-modified (free) EGCG will be less pronounced in vivo compared to in vitro
because of its low stability, which can be further influenced by extrinsic conditions such as type
of storage and intrinsic factors like Ca2+ and Mg2+ ions or antioxidant concentration [258].
Encapsulation of EGCG in polymeric carriers represents a promising strategy to increase its
bioavailability and release period. In fact, different types of polymeric carriers administered
orally or intravenously were demonstrated to significantly increase the stability and efficiency
of EGCG in cancer therapy [260, 261], showing their potential to be used also inside the IVD.
However, the disc-specific environment (high amount of proteoglycans, lower pH in
degenerated disc, low amount of cells) should be taken in account when choosing a carrier and
63
delivery method for EGCG. The efficacy of these respective EGCG slow-release systems will have
to be determined in vitro using cell or organ culture studies (by detecting inflammation) as well
as in appropriate in vivo models (by detecting pain sensation). Despite the remaining
challenges, EGCG offers several potential clinical advantages: it is globally available, it is
inexpensive to isolate, it was reported to be safe, well tolerated and clinically active
concentrations can be reached by its oral administration or by its modification [218, 219].
3.6 Conclusion
In this study, we examined the effects of the bioactive polyphenol from green tea,
Epigallocatechin 3-gallate, on IVD cells cultured in 2D and 3D in vitro and on IVD-related
radiculopathic pain in vivo. We have described its anti-inflammatory and anti-catabolic effects
and the benefit of EGCG in the reduction of both nociceptive and neuropathic disc-related pain.
We identified IRAK-1 - NF-κB/p38/JNK signaling pathways to be modulated by EGCG and we
provide evidence that this contributes to the observed effects. Although precise mechanisms of
EGCG action and its direct targets in IVD cells still need to be elucidated, we showed promising
therapeutic potential of EGCG in the treatment of disc-related inflammation and back pain in
degenerative disc disease.
3.7 Author’s contributions
OK performed cell culture experiments, statistical analysis and helped to draft the
manuscript. MS performed animal experiments, statistical analysis and helped to draft the
manuscript. JK participated in study design, provided clinical samples and medical scientific
input and helped to draft the manuscript. OH participated in study design, provided clinical
samples and medical scientific input and helped to draft the manuscript. SK helped with study
design and coordination and helped to draft the manuscript. SJF conceived funding of the
study, helped with study design and coordination and helped to draft the manuscript. KW
designed and coordinated the study and helped to draft the manuscript. All authors approved a
final version of manuscript. Authors have no competing interests.
3.8 Acknowledgement
Funding for this research project was provided by the European Union through a Marie
Curie action (FPT7-PITN-GA-2009-238690-SPINEFX) and Theodor und Ida Herzog-Egli
Foundation. The authors would like to thank Ms. Greutert for technical assistance.
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65
Chapter 4
The natural polyphenol epigallocatechin gallate
protects intervertebral disc cells from oxidative
stress
Olga Krupkova*, Junichi Handa*, Marian Hlavna, Juergen Klasen, Caroline
Ospelt, Stephen John Ferguson, Karin Wuertz-Kozak
* these authors contributed equally
Oxidative medicine and cellular longevity, 2016, Jan(9): 1-17.
66
67
4.1 Abstract
Oxidative stress-related phenotypic changes and a decline in the number of viable cells
are crucial contributors to intervertebral disc degeneration. The polyphenol epigallocatechin 3-
gallate (EGCG) can interfere with painful disc degeneration by reducing inflammation,
catabolism and pain. In this study we hypothesized that EGCG furthermore protects against
senescence and/or cell death, induced by oxidative stress.
Sub-lethal and lethal oxidative stress was induced in primary human intervertebral disc
cells with H2O2 (total n = 36). Under sub-lethal conditions, the effects of EGCG on p53-p21
activation, proliferative capacity and accumulation of senescence-associated β-galactosidase
were tested. Further, the effects of EGCG on mitochondria depolarization and cell viability were
analyzed in lethal oxidative stress. The inhibitor LY249002 was applied to investigate the
PI3K/Akt pathway.
EGCG inhibited accumulation of senescence-associated β-galactosidase, but did not
affect the loss of proliferative capacity, suggesting that EGCG did not fully neutralize
exogenous radicals. Furthermore, EGCG increased the survival of IVD cells in lethal oxidative
stress via activation of pro-survival PI3K/Akt and protection of mitochondria.
We demonstrated that EGCG not only inhibits inflammation, but also can enhance the
survival of disc cells in oxidative stress, which makes it a suitable candidate for the
development of novel therapies targeting disc degeneration.
Key words
EGCG, epigallocatechin 3-gallate, oxidative stress, intervertebral disc degeneration, senescence,
apoptosis, H2O2, PI3K/Akt pathway
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4.2 Introduction
Degenerative disc disease, characterized by spinal micro-instability and lower back pain,
is a result of multiple events like age-related degradation of extracellular matrix (ECM),
inflammation or trauma [262, 263]. During degeneration, the intervertebral disc (IVD)
undergoes morphological and functional changes such as calcification of endplates, disruption
of annulus fibrosus (AF) and loss of water in the nucleus pulposus (NP), which not only impairs
disc function, but also decreases nutrient supply and causes accumulation of cellular waste
products [99, 264, 265]. In such situation cells can compensate for impaired homeostasis e.g. by
activation of neovascularization factors, to enhance the availability of nutrients. However, a
catabolic environment and abnormal blood supply in the originally avascular tissue can
increase reactive oxygen species (ROS) production and oxidative stress [94, 266-269].
ROS are highly reactive molecules with unpaired electrons formed externally or in
mitochondria as a normal part of the aerobic metabolism. To ensure a properly maintained
redox balance, organisms require a complex, coordinated network of antioxidants from various
sources that control ROS generation [270]. Cellular responses to ROS depend on the
concentration and duration of ROS exposure as well as the cell type. As signaling molecules,
ROS modulate a variety of physiological events, including proliferation, differentiation, host
defense and wound healing [271]. On the other hand, extensive ROS exposure and/or
insufficient cellular anti-oxidant capacity cause deleterious damage, senescence and cell death
[38].
Cellular senescence is described as an irreversible stress-induced cell cycle arrest [272].
Senescence followed by tissue remodeling is beneficial in healthy tissue, where it contributes to
the elimination of damaged cells. Furthermore, it is a major protective mechanism against
carcinogenesis. However, at the same time, persistent senescence impairs tissue function and
leads to inflammation and premature degeneration [90]. Internal DNA damage (telomere
shortening) as well as multiple stress signals, including ROS, aberrant oncogene activation and
chemotherapeutic drugs, are able to induce premature senescence via activation of the p53-p21
and/or p16 Ink4a [273]. Although cell and tissue aging have been extensively investigated, the
precise mechanisms underlying the development of a senescence phenotype are still unknown
[272].
Extensive oxidative stress disrupts the integrity of mitochondria, which can further
elicit cell death through three major mechanisms: During apoptosis (1), cytochrome c is
released into the cytosol where it activates caspase 9, which allows executioner caspases 3, 6
and 7 to cleave their substrates [274]. Caspase-independent cell death mechanisms, such as
regulated necrosis (2) or autophagy (3), are equally important in various pathologies, including
IVD degeneration [96, 275]. Similarly to senescence, the biological purpose of apoptosis is to
69
eliminate damaged or sub-optimal cells [90], although both processes are regulated differently
[272]. During apoptosis, a variety of triggers can quickly converge to the executioner effectors
through a common mechanism. In contrast, senescence is typically a delayed progressive
process, during which various effector mechanisms can each contribute and collectively define
the phenotype [272].
The functionality of human IVDs as spinal shock-absorbers gradually decreases
throughout lifetime. Senescence of IVD cells, which not only positively correlates with age, but
also with the degree of disc degeneration, can be linked to oxidative stress [3, 40-47]. Oxidative
stress-induced senescence and death of IVD cells contributes to the local tissue inflammation
and the expression of matrix degrading enzymes, hence accelerating loss of proteoglycans [39].
Extensive production of mitochondrial ROS during IVD cell death may further impair disc tissue
homeostasis [92-96]. Apart from oxidative stress, also other factors, like non-physiological
mechanical loading, an acidic environment or auto-immune reactions, can alter the cellular
phenotype and enhance premature senescence and cell death in the IVD [276-278].
Despite the fact that IVD degeneration and back pain represents an economic burden,
only symptomatic therapies, like analgesics or invasive surgeries, are currently available. Such
therapies do not target the biological causes of disc degeneration and either offer only
temporary relief or entail risks and complications. The focus of current research is to restore the
disc function via prevention of premature aging, inhibition of cell death, and enhancement of
ECM maintenance with an ultimate goal to prevent low back pain [56, 265]. One approach to
compensate the loss of functional disc cells is to apply stem cells embedded in diverse
biomaterials mimicking disc tissue, which may support the old disc population and
“rejuvenate” the aging disc. However, the implanted cells can in some cases undergo incorrect
differentiation, can induce inflammatory reactions and may not survive due to prior catabolic
environment [17, 279]. Another approach is to enhance survival of the existing intrinsic disc cells
using various pharmacological agents such as growth factors or inhibitors of inflammatory
pathways [265, 280].
We have previously shown that epigallocatechin 3-gallate (EGCG), a polyphenol
naturally occurring in green tea, inhibits inflammatory responses in IVD cells in vitro and
exhibit analgesic activity against disc-related radiculopathy in experimental animals [18]. The
aim of this in vitro study was to (1) further investigate whether EGCG exhibits protective effects
in mild and high oxidative stress and to (2) investigate the molecular mechanism involved.
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4.3 Methods
DPPH radical scavenging activity assay
Antioxidant activity of EGCG was determined by measuring its scavenging capacity of
the 2, 2’-diphyenyl-1-picrylhydrazyl (DPPH) radical as previously described [281]. 100 μl of 10-300
μM EGCG was incubated with 500 μl of DPPH (250 μM in ethanol; D9132 Sigma, Buchs,
Switzerland) for 1 hour at room temperature (n = 3). DPPH radical scavenging activity, which
manifests as a decrease in absorbance, was measured at 517 nm with a spectrophotometer
(Infinite M200 PRO, TECAN Group AG, Männedorf, Switzerland). L-Ascorbic acid (A4403, Sigma)
and ethanol (02860, Sigma) in equal amounts were used as positive and negative control,
respectively. DPPH radical scavenging activity (%) was calculated as [negative control optical
density (OD) – sample OD]*100 / negative control OD.
Cell isolation and cell culture
The study was approved by the cantonal ethic committee (Kantonale Ethikkommission
Zürich EK-16/2005). After informed consent was granted, human NP tissue (grade III-V) was
removed from donors undergoing spinal surgeries for degenerative disc disease or disc
herniation (n = 36). Details about the donors for this study are listed in Table 1. The tissue was
enzymatically digested using a mixture of 0.2 % collagenase NB4 (17454, Serva, Heidelberg,
Germany) and 0.3 % dispase II (04942078001, Roche, Basel, Switzerland) for 4-8 hours at 37 °C
and isolated primary cells were seeded in Dulbecco’s Modified Eagle’s Medium (DMEM/F12,
D8437, Sigma, St. Louis, MO, USA), supplemented with 10 % fetal calf serum (FCS, F7524, Sigma),
penicillin (50 units/mL), streptomycin (50 μg/mL) and ampicillin (125 ng/mL, 15240-062, Gibco,
Carlsbad, CA USA) and sub-cultured up to passage 3 using 1.5 % trypsin (15090- 046, Gibco).
Adherent cells in passage 1-3 were used for experiments. Due to the small biopsy size, low
proliferation rate and dedifferentiation of primary IVD cells in monolayer, a single donor cannot
be used for all required experiments. Therefore, different donors were randomly assigned to
the specific experiments. Cells were seeded on 6-well plates at a density of 1 × 105 cells per well
for all senescence experiments. Cells were seeded on 12-well plates at a density of 1 × 105 cells
per well for all lethal oxidative stress experiments, except JC-1 staining assay, which was
performed on 6-well plates. After seeding, cells were left to adhere overnight.
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Table 1. Intervertebral disc pathologies used for primary cell cultures. DDD = degenerative disc disease, PI/A = Propidium iodide/Annexin staining, MTT = MTT assay, MTT (LY) = MTT assay with LY, MTT (I) = MTT assay with insulin, uk = unknown.
Donor information Experiments
Number Gender, Age Level Pathology, Grade
Sub-lethal oxidative stress Lethal oxidative stress
1 M, 35 L4/L5 Herniation IV
H2O2 sensitivity study, p21 time course, senescence validation 10 days: additive effect
H2O2 sensitivity study PI/A
2 F, 77 L4/L5 DDD III
p21 time course 10 days: additive effect
JC-1, MTT
3 F, 53 L4/L5 DDD IV
H2O2 sensitivity study, p21 time course, senescence validation 10 days: additive effect
H2O2 sensitivity study PI/A, MTT
4 F, 39 L5/S1 Herniation V
H2O2 sensitivity study senescence validation 10 days: antioxidant
H2O2 sensitivity study PI/A, MTT)
5 F, 31 L4/L5 Herniation uk
H2O2 sensitivity study senescence validation
H2O2 sensitivity study JC-1, PI/A, MTT
6 F, 64 L4/L5 DDD III
H2O2 sensitivity study senescence validation
H2O2 sensitivity study JC-1, PI/A, MTT
7 F, 53 L5/S1 Herniation IV
- JC-1
8 M, 50 L4/5 DDD III
- JC-1
9 F, 54 L5/S1 Herniation V
10 days: recovery , RT qPCR -
10 M, 64 L5/S1 Herniation III
10 days: recovery , RT qPCR -
11 M, 44 L5/S1 Herniation IV
10 days: antioxidant, RT qPCR -
12 M, 56 L4/L5 Herniation III
10 days: recovery , RT qPCR -
13 F, 51 C4/5 Herniation III
10 days: antioxidant -
14 F, 42 L3/L4 Herniation III
10 days: antioxidant -
15 F, 35 L5/S1 DDD, Herniation IV
10 days: recovery -
16 M, 41 L4/L5 DDD III
10 days: additive effect -
17 M, 40 L5/S2 DDD, Herniation V
10 days: antioxidant -
18 M, 50 L4/L5 DDD, Herniation III
10 days: additive effect -
19 F, 42 L3/L4 Herniation III
10 days: recovery -
20 M, 43 L5/S1 DDD, uk
- JC-1, bright field, MTT, MTT (I)
21 F, 51 L4/L5 DDD IV
- JC-1, bright field, MTT, MTT (LY), MTT (I)
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Experimental design and treatments
Oxidative stress was induced with H2O2 (V800211, Sigma), which initiates the generation
of ROS that are able to attack all types of biomolecules [282, 283]. The active concentration of
H2O2 was selected based on the results of a preliminary H2O2 sensitivity study (n = 5). Increasing
concentrations of H2O2 (10-200 µM) were applied for 24 hours and cellular responses were
tested by MTT assay. The lethal concentration of H2O2 (100 µM and 200 µM for 24 hours) as well
as the sub-lethal concentration of H2O2, which can induce senescent changes without
significant cell death (50 µM H2O2 applied for 2 hours, followed by recovery period), were
identified. FCS-free media was used during all oxidative stress treatments to rule out the
possible interaction of radicals with FCS components. Previously reported non-toxic EGCG
concentrations (5 µM and 10 µM) were applied in this study [18].
22 M, 47 L4/L5 DDD, Herniation III
- JC-1, bright field, MTT
23 F, 44 L4/L5 DDD III
- JC-1, bright field, MTT, MTT (LY), MTT (I)
24 F, 43 L5/S1 DDD, Herniation III
- JC-1, bright field, MTT, MTT (LY), MTT (I)
25 M, 47 L4/5 DDD, Herniation III
- Lethal: MTT (LY), MTT (I)
26 M, 39 L4/5 DDD, Herniation IV
- Lethal: MTT (LY), MTT (I)
27 M, 21 L5/S1 Herniation III
- Lethal: MTT (LY), MTT (I)
28 F, 43 L4/5 DDD, Herniation IV
- Lethal: MTT (LY), MTT (I)
29 M, 33 L5/S1 Herniation III
- Lethal: WB
30 F, 39 L5/S1 DDD, Herniation III
- Lethal: MTT (LY), WB, MTT (I)
31 F, 39 L4/5 DDD, Herniation IV
- Lethal: MTT (LY), WB, MTT (I)
32 uk
uk - Lethal: MTT (LY), WB
33 M, 55 L3/4 DDD III
- Lethal: WB
34 F, 56 L3/4 Herniation IV
- Lethal: MTT (LY), WB
35 M, uk L4/5 Herniation uk
- Lethal: MTT (LY)
36 uk uk - Lethal: MTT (LY)
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The effect of EGCG (E4143, Sigma) on premature senescence was tested in three
different experimental setups to identify mechanism of EGCG’s action (Table 2): (1) to test its
antioxidant activity, 10 µM EGCG was applied in the stress phase simultaneously with H2O2, (2)
to test its effects on post-stress processes, 10 µM EGCG was added only in the recovery phase
and (3) to combine its potential benefits in both phases, 5 µM EGCG was added in the stress
phase and later again in the recovery phase.
The effect of 10 µM EGCG on cell death was tested directly in the stress phase, without
recovery period (Table 2). To further test the involved mechanism, LY294002 – an inhibitor of
the PI3K/Protein kinase B (Akt) pathway (LY, L9908, Sigma) – was applied at a concentration of
10 µM, along with EGCG (n = 10). PI3K/Akt activator insulin (I9278, Sigma) was used at a
concentration of 0.5 µl/mL to confirm functionality and specificity of LY in our experiments (n =
10).
Table 2. Experimental design
Sub-lethal oxidative stress – induction of premature senescence
Experimental setup Stress phase (2 hours)
Recovery phase (up to 15 days)
Tested EGCG effect
1. Antioxidant 50 µM H2O2 + 10 µM EGCG
- ROS neutralization
2. Recovery 50 µM H2O2
+ 10 µM EGCG interaction with post-stress signaling (e.g. changes in gene expression)
3. Combined 50 µM H2O2 + 5 µM EGCG
+ 5 µM EGCG ROS neutralization interaction with post-stress signaling
Lethal oxidative stress – cell death induction
Experimental setup Stress phase (24 hours)
Tested EGCG effect
1. Survival 100 and 200 µM H2O2
+ 10 µM EGCG pro-survival antioxidant
In vitro model system of premature senescence
Premature senescence was induced with sub-lethal H2O2. After seeding, cells were
starved in FCS-free medium for 2 hours before applying 50 µM H2O2 for another 2 hours. As a
next step, media was replaced by complete media (+ 10% FCS) to let the cells recover from
oxidative stress. Premature senescence was monitored for 15 days with media exchange on day
5 and 10. To verify senescence-associated changes, various tests were performed: Senescence-
associated β-galactosidase activity was tested on day 1, 5, 10 and 15 post-stress (n = 5). The
number of viable cells was determined by Trypan blue exclusion test on day 8 and 15 post-stress
(n = 5). Cellular metabolic activity was measured by MTT assay and expression/activity of
senescence-associated proteins p21 and p53 was analyzed by immunoblotting on day 15 post-
stress (n = 5). In separate experiment on day 8, trypsin-detached cells were reseeded again to
check their ability to adhere, which reflects general cellular fitness.
74
Senescence-associated SA β-galactosidase assay
Senescence-associated β-galactosidase (SA β-gal), which accumulates in aging cells,
serves as a reliable marker of senescence in vitro [284]. Senescence was induced with 50 µM
H2O2, followed by EGCG treatments as described above (n = 5 for each experimental setup). For
SA β-gal analysis, staining solution containing 40 mM Na3citrate x 2H2O + 5 mM K4Fe(CN)6 + 5
mM K3Fe(CN)6 + 0.15 M NaCl + 2 mM MgCl2 in H2O was prepared and its pH was adjusted to 6 by
adding 0.5 M NaH2PO4 (all from Sigma). X-gal (11680293, Roche) was dissolved in
dimethylformamide (33120, Sigma) to prepare a 20 mg/mL stock solution. Adherent cells were
washed twice with PBS and fixed with 3% paraformaldehyde (30525894, Acros, Geel, Belgium)
in PBS for 5 minutes at RT. After the fixation, cells were washed twice with PBS and mixture of
X-gal + staining solution in a 1:20 ratio was added. After 18 hours, the staining solution was
removed, cells were washed two times in distilled water and passed through graded ethanol
(75%, 95%, 99%), 1 minute each. After air drying, micrographs were taken from 3 random spots
of each well using light microscopy. The number of all cells (minimum 300) and SA β-gal
positive cells was counted by processing images with GIMP (http://www.gimp.org/) and
ImageJ (http://imagej.nih.gov/ij/). The percentage of SA β-gal positive cells in each treatment
group was calculated as the number of SA β-gal positive cells/number of all cells*100. SA β-gal
positive cells in the treatment groups were displayed relative to the control groups, due to the
high inter-donor variability in basal SA β-gal staining.
Trypan blue exclusion test
Trypan blue staining, which distinguishes viable and dying cells, was performed to
analyze the total number of viable cells at the end of each experiment. Senescence was induced
with 50 µM H2O2 and EGCG treatments were performed as described above (n = 5 for each
experimental setup). Cells were harvested using 1.5% trypsin into the complete media, an
aliquot was mixed 1:1 with 0.4% Trypan blue dye (93595, Fluka) and the cell suspension was
immediately analyzed on the grids of the hemacytometer (DHC-N01, DigitalBio). The absolute
number of non-stained (viable) cells was determined in each group to make a comparison with
the total number of seeded cells (1 × 105 cells per well). Non-viable cells, which took up Trypan
blue dye, were excluded from the analysis.
Metabolic activity measurement
Metabolic activity, which reflects cellular viability, was determined using the MTT assay.
Following seeding, sub-lethal or lethal oxidative stress was applied and the treatments were
performed as described above (n = 5 for sub-lethal oxidative stress experiments, n = 10 for
lethal oxidative stress experiment, n = 10 for inhibition experiments). After 24 hours or 10 days,
fresh MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, M5655, Sigma)
75
solution in media (0.5 mg/mL) was added and kept for 3 hours at 37°C. MTT was discarded, cells
were lysed in DMSO (D8418, Sigma) and the absorbance was measured at 565 nm. Metabolic
activity was calculated relative to the untreated control (100 %).
Immunoblotting
Treatments were performed as described above. Cells were harvested after 15 min
treatment for lethal oxidative stress experiments (n = 5) or after 10 days and 15 days for sub-
lethal oxidative stress experiments (n = 5). Whole cell lysates were prepared in RIPA buffer
(89900, Thermo Scientific, Waltham, MA, USA) according to producer’s instructions, mixed
with Laemmli buffer (S3401, Sigma), heated (99°C, 5 minutes), and then loaded onto 12% SDS
polyacrylamide gels. Separated proteins were transferred onto polyvinylidene difluoride (PVDF)
membranes (RPN303F, GE Healthcare, Little Chalfont, UK) and membranes were blocked in 5%
non-fat milk in Tris-buffered saline-Tween (TBS-T) for 1 hour at room temperature. Primary
antibodies were applied overnight at 4°C. After washing in 1% non-fat milk in TBS-T (3 × 10 min),
membranes were incubated with a secondary antibody conjugated to horseradish peroxidase
(HRP) for 1 hour at room temperature and washed in 1% non-fat milk in TBS-T (3 × 10 min).
Visualization was performed on medical X-ray film (28906836, GE Healthcare), using a
chemiluminescence kit West Dura (34076, Thermo Scientific) and films were scanned and
processed by GIMP. Tubulin was used as a loading control. Antibodies and dilutions: phospho-
p53 (Ser15) 1:1000 (9284, Cell Signaling, Danvers, MA, USA); p21 1:1000 (2947, Cell Signaling);
phospho-Akt (Ser473) 1:1000 (9271, Cell Signaling); Akt 1:1000 (9272, Cell Signaling); p16 Ink4a
(p16) 1:1000 (ab108349, Abcam); α-Tubulin 1:1000 (2144, Cell Signaling); mouse anti-rabbit IgG
HRP 1:5000 (7074, Cell Signaling).
Analysis of mitochondrial transmembrane potential (JC-1 staining)
JC-1 fluorochrome (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine
iodide) allows to monitor changes in mitochondrial transmembrane potential, which precedes
cell death. In physiological conditions, JC-1 accumulates in the intact mitochondrial membrane,
where it forms pronounced red aggregates. Once mitochondrial membrane potential is
disturbed, red fluorescence decreases, which is accompanied by an increase in green
fluorescence of the free JC-1 form. Substantial increase in the green fluorescence (= reduced
red/green ratio) suggests a breakdown of mitochondrial potential and activation of cell death.
Lethal oxidative stress was applied and EGCG treatment was performed as described
above (n = 10). After 24 hours, 1 µg/mL of JC-1 (T0046, Chemodex) diluted in FCS-free media was
added to the cells and kept at 37°C in the dark for 30 minutes. Then, cells were washed (3 × PBS)
and images of 3 random spots per well were taken immediately with a fluorescence microscope
76
(Olympus IX51, Volketswil, Switzerland). The ratio of red/green fluorescence was calculated
using ImageJ analysis (http://imagej.nih.gov/ij/).
Propidium iodide/Annexin V-FITC staining
The membrane of living cells is impermeable for Propidium iodide (PI), a red fluorescent
dye which intercalates into DNA. Annexin V-FITC (A) binds membrane phosphatidylserine,
which is exposed to the extracellular environment as one of the early apoptotic events. Flow
cytometry analysis of PI/A staining allows distinguishing viable cells (PI-negative/A-negative),
cells in early apoptosis (PI-negative/A-positive) and dead cells (PI-positive/A-positive) [285].
Lethal oxidative stress was applied and EGCG treatment was performed as described
above (n = 5). After 24 hours, supernatants were collected into pre-chilled tubes and placed on
ice. Cells were washed with PBS, detached from cell culture wells with 1.5% trypsin and
transferred into the corresponding pre-chilled tubes with matching supernatants. All the
further steps were performed on ice. Cell-supernatant mixtures were centrifuged (250 × g/5
min, 4°C), supernatants were discarded and 400 µL of Annexin Binding Buffer (556454, BD
Biosciences, Franklin Lakes, NJ, US) was added to the pellets and mixed. After the addition of 1
µL of PI (556463, BD Biosciences) and 2.5 µL of Annexin (556420, BD Biosciences), tubes were
kept in the dark for 15 minutes. Flow cytometry was performed immediately using the
cytometer FACS ARIA III (BD Biosciences). Results were analyzed with FlowJO
(http://www.flowjo.com/) and are displayed as percentage of viable cells (PI-/A-) in each
treatment group. Unstained cells, cells stained with Annexin V-FITC alone and cells stained
with PI alone were used as controls to set up quadrants.
Gene expression analysis (qRT-PCR)
The effect of sub-lethal oxidative stress on the activation of senescence-associated
secretory phenotype (SASP) was tested by means of gene expression of interleukin 6 (IL-6),
interleukin 8 (IL-8), matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 3 (MMP3), and
matrix metalloproteinase 13 (MMP13). These genes are typically expressed in a pro-
inflammatory and catabolic environment during inflammation-related disc degeneration [82,
229]. IVD cells were seeded on 6-well plates and senescence was induced as described above (n
= 4). After 24 hours, RNA was extracted with the Trizol/chloroform method according to the
manufacturer instructions (15596-018, Invitrogen, Carlsbad, CA, USA) and 1 μg was reverse
transcribed to cDNA using a reverse transcription kit (4374966, Applied Biosystems). cDNA was
then mixed with primers and master mix (4352042, Applied Biosystems) and gene expression
was measured using real-time PCR. The following primers were used: TATA box binding protein
(TBP) Hs00427620_m1, IL-6 Hs00174131_m1, IL-8 Hs00174103_m1, MMP1 Hs00233958_m1, MMP3
Hs00968308_m1 and MMP13 Hs00233992_m1. Data was analyzed with the comparative Cq
77
method (2-ΔΔCq, housekeeping gene TBP). Results are presented as a fold change relative to the
untreated control group.
Statistical analysis
A power calculation (80% power) determined the required sample size (n = 5). Data
normality was tested by Schapiro-Wilk test. Statistical significance between two groups was
analyzed using Student’s t-test. Statistical significance between more than two groups was
evaluated by ANOVA with Tukey post-hoc test. The number of donors used for each experiment
is listed in the methods section. Mean and SEM are displayed in graphs and asterisks represent
a significance level of p ˂ 0.05.
4.4 Results
Validation of the in vitro model system of premature senescence of IVD cells
Based on the sensitivity study, 50 µM H2O2 applied for 2 hours and followed by a
recovery period up to 15 days was chosen to induce premature senescence of IVD cells without
significant cell death (Supplementary figure 1). Analysis of senescence-associated changes was
performed on day 1, 5, 8, 10 and 15 post-stress. In the H2O2 treatment group, the number of SA β-
gal positive cells increased significantly from day 1 (12.67±2.05%) up to day 15 (49.53±4.58%)
post-stress, compared with the untreated controls (Figure 1A). Representative images of SA β-
gal staining on day 8 and reseeded cells on day 9 are shown (Figure 1B). Reseeded cells in the
H2O2 treatment group were able to adhere, which confirmed a good general cellular fitness;
however, altered cellular morphology suggested an aging cell population.
Phosphorylation of the stress-induced protein p53 (Ser15) and enhanced expression of
the cell cycle inhibitor p21 on day 15 in the H2O2 treatment group indicated oxidative damage
(Figure 1C). In order to compare the replicative potential of stressed and control cells, the living
cells were counted on day 8 and 15 by Trypan blue exclusion test. Cell number on day 8 in the
H2O2 group (1.53±0.16 × 105 cells/well) was significantly lower than in the control group
(2.63±0.18 × 105 cells/well), confirming oxidative stress-induced loss of proliferative capacity.
Cell death in the H2O2 group on day 8 did not occur, as the number of cells was higher than the
cell seeding density on day 0 (1 × 105 cells/well, marked as red line) (Figure 1D). However, on day
15, the number of cells in the H2O2 treatment group decreased below the seeding density
(0.68±0.13 × 105 cells/well versus 1 × 105 cells/well, marked as red line), indicating that the cells
slowly started to die from the ROS exposure (Figure 1E). As cell death may hamper data
interpretation, all subsequent experiments were performed for a maximum 10 days.
78
The expression of inflammatory genes in the H2O2 treatment group, measured on day 1,
was not significantly elevated (Supplementary figure 1).
Figure 1. In vitro model system of stress-induced premature senescence. Sub-lethal oxidative stress (50 µM H2O2) with subsequent recovery period activated premature senescence of IVD cells in vitro. (A) Percentage of SA β-gal-positive cells in the H2O2 treatment group gradually increased during 15 days post-stress (n = 5). (B) Upper part: representative images of SA β-gal staining of the untreated (ctrl) and the H2O2-treated cells on day 8 post-stress, showing senescent (blue) cells. (B) Lower part: representative images of reseeded cells on day 9, confirming general cellular fitness. (C) Phosphorylation of p53 (Ser15) and expression of p21 in the H2O2 treatment group on day 15 post-stress indicated cellular senescence. (D, E) Proliferative capacity, displayed as number of cells on day 8 and 15 post-stress, was reduced in the H2O2 groups. On day 15, the number of cells in the H2O2 treatment group decreased below the seeding number (1 × 105 cells per well, depicted as red line), suggesting ongoing cell death. Asterisks indicate statistical significance at p < 0.05 (ANOVA, Tukey post-hoc, Student’s t-test).
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Figure 2. As an antioxidant, EGCG inhibited senescence-associated β-galactosidase accumulation. (A) EGCG exhibited increasing 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity between 10 and 100 µM, which confirmed its antioxidant properties. Ascorbic acid in the same concentration was used as positive control (n = 3). (B-D) Oxidative stress was induced with 50 µM H2O2 for 2 hours and cellular senescence was measured during following 10 days. 10 µM EGCG inhibited SA β-gal accumulation when added to the oxidative stress phase, when its antioxidant activity was confirmed (n = 5). (C) 10 µM EGCG added to the recovery phase did not influence SA β-gal accumulation compared to the H2O2-only group (n = 5). (D) EGCG combined in both phases (5+5 µM) did not significantly inhibit SA β-gal accumulation, although a trend is visible (n = 5). Asterisks indicate statistical significance at p < 0.05 (ANOVA, Tukey post-hoc).
EGCG inhibited senescence-associated β-galactosidase accumulation in sub-lethal oxidative
stress
Previously reported antioxidant properties of EGCG were confirmed using 2,2-diphenyl-
1-picrylhydrazyl (DPPH) radical assay. EGCG exhibited dose-dependent radical scavenging
activity between 10 and 100 µM. As common antioxidant, ascorbic acid at the same
concentrations was used as positive control (Figure 2A) [286]. Apart from its antioxidant effects,
EGCG may also possess other beneficial functions. Therefore, the effect of EGCG on the
accumulation of SA β-gal was tested in 3 different experimental setups displayed in Table 2,
namely (1) antioxidant activity by adding EGCG in the stress phase, (2) activity on post-stress
signaling by adding EGCG in the recovery phase and (3) potential combined effect by adding
EGCG in both phases.
The percentage of SA β-gal positive cells was evaluated on day 1, 5, and 10 post-stress.
EGCG significantly inhibited the accumulation of SA β-gal on day 5 and 10 when added during
the stress phase, indicating that an antioxidant mechanism is involved (Figure 2B). EGCG did
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not influence the rate of SA β-gal accumulation during the recovery phase, suggesting that the
interaction of EGCG with post-stress signaling is less relevant (Figure 2C). The presence of EGCG
in both phases did not provide significant protection against oxidative stress, which is most
likely caused by insufficient ROS neutralization, as lower concentrations of EGCG (5 µM) were
used in the stress phase of this setup. However, an apparent trend towards statistical
significance was detectable (Figure 2D).
EGCG did not protect IVD cells from loss of proliferative capacity in sub-lethal oxidative stress
The effects of EGCG on proliferation and expression of the senescence markers p53 and
p21 were also tested in each experimental setup: (1) EGCG as an antioxidant, (2) EGCG in
recovery and (3) combined effects. In order to compare the proliferative capacity of H2O2-
trerated and H2O2+EGCG-treated cell populations, the number of viable cells on day 10 of
recovery period was determined in each treatment group. Untreated and EGCG-treated cells
proliferated, but all stress groups showed significantly lower cell numbers, regardless of the
presence of EGCG (Figure 3A-C). On day 10 post-stress, p53 was slightly phosphorylated in all
stress groups. Expression of p21 was induced by H2O2, but also not affected by the presence of
EGCG, hence indicating that exogenous ROS were not entirely neutralized by 10 µM EGCG
(Figure 3D-F).
As both non-senescent and senescent cells are metabolically active, detected metabolic
activity on day 10 corresponds to the number of cells in each treatment group: stress groups
showed significantly lower metabolic activity than untreated controls. No significant difference
between the H2O2 and H2O2+EGCG treatment groups was detected in either experimental
setup, confirming that EGCG did not protect IVD cells from loss of proliferative capacity (Figure
3G-I). Metabolic activity of cells on day 10 is displayed relative to the control group (100%).
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Figure 3. EGCG did not influence reactive oxygen species-induced loss of proliferative capacity. Senescence of IVD cells was induced with 50 µM H2O2 for 2 hours and proliferative capacity was evaluated on day 10 post-stress. (A-C) Over a period of 10 days the H2O2-treated cells proliferated significantly less than the cells in control groups. The number of cells in the H2O2 and H2O2+10 µM EGCG treatment groups did not significantly differ in either experimental setup (n = 5). (D-F) The activity of p53 and the expression of p21 was not significantly affected by EGCG in either experimental setup (n = 5). (G-I) Metabolic activity in the H2O2 and the H2O2+EGCG treatment groups did not significantly differ, indicating reduced proliferative capacity in all stress groups (n = 5). Asterisks indicate statistical significance at p < 0.05 vs. control group (ANOVA, Tukey post-hoc).
EGCG protected IVD cells from lethal oxidative stress via inhibition of mitochondrial
membrane depolarization
Based on the sensitivity study, 100 µM and 200 µM H2O2 applied for 24 hours was
identified to trigger significant cell death (Supplementary figure 1). As such ROS doses may
induce excessive mitochondria membrane depolarization and unrepairable damage, the
general pro-survival effect of EGCG can be tested.
Extensive oxidative stress (100 µM H2O2) significantly decreased the metabolic activity
of IVD cells (51.49±5.64%), compared with the control group (100%), while the addition of EGCG
reversed this effect (80.58±4.31%) (Figure 4A). More specific PI/A staining confirmed
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significantly higher number of living cells in the H2O2+EGCG treatment group (52.91±8.02%)
compared to the H2O2-only group (28.31±5.83%) (Figure 4B).
During lethal oxidative stress mitochondria loses its transmembrane potential, which
leads to the release of mitochondrial ROS and serves as a pro-apoptotic signal. EGCG may
possibly interact with ROS on the mitochondrial membrane and prevent further ROS leakage.
To test this hypothesis, the JC-1 dye, which forms red aggregates in intact mitochondria, was
used. H2O2 in a concentration of 100 µM (Figure 5A) and 200 µM (Figure 5B) caused extensive
mitochondrial membrane depolarization, while addition of EGCG prevented this effect. The
red/green fluorescence ratio of the untreated (10.19±4.54) and the EGCG-treated cells
(8.15±3.29) was significantly higher than the ratio of both H2O2 treatment groups (100 µM:
1.85±0.96, 200 µM: 0.09±0.07). Addition of EGCG significantly increased the ratio in H2O2
treatment groups to 6.05±1.14 (100 µM) and 0.63±0.2 (200 µM) (Figure 5D). Membrane blebbing
and nuclear shrinkage was detected in the H2O2+EGCG treatment group, confirming that the
cells were not completely protected from deleterious ROS and cell death was possibly only
delayed (Figure 5C).
Figure 4. EGCG significantly inhibited reactive oxygen species-induced cell death. Cell death was induced by lethal concentration of H2O2 (100 µM) applied for 24 hours. (A) 10 µM EGCG significantly reversed the detrimental effects of H2O2 on metabolic activity, measured by MTT assay (n = 10). (B) 10 µM EGCG also significantly increased cell viability in oxidative stress, as measured by Propidium Iodide/Annexin staining (n = 5). (C) Visualization of representative Propidium Iodide/Annexin V staining measured by flow cytometry. Asterisks indicate statistical significance at p < 0.05 (Student’s t-test).
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Figure 5. EGCG inhibited the loss of mitochondrial membrane potential. Cell death was activated by lethal concentrations of H2O2 (100 µM, 200 µM) applied for 24 hours. (A) 10 µM EGCG significantly inhibited mitochondrial membrane depolarization induced with 100 µM H2O2 (n = 10). (B) 10 µM EGCG significantly inhibited mitochondrial membrane depolarization induced with 200 µM H2O2 (n = 10). (C) Cellular morphology of the H2O2+EGCG treatment group was different from the control group: signs of membrane blebbing and nuclear condensation indicated that cell death was not completely inhibited (n = 5). (D) Ratio of red/green fluorescence showing the loss of mitochondrial membrane potential in the H2O2 treatment groups. Asterisks indicate statistical significance at p < 0.05 (ANOVA, Tukey post-hoc).
EGCG increased survival of IVD cells in lethal oxidative stress via PI3K/Protein kinase B (Akt)
activation
To further explore the mechanism underlying action of EGCG, activity of the pro-
survival PI3K/Akt pathway was studied. As shown by immunoblotting, EGCG activated Akt
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under oxidative stress conditions (Figure 6A). The biological relevance of this finding was tested
using the PI3K/Akt-specific inhibitor LY294002 (LY). Depending on the type of stimulus, Akt can
activate various downstream signaling pathways inducing proliferation, glucose metabolism or
survival. Akt activator insulin, which stimulates cell proliferation, was used as positive control
to confirm the activity and specificity of LY. As expected, insulin modestly increased the
proliferation of IVD cells (117.79±2.91% versus 100% in control), whereas the addition of LY
abolished this effect (108.16±2.86%), hence confirming the activity of LY for further PI3K/Akt
pathway tests (Supplementary figure 1).
PI3K/Akt inhibition with LY did not significantly alter the pro-survival effect of EGCG at
24 hours, as measured by MTT assay (Figure 6B). However, at longer time point (48 hours), the
difference in the metabolic activity between the H2O2+EGCG (80.01±8.93%) and the
H2O2+EGCG+LY treatment group (34.81±4.69%) became significant (Figure 6C), indicating that
EGCG-based activation of the PI3K/Akt pathway plays indeed an important role in the survival
of IVD cells under the oxidative stress.
Figure 6. PI3K/Akt is important for the protective function of EGCG under the lethal oxidative stress. Cell death was activated by 200 µM H2O2. (A) After 15 minutes of co-treatment, 10 µM EGCG activated PI3K/Akt (n = 5). (B) After 24 hours, no significant difference in metabolic activity between the H2O2+EGCG and the H2O2+EGCG+LY groups was detected (n = 5). (C) After 48 hours, LY completely abolished the protective effects of EGCG, underlying the importance of the PI3K/Akt pathway in the survival of IVD cells
(n = 5). Asterisks indicate statistical significance at p < 0.05 (ANOVA, Tukey post-hoc).
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4.5 Discussion
A stress-related decrease in the number of viable cells and phenotypic changes of living
cells are two major events in the process of IVD degeneration that warrant therapeutic
targeting [276]. Oxidative stress-induced senescence and cell death in vitro can represent
mechanisms involved in human disc aging in vivo [3, 39, 41]. We hypothesized that the natural
polyphenol EGCG can inhibit oxidative stress-induced senescence and/or cell death of IVD cells
and we investigated which mechanisms may be involved in its action.
In vitro as well as in vivo, EGCG can directly interact with ROS or operate in an indirect
manner by inhibiting ROS generating enzymes or by chelating potentially pro-oxidant metal
ions [31]. Nevertheless, antioxidant activity is not the only biological effect of EGCG. In fact,
many of its health-beneficial functions are independent of redox reactions. Depending on the
cell type, EGCG can interact with proteins and phospholipids in the plasma membrane, regulate
signal transduction and gene expression, DNA methylation, modulate mitochondrial function,
and autophagy [48].
Results of the radical scavenging assay confirmed the previously reported antioxidant
activity of 10 µM EGCG, which can neutralize 32.89±6.39% of 250 µM DPPH radicals. Although
higher concentrations of EGCG exhibited better ROS trapping, they were not used in this study
to avoid possible unfavorable interactions with cell culture media components. Dose-
dependent oxidation of polyphenols and subsequent ROS generation was reported as common
artifact of in vitro cell culture [287, 288]. The thus induced toxicity has no significance in vivo,
because of the presence of plasma antioxidants, but can interfere with research hypotheses via
increasing unspecific stress in cell culture. From this point of view, 10 µM EGCG was considered
active, yet experimentally safe.
We developed an in vitro model system of premature senescence of IVD cells, induced
by sub-lethal oxidative stress. Due to the vastly heterogeneous nature of the senescence
phenotype, the use of various markers is required to precisely identify senescence in a given cell
population [272]. The expression of certain markers, such as p21, can also increase during in
vitro cell culture as an artifact (Supplementary figure 1). Senescent cells commonly undergo cell
cycle arrest, start expressing SA β-gal, exhibit a senescence-associated secretory phenotype
(SASP) and activate p53-p21 and/or p16-RB pathways [40, 90]. Morphology of senescent cells
can differ from their young counterparts: cells become bigger, with prominent nucleoli and
numerous cytoplasmic vacuoles [289]. Sub-lethal doses of the widely used oxidative stress
inducer H2O2 inhibited proliferation and activated p53-p21 pathway and SA β-gal accumulation
in IVD cells, proving the suitability of the system to test potential anti-senescence compounds.
EGCG did not influence the progress of senescence when applied during the recovery
phase, suggesting no interaction with post-stress signaling. During recovery after oxidative
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stress, cells can undergo several changes, one of which can be the activation of gene expression
towards the senescence-associated secretory phenotype (SASP). The SASP is mediated e.g. by
the transcription factor nuclear factor κB (NF‑κB) and many other redox-sensitive transcription
factors, which can induce the expression of pro-inflammatory cytokines, proteases and
catabolic enzymes [90, 270, 280]. We have shown before that EGCG inhibited IL-1β-activated
inflammatory responses in the IVD [18], implying that EGCG may act via similar mechanisms
also in the ROS-activated SASP. However, sub-lethal oxidative stress in our settings did not
increase the expression of inflammatory markers (IL-6, IL-8) and catabolic enzymes (MMP1,
MMP3, MMP13). Therefore, the effect of EGCG on SASP could not be evaluated (Supplementary
figure 1). Previously, stress-induced SASP was activated in IVD cells under different culture
conditions [40], in our setup either longer time point or higher ROS concentration will be
needed.
As an antioxidant, 10 µM EGCG inhibited accumulation of SA β-gal, but did not protect
IVD cells from loss of proliferative capacity, suggesting that the exogenous radicals were not
entirely neutralized. The increase in SA β-gal expression/activity results from the need to
compensate for the accumulation of damaged macromolecules and organelles in lysosomes. As
the expression of SA β-gal is not required for senescence, we hypothesize that EGCG in
antioxidant setup can ameliorate the increase in lysosome dysfunction, rather than inhibiting a
complex senescence phenotype. In accordance, EGCG has been shown to promote
macroautophagy and lysosome recycling of accumulated intracellular materials [290, 291].
Nevertheless, the role of EGCG in enhancing cellular waste-disposal mechanisms is still
controversial and presumably cell-type specific.
Since EGCG inhibited SA β-gal accumulation in antioxidant setup and not in recovery
setup, one could expect similar responses when combining EGCG in both stress and recovery
phase. However, in the third setup, SA β-gal assay showed only a trend towards the statistical
significance, possibly because lower EGCG concentration was applied in the stress phase, in
order to avoid potential toxic effects mentioned above [292].
As p16 is another important senescence marker, its expression was initially tested in IVD
cells treated with EGCG in antioxidant settings, on day 10 (n = 4). Because of the fact that p16
expression in H2O2-treatment groups was detected only in 1 out of 4 donors, the experiments
on p16 expression were not continued (Supplementary figure 2). In fact, the relative
contributions of the p53-p21 and p16-RB pathways to the senescence growth arrest may vary,
depending on the type and level of stress [91]. Moreover, in different cell types the transition of
temporal to stable cell-cycle arrest can involve either an activation of p21, or p16, or an
activation of both [91]. Human IVD cells are generally slowly proliferating and responding cell
type, in which ROS can possibly induce p16 with delayed kinetics. p16, if activated, subsequently
provides a second barrier to stop cell proliferation and plays a role in maintaining senescence,
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rather than in its initiation, as suggested before [293, 294], which may explain its low
expression in our study.
To test whether EGCG can influence the expression of p16, SAOS-2 cells, with
constitutive expression of p16, were treated with 10 µM EGCG for 24 hours. This experiment
showed no influence of EGCG on the expression of p16 in SAOS-2 (Supplementary figure 2).
Lethal oxidative stress causes unrepairable damage on mitochondrial membranes,
leading to extensive ROS leakage and quick activation of death signals [295] (Supplementary
figure 3). Two independent viability assays revealed that EGCG can significantly increase
survival of IVD cells in lethal oxidative stress. As a primary target of oxidative stress,
mitochondria quickly lose their membrane integrity. We demonstrated that EGCG can delay
the onset of mitochondrial membrane depolarization, hence protecting mitochondria from ROS
leakage. Similar antioxidant effects of EGCG were previously described, e.g. in the mice liver-
injury in vivo model [296] or in the rat retinal primary cells in vitro [297].
As an exogenous oxidizing agent, H2O2 produces hydroxyl radicals, which can efficiently
react with all cellular components. On the other hand, damaged mitochondria generate mainly
superoxide, followed by lower amounts of hydrogen peroxide and hydroxyl radicals [298, 299].
Therefore, EGCG can affect mitochondrial ROS leakage by being a good superoxide anion
radical scavenger [299], but it may neutralize other ROS species less efficiently (Supplementary
figure 3). As previously reported, EGCG can also activate superoxide dismutase expression,
another mechanism to counteract exclusively superoxide anion radicals and to protect
mitochondria [300, 301].
Interestingly, EGCG activated PI3K/Akt under lethal oxidative stress, an ability that was
important for IVD cell survival. We suggest that the reason why EGCG alone did not activate
PI3K/Akt pathway can be an absence of the stress signal (H2O2). In non-stressed cells, the
activity of pro-survival pathways (such as PI3K/Akt) is negatively regulated by their inhibitors
(such as PTEN). In stressed cells, the activity of intracellular kinase inhibitors can be reduced
[302] in order to facilitate activation of the pro-survival kinases. However, the pro-survival
kinases can fully function only upon external activation [303]. Interestingly, it has been shown
that EGCG interacts with various membrane receptors [48, 304]. We therefore hypothesize that
EGCG-mediated interaction upstream of Akt, in combination with stress-mediated reduction in
the activity of intracellular kinase inhibitors, can lead to complete PI3K/Akt activation, hence its
pro-survival effects in H2O2-treated cells.
Previously, Akt gene expression has been correlated with increased disc cell proliferation
in vitro [305] and lumbar disc herniation in vivo [306]. More importantly, active PI3K/Akt
signaling enhanced pro-survival capacity of disc cells in vitro [307] and antagonized disc
degeneration in vivo [308]. The PI3K/Akt pathway can therefore represent another attractive
therapeutic goal in disc degeneration, which can be targeted by EGCG.
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In this study, 2D cell culture was employed to ensure applicability of cell analysis assays.
Adherent in vitro system, in contrast to 3D, reduced cell loss during analyses, which is
particularly important for stress experiments. The 2D system also allowed quick and precise
staining of organelles in living cells. While results are very clear, it remains open whether cells
react similarly in their natural 3D environment. This study is furthermore limited by the inter-
donor variability of disc tissue biopsies, originating from differences in age and degeneration
grades [309]. Nevertheless, the use of primary cells during first three passages represents a
system more close to human tissue than a stabilized cell line. The inter-donor variability issue
can be corrected by displaying results relative to a control group, instead of using absolute
numbers. The main third limitation is that the effect of EGCG during the recovery phase of
senescence was tested in complete media, which could possibly influence the activity of EGCG.
4.6 Conclusions
This study showed that EGCG can counteract oxidative stress-induced changes of IVD
cells in vitro via protection of the mitochondrial membrane from depolarization. In oxidative
stress, EGCG also activated PI3K/Akt as an important pro-survival mechanism of IVD cells. As
this study did not provide direct evidence whether and how the EGCG-activated Akt and
mitochondria protection are linked, investigation of this coupling in IVD cells will be an
objective of future research.
Together with previously reported anti-inflammatory, anti-catabolic and analgesic
effects of EGCG our findings can be further used for the development of novel therapies
targeting consequences of oxidative stress and neovascularization in degenerative disc disease.
4.7 Authors’ contributions
OK carried out sub-lethal stress experiments, participated in lethal stress experiments,
contributed to study design and drafted the manuscript. JH participated in lethal stress
experiments, performed pathway analysis and statistical analysis. MH performed gene
expression experiments and statistical analysis. JK participated in study design, provided
clinical samples and medical scientific input. CO conceived funding, participated in study
design and provided scientific input. SJF conceived funding, helped with study design,
coordination and helped to draft the manuscript. KW conceived funding, designed and
coordinated the study and helped to draft the manuscript. All authors approved a final version
of the manuscript.
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4.8 Acknowledgement
The authors would like to thank the Herzog-Egli Foundation and the Mäxi Foundation
for financial support, Sonia Rossi for preliminary H2O2 tests and Helen Greutert and ETH FACS
core facility for technical support. The authors have no conflict of interests.
4.9 Supplement
Supplementary figure 1. H2O2 sensitivity study, p21 expression during cell culture period, the effect of H2O2 on senescence-associated secretory phenotype and confirmation of the LY294002 activity/specificity. IVD cells were treated with different concentrations of H2O2 to determine the cellular metabolic activity by MTT assay. (A) After 24 hours 50, 100 and 200 µM H2O2 caused a significant decrease in the metabolic activity. Thus concentrations 100 and 200 µM were selected for lethal oxidative stress induction (n = 5). (B) Metabolic activity of IVD cells treated with 50 µM H2O2 for 2 hours did not change within 3 days, therefore this concentration was chosen for sub-lethal oxidative stress induction (n = 5). (C) The expression of p21 on day 1, 5, 10 and 15 was tested by immunoblotting, with
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etoposide (10 µM, 72 hours) as a positive control. p21 expression increased during the cell culture period as an artifact of the in vitro environment (n = 3). (D) Sub-lethal oxidative stress (50 µM H2O2, 2 hours) did not increase the expression of inflammatory markers (IL-6, IL-8) and catabolic enzymes (MMP1, MMP3, MMP13) on day 1 post-stress (n = 4). (E) Akt activator insulin at 0.5 µl/mL increased proliferation of IVD cells whereas addition of 10 µM LY294002 (LY) abolished this effect, confirming functionality of LY for further tests involving Akt. Asterisks indicate statistical significance p < 0.05 (ANOVA, Tukey post-hoc).
Supplementary figure 2. The expression of p16 Ink4a in oxidative stress-induced senescence of IVD cells. Senescence was induced with 50 µM H2O2 for 2 hours and 10 µM EGCG was added directly to the stress phase (antioxidant experimental setup). The expression of p16 was measured on day 10 post-stress with SAOS-2 cells as positive control. (A) The expression of p16 is constitutively active in SAOS-2 cells. The expression of p16 was not induced by H2O2 in 3 out of 4 donors, therefore the effects of EGCG could not be studied (n = 4). (B) The constitutive expression of p16 in SAOS-2 cells is not affected by 10 µM EGCG.
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Supplementary figure 3. The role of reactive oxygen species and mitochondria in senescence and apoptosis of IVD cells (schematic). (A, C) Sub-lethal oxidative stress causes damage of cellular macromolecules, which activates the p53-p21 pathway and growth arrest, without significant mitochondrial depolarization. During growth arrest, cells can repair the damage or irreversibly enter senescence, depending on various factors, such as activity of cellular antioxidants and inflammatory pathways. (B, D) Lethal oxidative stress leads to extensive mitochondrial membrane depolarization and subsequent mitochondrial ROS leakage, which induces apoptosis. Based on our data, EGCG can protect mitochondrial membrane from ROS leakage and prevent subsequent activation of cell death cascade. (C, D) The ratio of red/green fluorescence, with a loss of mitochondrial membrane potential in the H2O2
treatment groups is shown. Data presented in (D) are replicated from Figure 5D to emphasize the difference between the mitochondrial membrane potential in senescence and apoptosis. Asterisks indicate statistical significance at p < 0.05 (ANOVA, Tukey post-hoc) (n = 5).
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Chapter 5
An inflammatory nucleus pulposus tissue
culture model to test molecular regenerative
therapies: Validation with epigallocatechin 3-
gallate
Olga Krupkova, Marian Hlavna, Julie Amir Tahmasseb, Joel Zvick, Dominik
Kunz, Keita Ito, Stephen J. Ferguson, Karin Wuertz-Kozak
Under review in the International Journal of Molecular Sciences
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5.1 Abstract
Organ cultures are practical tools to investigate regenerative strategies for the
intervertebral disc. However, most existing organ culture systems induce severe tissue
degradation with only limited representation of the in vivo processes. The objective of this
study was to develop a space- and cost-efficient tissue culture model, which represents
degenerative processes of the nucleus pulposus (NP). Intact bovine NPs were cultured in a
previously developed system using Dyneema jackets. Degenerative changes in the NP tissue
were induced either by the direct injection of chondroitinase ABC (1–20U/mL) or by the
diffusion of IL-1β and TNF-α (both 100ng/mL) from the culture media. Extracellular matrix
composition (collagens, proteoglycans, water, DNA) and the expression of inflammatory and
catabolic genes were analyzed. The anti-inflammatory and anti-catabolic compound
epigallocatechin 3-galalte (EGCG, 10µM) was employed to assess the relevance of the
degenerative NP model. Although a single injection of chondroitinase ABC reduced the
proteoglycan content in the NPs, it did not activate cellular responses. On the other hand, IL-1β
and TNF-α significantly increased the mRNA expression of inflammatory mediators (IL-6, IL-8,
iNOS, PTGS2) and matrix metalloproteinases (MMP1, MMP3, MMP13). The cytokine-induced
gene expression in the NPs was ameliorated with EGCG. This study provides a proof of concept
that inflammatory NP cultures, with appropriate containment, can be useful for the discovery
and evaluation of molecular therapeutic strategies against early degenerative disc disease.
Keywords
nucleus pulposus, organ culture model, degenerative disc disease, cytokines, chondroitinase
ABC, epigallocatechin gallate, inflammation, extracellular matrix
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5.2 Introduction
The intervertebral disc (IVD) consists of three structurally and functionally different
tissues: the outer annulus fibrosus (AF), the inner nucleus pulposus (NP), and two cartilaginous
endplates that connect the disc with the adjacent vertebrae [1, 56]. IVD degeneration is caused
by a combination of many events throughout a lifetime, including non-physiological loading,
endplate calcification, inflammation, immune reactions, cell senescence, and cell death [1-3],
and occurs initially and predominantly in the NP.
The inflammatory cytokines interleukin-1 beta (IL-1β) [82, 84] and tumor necrosis factor
alpha (TNF-α) [28] are key molecules involved in IVD degeneration. The events that promote
their production by NP and AF cells, especially in the absence of trauma or herniation, can be
chronic stress, spinal infection or free swelling, i.e. during herniation [2, 81, 310]. IL-1β and TNF-α
bind to their cell surface receptors (e.g. IL-1R1, TNFR1), which activate the signaling mediators
NF-κB, JNK, and p38 MAPK, resulting in the transcription of inflammatory and catabolic genes
such as interleukins (IL-1α, IL-1β, IL-6, IL-8), matrix metalloproteinases (MMP1, MMP3 and
MMP13), aggrecanases (ADAMTs) and other relevant molecules that can promote inflammation
and pain (PTGS2, NGF, iNOS) [2, 18, 86, 88]. In addition to the catabolic and pro-inflammatory
effects, IL-1β and TNF-α can influence disc cell senescence, autophagy and the expression of
genes involved in proliferation [80]. MMPs and aggrecanases further promote degradation of
the extracellular matrix (ECM) [85-87]. Changes of the NP ultimately result in a loss of disc
hydration, disc height and load-bearing capacity at later stages, when the AF is also affected [4,
60]. These accelerated pathological processes with structural failure and the development of
low back pain, e.g. due to nerve compression and/or chemical irritation, are together termed
degenerative disc disease, a distinct entity to the process of physiological ageing [4-7].
Despite an increasing prevalence of discogenic back pain and the consequently high
economic burden [9], only therapies targeting symptoms are currently available [10, 11]. Most of
the current research on novel treatments focuses on restoring the function and homeostasis of
degenerated discs via inhibition of inflammation, prevention of premature ageing, and
augmentation of the ECM content. Cell therapies (e.g. mesenchymal stem cells) can support
the existing cell population and “rejuvenate” the aging disc [15, 16]. However, newly implanted
cells are at risk of undergoing apoptosis, due to the surrounding inflammatory and catabolic
environment [17]. Inflammation, catabolism and matrix degradation in the disc can be
controlled using molecular therapeutics, such as anti-inflammatory compounds [18, 19], growth
factors [20, 150] and N-terminus of Link protein (LinkN) [21]. Application of growth factors like
bone morphogenetic proteins (BMP-2, BMP-7), insulin growth factor (IGF-1) or tumor growth
factor (TGF-β3) can induce the formation of new matrix and improve the mechanical properties
of the disc [147]. However, drawbacks of exogenous growth factors are their short half-life
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(hours to days) and possibly only transient effects. Another complication is their large size,
which can limit their diffusion through the NP [109].
Diverse signaling pathways are deregulated in disc degeneration. Therefore, small
molecules with multiple effects, targeting both survival and function of resident disc cells, are
promising therapeutic candidates. Compounds derived from traditional medicinal plants act on
several signal transduction and apoptotic cascades at the same time, while having minimal
side effects [22]. We have shown before that polyphenols such as resveratrol [170], curcumin
[171], triptolide [173] and epigallocatechin 3-gallate (EGCG) [18, 311] exhibit anti-inflammatory,
anti-catabolic and antioxidant activity in disc cells. Although more research is needed to fully
understand the effects of polyphenols on the integrity of the IVD, it has been suggested that
they can delay degeneration of the NP or even reverse it. Polyphenols can also enhance disc
nutrition via improving vascular health [174] and relieve radicular pain [18, 170].
A number of organ culture models have been developed over the past years to facilitate
testing of novel regenerative treatments. Unlike cell cultures, organ culture models retain
native ECM and allow for mechanical loading [23]. Induction of degeneration in these models
commonly is achieved by injection of matrix degrading enzymes such as trypsin [24, 25] and
papain [312, 313]. Enzymatic tissue damage allows evaluation of the efficiency of cell therapies,
but is less efficient for testing molecular therapeutics. Cells injected or implanted within a
supportive scaffold can fill in the missing ECM, proliferate, produce new matrix and restore
tissue properties. However, while ECM degradation is often extensive, and thus higher than
clinically observed, no or limited cellular inflammatory and catabolic reactions have been found
in these enzymatic models. This suggests that in vivo disc degeneration is not fully mimicked.
Mechanical damage such as endplate fracture [26] or stab incision in animal models [314] can
activate cellular responses in the disc more reliably than non-physiological proteolytic enzymes.
Recently, a stab injury model, based on motion segments isolated from transgenic animals
with an NF-κB-luc reporter, was developed. Longitudinal monitoring of NF-κB activity in this
model can provide insights into the mechanisms that regulate IVD inflammatory responses to
stab injury [315]. At the same time, physiologically relevant cytokine-induced inflammatory
organ culture models, without involvement of mechanical damage, have emerged [27-29].
Whole disc organ cultures with biologically relevant ECM degradation and inflammatory
responses are certainly most suitable for testing cell therapies and regenerative compounds.
However, although whole-disc organ cultures have many advantages, they typically do not
allow for high throughput experiments due to space-related demands and sample
heterogeneity. For this reason, a nucleus pulposus (NP) culture in Dyneema jackets has been
recently developed [30], (further referred to as “NP culture”). The Dyneema jackets (also called
“artificial AF”) restrain swelling, while maintaining diffusion of nutrients from culture media.
This bovine NP culture was previously shown to preserve a glycosaminoglycan (GAG) content
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and gene expression pattern similar to native tissue over sustained periods, which makes it
suitable for further use [30]. The objective of this work was to adapt the existing NP culture as a
novel platform for testing regenerative treatment strategies.
We hypothesized that (1) physiological degradation of the ECM can be induced by injection
of the biologically relevant enzyme chondroitinase ABC and that (2) degenerative responses can
be activated by diffusion of IL-1β and TNF-α from culture media, hence allowing for the creation
of a suitable testing platform for novel disc therapeutics. (3) Furthermore, we hypothesized that
the anti-inflammatory and anti-catabolic compound EGCG will ameliorate this degenerative
response. Using bovine caudal NPs for preclinical testing is not only cost-effective, but also in
accordance with the 3R animal welfare recommendations.
5.3 Methods
Bovine nucleus pulposus tissue culture
NP cultures were prepared as previously described, with minor changes [30]. Bovine tails
were obtained from the local slaughterhouse (18-24 month old cows), muscles and fat were
removed and vertebrae were cut to obtain individual motion segments of the first 4 – 5 intact
proximal discs. The motion segments were sterilized with betadine for 20 minutes and further
washed under sterile conditions with 70% ethanol. Intervertebral discs were cut transversally,
close to the endplate, and nucleus pulposus tissue was isolated using an 8-mm biopsy punch
(8437995, Polymed) and scalpel (Scheme 1A). NP biopsies were placed in 1.5 mL tubes and
weighed.
For NP pre-shrinkage, dialysis membranes (Spectrapor 15 kDa MWCO/18 mm, 734-0507,
VWR) were washed in PBS, opened with tweezers and one NP each was pushed inside using a
custom-made cylindrical metal device. The Spectrapore membranes with NPs were then placed
into membrane clips, closed and allowed to shrink in a 6-well plate with 5 ml of 30% w/v
polyethylene glycol (PEG) solution in PBS for 100 minutes (Scheme 1B). After that, the
membrane clips were opened, the membranes were cut open and the NP biopsies were
weighed again. NP biopsies were placed on a regenerated cellulose membrane (97010-104
RC58, 0.2 µm pore size, GE) and folded into the membrane. Then NPs were inserted into the
custom-made Dyneema jacket (UHMWPE fiber, Dyneema, DSM, The Netherlands). The open
end of the jacket was twisted once and turned inside-out again over the sample. This step was
repeated once more to create three layers of the jacket, preventing excessive swelling. A sterile
needle and thread was used to suture the jacket closed (Scheme 1C).
The encapsulated NP cultures were placed into 6-well plates with 10 mL of DMEM/F-12
(D8437, Sigma, St. Louis, MO, USA) supplemented with 10% FCS (F7524, Sigma), penicillin (100
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units/mL), streptomycin (100 μg/mL) and ampicillin (250 ng/mL, 15240-062, Gibco). 2% FCS was
used for experiments with IL-1β and TNF-α. NP tissues were cultured for up to 21 days. At each
time point (day 3, 7, 14, 21), NP cultures were harvested for analysis, weighed, immediately
frozen in liquid nitrogen and stored at -80°C.
Scheme 1. Preparation of bovine NP cultures. (A) Bovine tails were dissected and NP tissue was isolated using an 8-mm biopsy punch. (B) NP tissue was placed in 30% w/v PEG solution (100 minutes) to reduce water content. (C) NPs packed in regenerated cellulose membranes and Dyneema jackets were sutured and placed in culture media.
Induction of nucleus pulposus degeneration
To induce early degenerative changes of NP tissue, two approaches were tested (1) enzyme
treatment and (2) cytokine treatment. In the first approach, NPs were injected with
chondroitinase ABC (1 - 20 U/mL), which selectively removes chondroitin-4-sulphate, chondroitin-
6-sulphate and dermatan sulphate, the major components of proteoglycans [316].
Chondroitinase ABC is a known inducer of disc degeneration in vitro and in vivo [316, 317].
However, it is not known whether chondroitinase ABC can activate inflammatory responses in NP
tissues. Lyophilized chondroitinase ABC from Proteus vulgaris (120 kDa, C3667, Sigma) was diluted
according to the producer’s instructions in PBS and 1% BSA to 20 U/mL and stored as a stock
solution at -20ºC until use. 20 µl of 1, 5, 10 or 20 U/mL chondroitinase ABC was injected with a 27G
needle in the center of the NPs before packing in Dyneema jackets. In the second approach,
human recombinant TNF-α (17 kDa, 100 ng/mL, PHC3016, Gibco) and bovine recombinant IL-1β (31
kDa, 100 ng/mL, RBOIL1BI, Pierce) were added to the culture media on day 0 and 7. Re-swelling of
the NP in the Dyneema jacket to its initial weight is thought to allow the media with IL-1β and
TNF-α to actively enter the NP tissue. Samples were collected for analyses of degenerative
responses and ECM composition on days 0 (native), 3, 7, and 14. Epigallocatechin 3-gallate (EGCG,
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10 µM), which inhibits inflammatory and catabolic responses in IVD cells in vitro [18], was selected
to test the relevance and applicability of the degenerative NP culture model for therapeutic
testing. The experimental setup is depicted in Scheme 2. The number of NP cultures used for each
experimental group, treatment, and type of analysis are listed in Table 1.
Scheme 2. Experimental setup. (A) NP tissues were cultured up to 21 days to confirm the system’s stability. (B) Chondroitinase ABC was injected into NP cultures. (C) TNF-α and IL-1β were added into the culture media. Gene expression and extracellular matrix (ECM) composition were analyzed on days 0, 3, 7, 14, and/or 21. Histological analysis was performed to confirm GAG depletion in the enzyme treatment group. Epigallocatechin gallate was used to validate cytokine treatment group.
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Table 1. Number of NP cultures used for each experimental group, treatment, and type of analysis. ECM = extracellular matrix.
Experimental groups Analyses Treatments Gene expression ECM composition Native tissue n = 8
n = 5
Stable NP culture Day 3: n = 8 Day 7: n = 8 Day 14: n = 8 Day 21: n = 8
Day 3: n = 4 Day 7: n = 4 Day 14: n = 4 Day 21: n = 4
Enzyme treatment 1. PBS 2. Chondroitinase ABC 1 U/mL 3. Chondroitinase ABC 5 U/mL 4. Chondroitinase ABC 10 U/mL 5. Chondroitinase ABC 20 U/mL
Day 3: n = 4 / treatment Day 7: n = 4 / treatment Day 14: n = 4 / treatment
Day 3: n = 7 / treatment Day 7: n = 5 / treatment Day 14: n = 5 / treatment
Cytokine treatment 1. Control 2. TNF-α + IL-1β (100 ng/mL) 3. EGCG (10 µM) 4. TNF-α + IL-1β + EGCG
Day 3: n = 5 / treatment Day 7: n = 5 / treatment Day 14: n = 7 / treatment
Day 3: n = 4 / treatment Day 7: n = 4 / treatment Day 14: n = 4 / treatment
Total n = 321 NPs
RNA isolation
RNA was extracted with the classical TRIzol/chloroform method, but optimized for bovine
discs. Each bovine NP sample was quickly ground and pulverized in liquid nitrogen using
custom-made grinders. The NP powder was transferred into 1 mL of TRIzol (15596018, Thermo
Scientific) and the mixture was further homogenized with a polytron 4 times/20 seconds
(POLYTRON® PT 10/35 GT). During homogenization, samples were kept on ice. Homogenized
samples were transferred to 15 mL tubes with 9 mL of fresh TRIzol, mixed and left at room
temperature for 5 minutes. Then 2 mL of chloroform (C7559, Sigma) was added, samples were
vortexed and left 10 minutes on a shaker. After shaking, samples were centrifuged (4000g/10
minutes) and the upper phase (5 mL) was transferred into new 15 mL tubes before adding 10 mL
of 2-propanol (I9516, Sigma), followed by mixing, vortexing and centrifugation after 5 minutes
at room temperature (4000g/10 minutes). Isopropanol was discarded, 1 mL of 70% ethanol
(51976, Sigma) was added, and samples were transferred into 2 mL tubes and centrifuged
(7500g/5 minutes). If the pellet contained white proteoglycans, samples were thoroughly
vortexed for 2 minutes and a second TRIzol/chloroform isolation in smaller volume (2 mL tube)
was performed for additional purification. Purified samples were centrifuged (7500g /5
minutes), ethanol was aspirated and the pellets were dried for 5 minutes at room temperature.
Dry pellets were then mixed with 30 µL of RNase-free water and immediately frozen at -80°C
until further analysis.
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Gene expression (RT-qPCR)
As RNA yields were low, due to the low cellularity of the tissue and the high amount of
proteoglycans [318], the whole RNA solution (30 µL) was reverse transcribed to cDNA using a
reverse transcription kit (4374966, Applied Biosystems). Resulting cDNA was diluted 3-5 times
in RNase free water, mixed with primers and master mix (4352042, Applied Biosystems) and
gene expression was measured by real-time PCR (CFX96 Touch™ Detection System, Biorad).
Bovine TaqMan primers for interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1β), tissue
inhibitor of matrix metalloproteinases (TIMP1), matrix metalloproteinase 1 (MMP1), matrix
metalloproteinase 3 (MMP3), matrix metalloproteinase 13 (MMP13), aggrecanase 1 (ADAMTS4),
prostaglandin synthase 2 (PTGS2), inducible nitric oxid synthase (iNOS), collagen 1, collagen 2
and aggrecan were employed to study the inflammatory and catabolic phenotype. S18, Actin
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as housekeeping genes
(Table 2). Obtained Cq values were analyzed by the comparative method (2-ΔΔCq) and displayed
as fold change to control or relative to IL-1β and TNF-α stimulation.
Table 2. TaqMan primers and sequence numbers used for qPCR analysis.
Gene Primer Seq. N. Gene Primer Seq. N. S18 Bt03225193_m1 ADAMTS4 Bt03224693_m1 GAPDH Bt03210919_g1 MMP1 Bt04259497_m1 Actin-β Bt03279174_g1 MMP3 Bt04259495_m1 Interleukin-6 Bt03211904_m1 MMP13 Bt03214052_m1 Interleukin-8 Bt03211908_m1 TIMP1 Bt03223720_m1 Interleukin-1β Bt03212741_m1 Collagen 1 Bt03214883_m1 iNOS Bt03249584_m1 Collagen 2 Bt03251861_m1 PTGS2 Bt03214492_m1 Aggrecan Bt03212186_m1
Extracellular matrix composition (glycosaminoglycans, hydroxyproline, DNA, water)
NP samples were dried at 60°C overnight and weighed to determine dry weight. The dried
samples were then digested in papain buffer (55mM Na-Citrate, 150mM NaCl, 5mM EDTA, pH
6) with 5 mM L-cystein and 125 µg/mL papain from papaya latex (P-3125, Sigma) at 60°C
overnight. The digested samples were used to quantify the content of sulfated
glycosaminoglycans (GAG), collagen (hydroxyproline) and DNA. GAGs were measured using
dimethylmethylene blue (DMMB) assay with chondroitin sulfate as standard (C6737, Sigma)
[319]. Samples were mixed with DMMB stock solution (10.5 mg DMMB, 2.5 ml absolute ethanol,
1.0 g sodium formate in 500 ml dH2O, pH 1.5) in a 96-well plate and the absorbance was
measured at 595 nm. The collagen content was analyzed with the hydroxyproline (HYP) assay
kit (MAK008, Sigma) and DNA was determined using the Quant-iT PicoGreen dsDNA assay kit
(P11496, Thermo Fischer) according to the manufacturer’s recommendations. The amounts of
GAG, HYP, DNA and water were expressed per mg dry or wet weight of the tissue (for stable NP
culture) or relative to the control (for degeneration groups) [30].
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Histology
After harvesting, NPs were fixed in 4% buffered paraformaldehyde (48 hours, 5
mL/sample). After fixation, specimens were washed, dehydrated and stored in 75% ethanol
[320]. Before paraffin embedding, samples were cut transversally into two halves. After
embedding, 3 µm sections were prepared with a microtome (CryoStar NX70, ThermoScientific)
and stained with Alcian blue/Picosirius red and Safranin O. Images were taken with a CCD
camera mounted on an Olympus IX51 microscope (Volketswil, Switzerland).
Statistical analysis
All data sets were tested for normal distribution with the Shapiro-Wilk test and found to
be normally distributed. A Student’s t-test (2 groups) and a one-way ANOVA with Tukey post-
hoc test (more than 2 groups) were performed to detect statistical significance of differences at
p < 0.05.
5.4 Results
Nucleus pulposus tissue in Dyneema jackets is stable over a period of 21 days
A stable amount of GAG, collagen, DNA and water reflects healthy extracellular matrix.
These parameters were determined in the NP cultures on day 3, 7, 14 and 21 and compared with
native tissue (day 0). The content of GAG, collagen, DNA and water in the NP cultures did not
significantly differ from the native tissue, confirming stability of the ECM [30] (Figure 1A-D).
Gene expression of MMP3 and TIMP1 increased on days 3 and day 7, which may reflect a tissue
response to culture conditions. Their expression was downregulated at later time points (Figure
1E). Genes for other matrix degrading enzymes (MMP1, MMP13) and interleukins (IL-6, IL-8, IL-1β)
were not detectable. In comparison with the native tissue, gene expression of aggrecan and
collagens decreased between day 0 and day 3, which can be explained by the absence of
physiological loading [30]. After this initial down-regulation, the expression of ECM genes
remained stable (Figure 1E). Average Cq ± SD values for genes expressed in the stable NP
cultures are listed in Table 3.
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Figure 1. Bovine NP culture preserves extracellular matrix over a period of 21 days. (A) Glycosaminoglycan, (B) collagen, (C) DNA and (D) water content on day 3, 7, 14 and 21 did not differ from the native values (day 0). Graphs show the percentage of dry or wet weight relative to the native tissue (mean ± SEM, n = 4 per group). (E) Gene expression of MMP3 and TIMP1 increased and subsequently returned close to the native levels. Gene expression of aggrecan, collagen 1 and collagen 2 was down-regulated after NPs were placed in culture, but remained stable thereafter. Graph shows fold change relative to the native tissue (mean ± SEM, n = 4-8 per group). *p < 0.05 vs. native tissue, #p < 0.05 vs. each other (ANOVA). Table 3. Average Cq ± SD values for all expressed genes in stable NP cultures (day 3).
Gene mean Cq ± SD Gene mean Cq ± SD
S18 28.24 ± 5.22 Collagen 2 26.92 ± 2.85
GAPDH 29.69 ± 1.78 Aggrecan 27.13 ± 3.33
Actin-β 30.02 ± 2.98 MMP3 33.65 ± 3.55
Collagen 1 35.52 ± 3.14 TIMP1 27.78 ± 2.49
Chondroitinase ABC reduces proteoglycan content in the NP
As chondroitinase ABC removes chondroitin sulfate from proteoglycans, a dose- and/or
time-dependent decrease in sulfated GAGs was expected. Compared with the PBS injection, the
injection of 20 U/mL chondroitinase ABC significantly reduced GAG content at all tested time
points (Figure 2A). No major alterations in collagen, DNA and water content were detected over
a period of 14 days (Figure 2B-D). Alcian blue/Picrosirius red and Safranin O staining confirmed
that 20 U/mL of chondroitinase ABC depleted proteoglycans, whereas 1 U/mL chondroitinase
ABC retained a macromolecular composition closer to the native tissue (Figure 2E-F). However,
RT q-PCR revealed no profound changes in the expression of MMP3 TIMP1 and ECM
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macromolecules. Chondroitinase ABC downregulated the expression of collagen 2 and
aggrecan, but the expression of collagen 1 remained unchanged (Figure 3A-C). Genes for other
matrix degrading enzymes (MMP1, MMP13) and interleukins (IL-6, IL-8, IL-1β) were not
detectable.
Figure 2. Chondroitinase ABC reduces proteoglycan content in the NP. (A) As expected, the glycosaminoglycan content significantly decreased in the NPs injected with 20 U/mL Chondroitinase ABC compared with the NPs injected with PBS (set as 1). (B) Collagens, (C) DNA content and (D) water content remained unchanged. Graphs show ECM composition relative to the PBS injection (mean ± SEM, n = 4 per group). (E) Alcian blue/Picrosirius red and (F) Safranin O staining confirmed that 1 U/mL chondroitinase ABC did not have major proteoglycan-degrading effects, whereas 20 U/mL reduced GAG content at all tested time points. Scale bar 100 µm. *p < 0.05 vs. PBS injection, #p < 0.05 vs. each other (ANOVA).
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Figure 3. Chondroitinase ABC does not activate cell reactions in the NP. (A-C) Injection of 1 - 20 U/mL chondroitinase ABC reduced gene expression of aggrecan and collagen 2 in the NP cultures on day 3, 7 and 14. Gene expression of MMP3 increased on day 3, but was downregulated at later time points. Gene expression of TIMP1 and collagen 1 remained close to the control levels. Inflammatory and catabolic markers were not detectable. Graphs show fold change relative to the PBS injection (mean ± SEM, n = 4). *p < 0.05 vs. PBS injection, #p < 0.05 vs. each other (ANOVA).
IL-1β and TNF-α induce gene expression of inflammatory and pain-related markers in the NP
Cytokines IL-1β and TNF-α are known activators of inflammatory and catabolic cascades in
the IVD and their expression may affect ECM composition at later stages. However, during 14
days of culture, IL-1β and TNF-α did not influence GAG, collagen, DNA and water content in the
NPs (Figure 4A-D). On day 3 and 7, cytokine-treated NPs expressed low levels of MMP3, TIMP1
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and ECM macromolecules, similarly to the stable and enzyme-treated NP cultures (Figure 4E).
Cytokines significantly induced the expression of pro-inflammatory interleukins (IL-6, IL-8),
collagenases (MMP1, MMP3, MMP13), their inhibitor TIMP1, and pain-related mediators (iNOS,
PTGS2) on day 14 (Figure 4F). These results indicate that a catabolic/inflammatory shift towards
a degenerative phenotype developed between day 7 and day 14. Changes in the expression of
the ECM macromolecules may suggest remodeling attempts in response to the inflammatory
stimuli. Average Cq ± SD values for all genes in all treatment groups are shown in
Supplementary table 1.
Figure 4. IL-1β and TNF-α induce an inflammatory and catabolic phenotype in the NPs. (A) Glycosaminoglycan, (B) collagen, (C) DNA and (D) water content in cytokine-treated NPs remained unchanged over a period of 14 days, when compared with the untreated group (set as 1). Graphs show ECM composition relative to untreated samples (mean ± SEM from n = 4). (E) Inflammatory and catabolic gene expression was not detectable on day 3 and 7, and the expression of MMP3, TIMP1 and ECM macromolecules were close to the control values (fold change). (F) On day 14, IL-1β and TNF-α significantly induced gene expression of interleukins (IL-6, IL-8), collagenases (MMP1, MMP3, MMP13) and their tissue inhibitor (TIMP1), aggrecanase 1 (ADAMTS4) and pain mediators (PTGS2, iNOS). Graphs show fold change relative to the untreated samples (mean ± SEM from n = 4-7). *p < 0.05 vs. untreated control, #p < 0.05 vs. each other (ANOVA).
Epigallocatechin 3-gallate ameliorates the inflammatory and catabolic phenotype in the NP
As a significant up-regulation of inflammatory and catabolic genes was detected in the IL-
1β and TNF-α-treated NP cultures on day 14, this experimental group was subjected to EGCG
treatment. Culture media was supplemented with EGCG (10 µM) on day 0 and day 7. Neither
EGCG alone nor in combination with IL-1β and TNF-α significantly influenced the composition of
the ECM (Figure 5A-D) or the gene expression of ECM macromolecules, but EGCG inhibited the
expression of inflammatory markers (IL-6, IL-8), catabolic enzymes (MMP1, MMP3), and pain
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mediators (iNOS, PTGS2). This not only provided evidence on the activity of EGCG in the NP
tissue, but also proved the suitability of the inflammatory NP cultures for evaluating the effects
of potential molecular therapeutics (Figure 5E).
Figure 5. Epigallocatechin 3-gallate ameliorates inflammatory and catabolic responses in the NPs. (A) Glycosaminoglycan, (B) collagen, (C) DNA and (D) water content in the NPs treated with EGCG, IL-1β and TNF-α, and their combination remained unchanged over a period of 14 days. Graphs show ECM composition relative to the untreated samples (mean ± SEM from n = 4). (E) On day 14, EGCG significantly reduced the expression of interleukins (IL-6, IL-8), collagenases (MMP1, MMP3) and pain mediators (PTGS2, iNOS) in cytokine-treated NPs. Gene expression of ECM components remained close to the control values. Graphs show mRNA expression relative to the cytokine-stimulated samples (mean ± SEM from n = 4-7). *p < 0.05 vs. IL-1β and TNF-α treatment (t-test).
5.5 Discussion
Throughout a lifetime, lumbar intervertebral discs undergo a morphological and functional
decline. Early signs of disc degeneration, caused by impaired exchange of solutes and activated
catabolic reactions, occur in the NP [110]. In some individuals, NP degeneration leads to internal
disc disruption and annulus fibrosus damage, which can result in low back pain. Although
discogenic back pain constitutes a rising socioeconomic burden for western countries,
therapies targeting its biological causes are lacking. The aim of this study was to develop an
organ culture model, which will allow preclinical screening of novel molecular therapeutics for
the degenerative nucleus pulposus.
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A previously validated system that employs bovine caudal NPs cultured in Dyneema jackets
(“artificial AF”) was used [30]. Although bovine caudal discs are not weight bearing, they have
been proposed as a suitable biological model for human disc degeneration, due to their close
size and similar in vivo pressure (0.1–0.3 MPa) [321]. The bovine NP culture potentially represents
a space-efficient and relatively inexpensive approach to screen for molecular therapeutics that
can be used in human medicine. Bovine and human NP cultures were used for investigating the
effects of ECM anabolics such as LinkN, notochordal cell-conditioned medium [148] and BMP-7
[322]. However, the stable bovine NP culture exhibits neither an inflammatory phenotype nor
ECM degradation. Inducing biologically relevant degenerative changes in this system will
provide a platform for testing compounds that not only induce ECM production, but also
interfere with inflammation, catabolism and pain on the cellular level.
Although validated, NP cultures may not completely preserve the native tissue
characteristics, as a decrease in GAG content and an increase in gene expression of MMPs have
been described, especially at later time points [30]. Therefore, our initial experiments aimed to
test NP culture stability over a period of 21 days, to evaluate its relevance for development
towards a degenerative phenotype. Similarly to the previously published study, we observed an
initial downregulation of ECM genes and a transient increase in the expression of MMP3, likely
caused by a lack of mechanical loading. As stabilization of these parameters followed, the NP
culture was considered suitable for optimization towards a degenerative model.
ECM degrading enzymes that are commonly used to induce proteoglycan depletion in the
NP are the protein hydrolases trypsin and papain. However, as there is no naturally occurring
proteolysis in the disc, these enzymes do not activate cell reactions such as the expression of
interleukins and MMPs, but induce excessive matrix degradation, with a low physiological
fidelity. On the other hand, enzymes that degrade extracellular matrix in a biologically relevant
manner can possibly induce cellular responses that are found during disc degeneration.
Recently MMP3, ADAMTS4, and HTRA1 were injected into bovine discs and cultured for 8 days.
Even though these enzymes did not affect matrix degradation and gene expression, they
reduced cellular metabolic activity. HTRA1 additionally caused a loss of disc height and was able
to induce MMP expression in vitro [323, 324]. In our study, chondroitinase ABC with lyase
activity was employed to facilitate GAG depletion. Chondroitinase ABC degrades
polysaccharides, reproducing age-related removal of chondroitin sulfate from aggrecan [49].
However, although injection of chondroitinase ABC accelerated degradation of GAGs in a
biologically relevant manner, it was not sufficient for activation of inflammation and
catabolism in the NP tissue. This suggests that ECM degradation is a consequence, rather than
a cause of degenerative disc disease and confirms previous observations that injection of the
ECM degrading enzyme alone does not entirely mimic the degenerative process. Therefore,
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chondroitinase ABC-injection into a NP culture is not entirely suitable when the aim is to create
a tissue culture system for testing bio-active compounds.
The involvement of pro-inflammatory cytokines in painful degenerative disc disease is well
established [2]. TNF-α and IL-1β can act as nociceptive triggers as well as induce the expression of
other potentially nociceptive interleukins (such as IL-6, IL-8, IL-12, IL-17) and direct pain mediators
(nitric oxide, prostaglandin E, substance P) [66, 80, 102-104, 106]. Degenerated discs with newly
formed nociceptive nerve fibers can thus be a primary source of back pain [61]. Moreover, disc
degeneration also can result in compression of nerve roots and spinal nerves. IL-1β, TNF-α and
other pain-mediating chemicals released from the NP can irritate dorsal root ganglions,
contributing to functional and structural nerve damage, radiculopathy and muscle weakness in
vivo [55, 66, 122]. Replicating such an inflammatory phenotype in organ cultures is therefore
needed for a successful drug discovery process.
In the study of Purmessur et al. (2013), TNF-α (200 ng/mL) activated the expression of
inflammatory and catabolic genes and accelerated degradation of aggrecan in bovine explants
[28]. Similarly, in another study, IL-1β up-regulated pro-inflammatory markers (IL-6, IL-8,
prostaglandin E2) and MMPs in punctured bovine discs, compared with disc organ cultures
treated with needle puncture alone [29]. In our study, a combination of IL-1β and TNF-α (100
ng/mL each) strongly induced the expression of interleukins (IL-6, IL-8), collagenases (MMP1,
MMP3, MMP13), aggrecanase (ADAMTS4) and molecules associated with pain sensation (iNOS,
PTGS2). The inflammatory and catabolic phenotype in our NP cultures fully developed on day 14,
possibly due to the dense proteoglycan network in initially healthy bovine NP tissue with lower
diffusivity, which can slow down cytokine penetration [109]. In smaller rat lumbar discs (± 0.5
cm in diameter), TNF-α and IL-1β activated the expression of MMPs and ECM changes already
after 3 days [50], most probably due to shorter diffusion distances [321]. Mechanical loading of
the bovine NP cultures in future studies may aid to promote cytokine penetration and activate
responses at earlier time points.
IL-1β and TNF-α-induced inflammation did not affect composition of the ECM over 14 days.
It has been shown before that healthy disc cells respond to stress by upregualtion of matrix
metalloproteinase inhibitors (TIMPs) [325], which inhibit the activity of MMPs in a 1:1
stoichiometry. In particular, TIMP-1 forms a complex with the catalytic domain of MMP-3,
reducing its activity in the NP tissue [323]. Therefore, the inflammation-related loss of ECM in
our model is expected to occur at later time points. Although ECM degradation has not been
observed in the cytokine-treated group, the current inflammatory bovine NP model can be used
to screen therapeutics against early stages of disc degeneration, before the loss of ECM occurs.
Another advantage of our bovine NP culture model is that molecular diffusion distances are
relevant to human NPs [321]. An interesting option would be to combine chondroitinase ABC
injection with cytokine treatment. Biologically relevant proteoglycan depletion, accompanied
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by inflammatory and catabolic cell reactions, could accurately reproduce more advanced stages
of NP degeneration. Possibilities to modulate various stages of degeneration in the NP cultures
are currently being investigated in our group.
In the past, we have identified the polyphenol epigallocatechin gallate (EGCG) as a
promising compound for the treatment of painful degenerative disc disease. EGCG inhibited
the expression of inflammatory markers (IL-6, IL-8, PTGS2, TLR2, iNOS), collagenases (MMP1,
MMP3, MMP13) and NGF in human IVD cells cultured adherently and in alginate beads [18].
EGCG also protected IVD cells from oxidative stress caused by exogenous radicals [311] and
moreover, locally delivered EGCG reduced radicular pain in a rat model of NP herniation [18].
Therefore, we selected this compound for validation of the new inflammation-induced NP
culture model of degeneration. EGCG treatment of NP cultures exposed to IL-1β and TNF-α
reduced the expression of interleukins, MMPs, iNOS and PTGS2, which confirmed our results
from previous in vitro tests. In addition to in vitro studies, the NP culture system revealed no
negative effects of EGCG on the extracellular matrix, further supporting future research on this
compound. As EGCG ameliorated the expression of inflammatory markers in the tissue, the NP
culture system proved suitable for preclinical testing of bio-active compounds and their
combinations as well as delivery systems. Although the effects of EGCG have been shown
before in cell culture studies, it is necessary to include testing on the tissue level. While cell
cultures are suitable to evaluate mechanism of action, they are less relevant to determine
overall efficacy of compounds, due to the possible effects on the extracellular matrix. As human
NP contains less than 1% cells [60], an organ culture study should be performed in order to
assess the clinical relevance of EGCG. To our knowledge, this is the first study presenting the
effects of EGCG on inflammatory intervertebral disc tissue.
In comparison with animal models, organ culture systems represent a cost-effective
alternative for discovery and testing of novel therapeutic compounds. Organ cultures can
bridge basic science with translational medicine and allow in vitro experiments while
maintaining cells in their native tissue [23]. NP cultures confined in Dyneema jackets exhibit
rather stable characteristics over sustained periods of time. Moreover, accessibility and low
costs of bovine tissue, as well as the possibility to modulate the phenotype from early to
advanced NP degeneration, make this model attractive. Although more research is needed, we
conclude that these advanced NP cultures can be suitable for the screening of novel
regenerative compounds and for revealing their mechanisms of action in the NP tissue.
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5.6 Conclusions
Organ culture models are valuable tools for studying therapeutic strategies against
degenerative disc disease. However, it is challenging to develop a cost-effective organ culture
model that contains all main hallmarks of degenerative disc disease and is at the same time
biologically relevant. In this study, an inflammatory NP culture model was established and
validated with the anti-inflammatory compound EGCG. This NP culture model expresses
inflammatory, catabolic, and pain-related genes that are involved in the progression of human
degenerative disc disease. As composition and molecular diffusion distances of the bovine NP
are comparable to the human NP, obtained results are applicable to human. Moreover, this
model is cost-effective and allows high throughput tests for molecular therapeutics. ECM
anabolic agents and compound combinations can be tested in the NP cultures, in which TNF-α
and IL-1β-induced inflammation will be combined with Chondroitinase ABC-induced loss of
GAG.
5.7 Author’s contributions
OK performed experiments (NP cultures, ECM analyses, RNA isolations, RT-qPCRs), data
analysis, helped with the study design, and drafted the manuscript. MH performed
experiments (NP cultures, ECM analyses, RNA isolations and RT-qPCRs) and helped with the
study design. JAT performed NP culture experiments and RNA isolations. JZ and DK performed
NP culture experiments and ECM analyses. SJF conceived funding, helped to draft the
manuscript and coordinated the study. KW conceived funding, designed and coordinated the
study and helped to draft the manuscript. KI introduced the NP culture systems and helped to
conceive the study aims. All authors approved a final version of manuscript.
5.8 Acknowledgement
Funding for this research project was provided by Sciex (Project 14.145) and the Mäxi
Foundation. The authors would like to thank Ms. Ladina Ettinger-Ferguson for histological
analysis and to acknowledge Simon Zollinger for the images of the bovine NP cultures. The
authors wish to confirm that there are no known conflicts of interest associated with this
publication.
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5.9 Supplement Supplementary table 1. Average Cq ± SD values for all expressed genes in inflammation group.
Condition Gene mean Cq ± SD Condition Gene mean Cq ± SD Control S18 27.49 ± 5.12 IL-1β S18 27.61 ± 5.19 GAPDH 27.74 ± 0.95 GAPDH 27.83 ± 1.17 Collagen 1 43.46 ± 3.43 Collagen 1 43.38 ± 3.43
Collagen 2 34.13 ± 3.29 Collagen 2 38.01 ± 3.55
Aggrecan 36.14 ± 3.53 Aggrecan 35.59 ± 4.35 MMP1 41.37 ± 8.87 MMP1 32.51 ± 12.11 MMP3 43.61 ± 2.91 MMP3 32.47 ± 8.21 MMP13 37.50 ± 10.18 MMP13 33.92 ± 5.44 TIMP1 35.71 ± 9.91 TIMP1 31.41 ± 2.94 IL-6 45 ± 0 IL-6 36.35 ± 4.49 IL-8 45 ± 0 IL-8 38.76 ± 4.12 PTGS2 43.13 ± 3.23 PTGS2 37.21 ± 2.21 ADAMPTS4 45 ± 0 ADAMPTS4 43.57 ± 2.85 iNOS 45 ± 0 iNOS 35.31 ± 1.54 Condition Gene mean Cq ± SD Condition Gene mean Cq ± SD EGCG S18 27.69 ± 4.49 IL-1β + EGCG S18 27.61 ± 5.25 GAPDH 27.91 ± 0.45 GAPDH 27.84 ± 1.14 Collagen 1 45 ± 0 Collagen 1 45 ± 0 Collagen 2 36.62 ± 5.01 Collagen 2 37.86 ± 4.09 Aggrecan 36.31 ± 4.43 Aggrecan 37.01 ± 3.71 MMP1 39.28 ± 11.15 MMP1 37.95 ± 8.34 MMP3 40.79 ± 5.05 MMP3 35.82 ± 7.13 MMP13 37.84 ± 8.86 MMP13 35.23 ± 8.58 TIMP1 36.31 ±7.25 TIMP1 32.83 ± 5.05 IL-6 41.62 ± 4.65 IL-6 38.06 ± 3.95 IL-8 44.41 ± 1.45 IL-8 42.25 ± 4.35 PTGS2 41.67 ± 3.83 PTGS2 38.94 ± 2.61 ADAMPTS4 42.76 ± 4.46 ADAMPTS4 43.33 ± 3.32 iNOS 42.77 ± 4.44 iNOS 38.31 ± 4.57
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115
Chapter 6
Stability of (–)-epigallocatechin gallate and its
activity in liquid formulations and delivery
systems
Olga Krupkova, Stephen John Ferguson, Karin Wuertz-Kozak
The Journal of Nutritional Biochemistry, 2016, Nov(37): 1–12.
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117
6.1 Abstract
(–)-Epigallocatechin gallate (EGCG) has become a popular disease-preventive
supplement worldwide because it may aid in slowing down the onset of age-related diseases
such as cancer, diabetes, and tissue degeneration. As largely demonstrated in cell culture
studies, EGCG possesses antioxidant properties and exhibits favorable effects on gene
expression, signal transduction and other cell functions. However, only limited effects have
been observed in experimental animals and human epidemiological studies. The inconsistency
between the biological activity of EGCG in cell cultures and in vivo can be attributed to its low
stability, which not only decreases its bioavailability, but also leads to the formation of
degradation products and pro-oxidant molecules with possible side-effects.
Understanding EGCG degradation kinetics in solution and in vivo is crucial for its
successful clinical application. Ambient conditions (pH, temperature, oxygen) can either
enhance or decrease the stability of EGCG, thus influencing its biological activity. Usage of
stabilizers and/or encapsulation of EGCG into particulate systems such as nanoparticles or
microparticles can significantly increase its stability. In this review, the effects of ambient
conditions, stabilizers and encapsulation systems on EGCG stability, activity and degradation
rate are illustrated.
Graphical abstract
Key words
Epigallocatechin gallate, stability, activity, degradation, encapsulation
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6.2 Biological effects of (–)-epigallocatechin gallate
Natural polyphenols, including catechins, have received considerable attention as
potential therapeutics against lifestyle diseases. In the past decade, a substantial contribution
to understanding various biological activities of (–)-epigallocatechin gallate (EGCG) in cell
cultures and in vivo has been made [218, 326].
EGCG (Mw = 458.372 g/mol) belongs to the family of catechins, which are polyphenols
found in tea (Camelia sinensis) and other plants as secondary metabolites. The principal
epicatechins in fresh tea leaves apart from EGCG are (–)-epicatechin (EC), (–)-epigallocatechin
(EGC), and (–)-epicatechin gallate (ECG). Their less active trans isoforms catechin, gallocatechin,
catechin gallate and gallocatechin gallate are present in lower amounts. Dry green tea contains
approximately 10% catechins, i.e. about 8-15 g per 100 g of dry leaves [327]. Classically, 4-5 g of
dry tea leaves is used for the preparation of a tea cup infusion, hence 400-500 mg catechins
can be ingested per cup of tea (300-400 mL) [327, 328]. However, only 0.1-1.1% of the orally
administered dose reaches the systemic circulation [329, 330]. EGCG is the most abundant and
active tea catechin. Structurally, EGCG has hydroxyl groups at carbons 3', 4', and 5' of the B ring
(3'4'5'-OH), and a gallate moiety esterified at carbon 3 of the C ring [326, 331], which enables
EGCG to exhibit antioxidant properties. Nevertheless, the chemical structure of EGCG makes it
susceptible to degradation via auto-oxidation [31].
EGCG has been traditionally regarded as beneficial because of its antioxidant effects,
which may aid in a number of clinical conditions such as cancer, obesity, atherosclerosis,
diabetes and neurodegeneration. These age-related diseases are collectively characterized by
increased production of reactive oxygen species (ROS) and/or insufficient cellular anti-oxidant
capacity [218, 332-336]. Although balanced cellular ROS modulate a variety of physiological
events including proliferation, differentiation, host defense and wound healing [271], they are
highly reactive. Extensive ROS exposure causes deleterious damage of proteins, lipids and DNA,
which leads to premature senescence and cell death [38]. The antioxidant activity of EGCG does
not only involve direct trapping of ROS [326, 337-342], but also inhibition of ROS production via
interaction with anti- and pro-oxidant proteins [296, 343-345], and chelation of potentially pro-
oxidant metal ions [31, 326, 346-349].
Several structures with the capability of donating hydrogen atoms and electrons appear
to be important in radical scavenging activity of EGCG: the trihydroxyl group in the B ring
(3'4'5'-OH) participates in electron delocalization and stabilization [339-341], the gallate moiety
at the 3 position in the C ring is associated with increased ROS and reactive nitrogen species
(RNS) scavenging properties [326] and the A ring was identified as an antioxidant site as well
[337, 338, 342].
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EGCG also has the ability to counteract the formation of ROS by inhibiting ROS-
generating enzymes (e.g. xanthine oxidase, cyclooxygenase and lipoxygenase [296, 343-345])
and to affect the production of nitric oxide via an interaction with nitric oxide synthase (NOS)
[350]. Importantly, EGCG may be able to activate superoxide dismutase expression, hence
providing yet another mechanism to inhibit ROS and to protect mitochondria [300, 301].
The described ability of catechins to chelate metal ions may contribute to their
antioxidant activity, specifically by preventing redox-active transition metals from catalyzing
free radical formation, e.g. in degenerated nerve tissue [346]. Chelating groups in EGCG are the
3,4-dihydroxyl groups on the B ring as well the gallate group, both of which are able to bind
several metals bearing strong positive charges (Fe3+, Al3+ and Cu2+) [326, 347, 348]. EGCG can
inactivate iron ions, therefore suppressing the superoxide-driven Fenton reaction, inhibiting
H2O2 formation and lipid peroxidation, which is believed to be a further source of harmful ROS
in degenerative diseases [349].
The ability of EGCG to inhibit free radicals and other reactive species in vitro is well
established. However, it is also known that its molecular structure allows fast degradation,
which complicates EGCG’s clinical application [52, 53]. Encapsulation methods, usually used to
increase stability of natural polyphenols [31, 51], can protect EGCG only for a short period of
time. Understanding conditions influencing EGCG stability and degradation can increase the
potential success of its novel therapeutic formulations. Detailed information on findings about
EGCG stability in biological systems and solution is provided in this review.
Pharmacokinetics
The most common route of administration is oral, as tea infusions or pills, but it is
known that orally ingested EGCG undergoes unfavorable metabolic changes in the
gastrointestinal (GI) tract and liver. Enzymatic transformation reactions start already in the
saliva, where EGCG is hydrolyzed by esterases [326]. As catechins are metabolized
predominantly in the intestine and liver, where glucuronidation and sulfation of the hydroxyl
groups and O-methylation of the catechol groups through UDP-glucuronosyltransferase (UGT),
phenolsulfotransferase (SULT) and catechol-O-methyltransferase (COMT) takes place [330, 351],
the free catechins as well as O-methylated and both O-methylated and glucuronidated
conjugates can be found in plasma, urine, and bile [352-354]. Conjugates still contain intact
catechol and gallate moieties and can have similar biological activity as their parent
compounds [334]. Catechins that are not absorbed in the small intestine and conjugated
catechins excreted in bile reach the colon where they can be metabolized by colonic bacteria
and reabsorbed [334, 354].
Interestingly, although EGCG seems not to be significantly affected by P450
oxidases/reductases, it belongs to the group of compounds that can inhibit or activate P450
120
enzymes hence modulating pharmacokinetics of co-administered drugs. For this reason,
caution is needed especially regarding the co-administration of drugs with small therapeutic
windows and drugs with considerable side-effects [224, 225, 355].
Cellular uptake of catechins occurs mainly by passive transport. The amount of catechin
incorporated is related to its affinity for biomembranes, which increases with catechin
hydrophobicity (i.e. uptake of EC/EGC < ECG/EGCG). Therefore the interaction of catechins with
lipid bilayers may partly govern their biological activity [356]. Intracellular amount of EGCG and
its metabolites is regulated by ATP-dependent efflux pumps with a broad substrate specificity,
by multidrug resistance-associated protein (MRP) efflux pumps [357], and by P-glycoprotein (P-
gp) [358], which actively pump EGCG to the extracellular or intestinal space and so decrease its
bioavailability.
Despite the limited bioavailability of catechins, which is attributed to their low stability
[331] and poor absorption within the GI tract [330, 359], elevated plasma levels of catechins
have consistently been found in humans following the consumption of green tea. Plasma
catechin concentration peaks 1 to 4 hours after ingestion of green tea or catechin supplements
and gradually decreases back to baseline within 24 hours [259, 329, 334, 352, 360]. After a single
dose of green tea or green tea extract, the concentration of individual catechins in rodent or
human plasma increase up to 1 μM [330, 361], but EGCG levels can be much higher in the
tissues that are in direct contact, such as oral cavity [362] and GI tract [334, 363, 364]. Catechin
metabolism is extensively reviewed in [326, 351].
Biological activity in cell cultures
EGCG not only possesses antioxidant activity as described above, but also exhibits
multiple other beneficial functions, which are independent from the antioxidant reactions and
have been demonstrated in cell culture studies.
As an active anti-inflammatory compound, EGCG inhibits phosphorylation of p38, JNK
and nuclear translocation of NF-kB, and therefore negatively regulates the expression of pro-
inflammatory genes and proteins in various cell types [34, 365-367]. Furthermore, EGCG reduces
accumulation of malfunctioning and abnormal proteins and other waste products during
cellular aging [290, 291, 368]. EGCG may also alleviate the detrimental effects of diabetes, as it
was shown to improve insulin secretory function and viability of β-cells under glucotoxicity, and
insulin resistance in skeletal muscle cells [369, 370]. Diverse EGCG activities are reviewed in
[371-374] and summarized in Figure 1.
Cells with features of genetic instability are affected by EGCG in a different way. It has
been shown that EGCG exhibits diverse toxic effects in cancer cell lines with deregulated
signaling, and therefore can contribute to the treatment of e.g. skin, breast and lung cancers or
tumors of GI tract, either alone or in combination with other anti-cancer compounds [375-377].
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EGCG also interacts with proteins and phospholipids in the plasma membrane, influencing
membrane receptor activity and consequent cellular responses, which can be particularly
useful in cancer chemoprevention [48, 304, 378].
Although bioactivity of EGCG has been widely linked to its antioxidant effects and
interactions with other molecules, increasing evidence suggests that EGCG can also function as
a pro-oxidant, hence promoting oxidative stress [326, 334, 338, 364, 379-382]. After contact with
components in cell culture media, EGCG can be oxidized to form semiquinone radicals,
superoxide, and hydrogen peroxide [342, 364, 380, 381, 383]. H2O2 generation, followed by
effects that can be abolished by antioxidant enzymes, was described particularly at higher
EGCG concentrations in vitro (˃ 50 µM) [384]. EGCG-mediated H2O2 generation is lower in the
presence of cells [288, 364, 380, 385], due to the action of cellular glutathione peroxidase and
catalase [288, 381].
Figure 1. Reported biological activity of EGCG in cell cultures. Oxidation of EGCG molecule leads to generation of various pro-oxidants with unfavorable effects. EGCG can exhibit antioxidant activity via several mechanisms, such as neutralization of reactive oxygen species (ROS), chelation of metals or modulation of ROS-regulating enzymes. EGCG also possesses redox-independent activity on multiple stages of cellular signal transduction and gene expression.
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Biological activity in vivo
Depending on the cell type, the therapeutically active, non-toxic concentration of EGCG
in vitro lies between 1 and 100 µM, but certain toxic effects may start to occur already at 50 µM.
Whether EGCG induces oxidative stress in vivo is however controversial as plasma proteins and
antioxidants can enhance its stability [386]. The anti-inflammatory, antioxidant and other
health-protective effects of EGCG have been continuously reported in animal models [387-390],
but in vivo studies describing its toxicity emerged as well [53].
The discrepancy can be explained by the fact that plasma antioxidants may not be
sufficient to balance the pro-oxidative effect of EGCG when applied in high doses. Cases of
hepatotoxicity and nephrotoxicity upon consumption of high doses of green tea-containing
dietary supplements and preparations (10-29 mg/kg/day) have been reported in experimental
animals and humans [53, 381, 391-393]. A single oral dose of 1500 mg/kg EGCG in CF-1 mice
increased plasma alanine aminotransferase and reduced survival by 85%. High-dose EGCG-
activated hepatotoxicity was associated with oxidative stress, including increased hepatic lipid
peroxidation, plasma 8-isoprostane, hepatic metallothionein and γ-histone 2AX protein
expression. EGCG also increased plasma interleukin-6 and monocyte chemoattractant protein
1. Long-term consumption of 1% EGCG in a diet for 6 weeks evoked elevated splenocyte and
macrophage inflammatory markers such as tumor necrosis factor-α, interleukin-6, interleukin-
1β, and prostaglandin E2, and disturbed immune cell populations in mice [394]. A single
intraperitoneal (IP) administration of 100 mg/kg EGCG into mice has generated hepatotoxicity,
whereas 150 mg/kg resulted in 100% mortality within 24 hours [392].
Animal studies unfortunately do not allow full extrapolation of a safe EGCG dose and
mode of application to humans. Differences in pharmacokinetics and bioavailability of EGCG
caused by different enzyme activities have been described between species, namely mice, rats,
and humans. As a result, different metabolites are produced: in mouse plasma, mostly
conjugates were identified, while in human plasma mainly free catechins were present after
oral administration. Results of epidemiological and animal studies involving EGCG are reviewed
in [326, 395].
6.3 Degradation of (–)-epigallocatechin gallate
Catechins can undergo significant degradation, not only in biological fluids, but also
during tea processing and storage, leading to the loss of their health-promoting effects.
However, some biological effects have been directly attributed to EGCG degradation products,
rather than to EGCG itself [380]. The instability of EGCG is caused by two major reactions,
epimerization and auto-oxidation [31, 396]. Rates of these two reactions are affected by factors
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like the presence of oxygen, antioxidants and EGCG concentration. Therefore, the stability and
biological activity of EGCG can be controlled by the physicochemical conditions of ambient
media.
Figure 2. Scheme of EGCG degradation pathways in various ambient conditions. In aqueous media EGCG can undergo epimerization, yielding (–)-gallocatechin gallate (GCG) with similar biological activities, or degrade via auto-oxidation, yielding dimers such as thenasinensin A that can further oligomerize. Epimerization is favored in mild acidic pH, above 40°C and in the presence of antioxidants. Auto-oxidation, which occurs at mild alkaline pH and temperatures below 40°C, is unfavorable as various radical intermediates can be formed.
Auto-oxidation
In solution, catechins rapidly degrade via auto-oxidation, involving a loss of hydrogen
atoms, generation of semiquinone radical intermediates, superoxide and formation of quinone
oxidized products, which may possibly have damaging consequences for cells and tissues [338,
351, 379, 397-400]. Subsequently, dimers theasinensin A and compound P2 can be identified as
major products of B-ring auto-oxidation in mild alkaline fluids with micromolar EGCG
concentrations, which is typical for cell culture. EGCG degradation is usually accompanied by
the color change of its aqueous solution from transparent to brown, indicating the presence of
structures with larger molecular weight, via polymerization of EGCG dimers [396]. In contrast
to auto-oxidation in aqueous media, which involves the B-ring, oxidation of EGCG in organic
solvents rather takes place at the D-ring [401].
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Epimerization
In millimolar EGCG concentrations and when auto-oxidation is prevented by
antioxidants, epimerization of EGCG to its trans epimer (–)-gallocatechin gallate (GCG) is
preferred [327, 380, 385, 396, 402, 403] (Figure 2). The epimerization of EGCG to GCG is more
favorable at pH below 5.5, at high concentrations (mM solution) or at temperatures above 50°C,
e.g. during autoclaving [396, 402, 403]. The conversion of EGCG to GCG occurs also under
anaerobic conditions and once EGCG is stabilized by the presence of antioxidants [380].
Catechin trans-epimers do not have toxic effects, but rather possess similar biological activities
to their cis- counterparts [339, 340, 404, 405]. Catechin epimerization can be reversible [402,
403].
Table 1. The stability of EGCG in aqueous solution. The initial concentrations of EGCG, its mean half-life, and its degradation products in various solvents are listed. GTC = green tea catechins, RT = room temperature, na = information not available. EGCG, 37 °C [328, 380, 385, 396] solvent additive initial concentration mean half-life (t1/2)
degradation product
McCoy’s 5a - 10 µM 30 minutes theasinensin A product 2
McCoy’s 5a HT29 cells 10 µM 130 minutes theasinensin A product 2
Ham’s F12 - 10 µM ˃ 30 minutes theasinensin A product 2
HBSS - 10 µM 30 minutes theasinensin A product 2
Na2HPO4 (50 mM)
- 20 µM 30 minutes theasinensin A product 2
Ham’s F12:RPMI (1:1)
- 20 µM 20 minutes theasinensin A product 2
Ham’s F12:RPMI (1:1)
KYSE 150 cells 20 µM 1 hour theasinensin A H2O2
PBS (0.05 M) - 218 µM ˃ 40 minutes na water (100 °C) - 1.09 mM ˃ 4 hours na PBS (60 mM, pH 7.4)
- 1.09 mM 1.5 hours na
EGCG, 50 °C [406] solvent additive initial concentration t = concentration
decreased to 90% degradation product
Glycerin - 10.9 mM 24.4 days na Transcutol P - 10.9 mM 22.8 days na
6.4 Stability of (–)-epigallocatechin gallate in solution
Since EGCG is prone to fast degradation, knowledge of the degradation kinetics is
necessary to predict its stability during thermal processing, storage as well as during
preparation and application of therapeutic formulations and food supplements [407] (Table 1).
Stability of EGCG is affected by parameters such as oxygen concentration [396, 407], pH [342,
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407-409], temperature [327, 408, 409], and ionic strength [409]. Stability of EGCG in aqueous
media depends also on its initial concentration [396, 409] and the presence of antioxidants,
which can prevent its auto-oxidation [385, 396, 409]. EGCG stability can be accurately
measured by high-performance liquid chromatography (HPLC) [409] and by various assays
using spectrophotometry [410].
Effect of concentration
The stability of EGCG in aqueous solution depends on the initial concentration [396,
400, 403, 409], with a clear difference between the micromolar and millimolar range
(Supplementary Table 1). The half-life of EGCG in water slightly increased from 30 minutes at 20
µM to 150 minutes at 96 µM [396]. After 2 days of storage at room temperature (RT), 54.5 µM
EGCG completely degraded, whereas 1.97 mM EGCG degraded only to 80% of its initial
concentration [409]. After 7 days of storage, 0.5 mM EGCG decreased to 80%, while 7 mM EGCG
solution remained stable [396].
Major degradation products of EGCG in micromolar concentrations are EGCG dimers. In
higher millimolar concentrations, however, dimers do not form, but EGCG epimerizes to GCG
[396, 403], suggesting that the catechin molecules are able to protect each other from auto-
oxidation [400].
The safe and active in vitro concentration of EGCG varies with cell type, but generally it
is less than 100 µM. Oral route of administration is highly affected by the composition of diet,
fasting conditions, and enzyme activities, therefore the bioavailability of EGCG can differ
between individuals. The maximum EGCG concentration in human plasma after oral
consumption was reported as 1 µM, but higher concentration can be achieved via other
administration routes or encapsulation [411].
Effect of pH
Upon oral administration, EGCG is exposed to variable pH levels, ranging from 1.5 in the
stomach up to 8.5 in the duodenum, where most of the absorption takes place. EGCG is rather
stable at lower pH of 2.0−5.5. Its instability particularly in the alkaline environment of the GI
tract has been reported as one of the reasons for its poor bioavailability [327, 328, 385, 400, 407-
409]. It was shown that 109 mM EGCG solution at pH 9 completely degraded after 1 day,
whereas a solution at pH 3 degraded only to 90% of its initial concentration at day 28 [406].
The degradation rate of EGCG is comparable to that of green tea catechin (GTC) extract, which
showed a pH-dependent rate of degradation as well [328, 397]. Long-term stability of GTC (4.4
mM) was examined at pH 4−5 for 6 months, which is the time desired for a storage of drinks
and liquid formulations, but already after one month, 30−78% of GTC was destroyed. The pH of
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solution also influenced the degradation rate of GTC during autoclaving: at pH 3, 95% of GTC
remained stable, whereas at pH 5 45% of GTC degraded [328] (Supplementary Table 2).
The acidic environment enhanced EGCG stability, but neutral and alkaline pH caused
auto-oxidation on the B-ring and produced theasinensin A and product P2 [385, 401].
Susceptibility to oxidation and formation of free radicals is caused by increased proton-
donating potential of EGCG in alkaline fluids [337, 406]. Aside from the stomach (pH ± 2.5),
acidic pH occurs also in various tumor tissues due to the cellular production of lactic acid and
ATP hydrolysis in hypoxic regions of tumors [412]. In contrast to the very low pH in the stomach,
the mildly acidic pH of tumors allows to prolong EGCG’s half-life, which may help to target
subpopulations of tumor cells that frequently escape conventional therapy [413]. A mildly acidic
pH is also present in degenerative tissues like cartilage [414] or intervertebral disc [206].
Effect of temperature
Temperature is a significant parameter governing EGCG stability and the mode of its
degradation (Supplementary Table 3). Low temperature can preserve the stability of EGCG: the
half-life of 109 mM EGCG solution at 4°C was 7 days, but decreased to 2 days when kept at RT
[406]. Similarly, upon storage at RT, 54.5 µM EGCG solution completely degraded by day 2,
while the same solution at 4°C degraded only to 60% of its initial concentration [409]. At 37°C,
10 µM EGCG solution degraded completely in 1 hour [385, 396], which was the most relevant
finding for cell culture experiments.
At temperatures below 50°C, EGCG underwent degradation via auto-oxidation while at
higher temperatures, epimerization was more profound [327, 396, 403, 406, 408, 409]. The
temperature 44°C was identified as crucial in aqueous system: below 44°C oxidation prevailed,
whereas above 44°C the epimerization from EGCG to GCG became prominent [415]. High
temperatures can be used to sterilize EGCG-containing formulations for long term storage.
After 20 minutes of autoclaving at 120°C, 75% of GTC with initial concentration 10.9 mM
remained in the solution and less than 12% of GCG was formed [327].
Effect of oxygen partial pressure and ionic strength
Oxygen partial pressure may have significant consequences for EGCG stability [327, 396,
407], as standard cell culture experiments are performed under the atmospheric pressure of
oxygen, which is 160 mmHg (21.1%). In the tissues, however, oxygen partial pressure drops to ˂
20 mmHg inside cells or within upper skin layer. Maximum tissue oxygen pressure can rise to
110 mmHg in alveoli and to 100 mmHg in arterial blood [416]. This shows that even the highest
values of tissue oxygen pressure are still lower than atmospheric oxygen pressure present
under the cell culture conditions, suggesting the degradation kinetics of EGCG may differ
between cell culture and in vivo situation. The majority of all the mentioned EGCG stability
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studies were performed under atmospheric oxygen partial pressure. Apart from that, the effect
of oxygen on the stability of EGCG was tested in phosphate buffer (pH 7.4) in the absence or
presence of nitrogen. In air-saturated buffer, EGCG underwent auto-oxidation; oxygen was
consumed and H2O2 was formed [380, 398]. Under low oxygen partial pressure, generated by
extensive flushing of buffer solution with nitrogen gas before adding EGCG, EGCG was greatly
stabilized, with 95% remaining after 6 hours. No dimers were detected, but GCG epimers were
formed [327, 396]. Indeed, EGCG was shown to be rather stable in blood, where no EGCG dimers
were present, which may reflect a combined effect of low oxygen partial pressure and presence
of plasma antioxidants [380].
The activity of EGCG is also likely to be dependent on the salt concentration of ambient
medium [356]. As degradation and transformation of EGCG increased with rising ionic strength
of the solvent, EGCG aqueous solution in the lowest buffer capacity possible may be a way to
avoid accelerated degradation [328, 406, 409]. On the other hand, the incorporation of EGCG
into the lipid bilayers was accelerated as the salt concentration increased [356] (Supplementary
table 4).
6.5 Stabilization of (–)-epigallocatechin gallate for clinical
application
Antioxidants
The presence of reducing agent is essential for the stability of EGCG in aqueous solution
[409]. In buffer or cell culture media, such as McCoy’s 5A, Ham’s H-12 or RPMI, the half-life of
micromolar EGCG is about 30 minutes [380, 385, 396], but addition of antioxidants can
significantly stabilize EGCG and increase its half-life to more than 24 hours. Antioxidants
inhibit formation of EGCG dimers, so epimerization of EGCG to GCG prevails [380].
In the presence of superoxide dismutase (SOD), which catalyzes the reduction of
superoxide anions to hydrogen peroxide, no superoxide-mediated auto-oxidation took place,
but GCG was formed instead [396]. Ascorbic acid (AA) was shown to stabilize EGCG at various
conditions in aqueous buffers preventing auto-oxidation, because it was preferentially oxidized
in place of the catechin [327, 400, 409, 417]. However, in the presence of sucrose, AA greatly
destabilized GTC [328] (Table 2).
Amphiphilic compounds and other stabilizers
Amphiphilic compounds are used to partition the phases in an immiscible biphasic
system consisting of aqueous and organic solvents. Knowledge of the degradation kinetics of
EGCG in the presence of amphiphilic compounds is important e.g. for transdermal drug delivery
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formulations, where organic permeation enhancers are used to promote drug migration
through the skin.
EGCG (10.9 mM) was tested in solutions of permeation enhancers propylene glycol,
polyethylene glycol 200, glycerin, and Transcutol P [406]. EGCG exhibited low stability in
propylene glycol and polyethylene glycol 200, whereas a higher stability was observed in
glycerin and Transcutol P, supported by the lack of brown discoloration for a period of one
month. Accelerated degradation studies conducted at 50°C revealed only a 10% decrease of the
total EGCG amount at day 23-24, showing a much higher stability in Transcutol P and glycerin
compared with aqueous media [406].
The effect of antioxidants propyl gallate (Pgal), AA, and butylated dihydroxytoluen (BHT)
on the stability of EGCG in glycerin and Transcutol P was also tested. In the presence of
amphiphilic compounds, AA did not have any significant stabilizing effect. Both Pgal and BHT
increased the stability of EGCG in glycerol, but decreased its stability in Transcutol P.
Combination of BHT and Pgal however significantly destabilized EGCG, most likely due to their
molecular interactions [406].
Additives and stabilizers in food, drinks and oral formulations can also influence the
stability of EGCG (Table 2). Several critical structural features of proteins for binding to
flavonoids, like the amino-acid composition and conformation, have been highlighted in the
literature [418]. For example milk, which is commonly added to the tea to reduce an astringent
taste, is able to complex flavonoids. Authors of several clinical studies on tea ingestions
concluded that milk might adversely affect tea catechin absorption [326]. Nevertheless,
interaction with milk proteins did not seem to impair catechin stability [419].
β-casein and gelatin were suggested as the most promising carriers for EGCG due to
their high EGCG affinity [418], but the stability of EGCG in the presence of β-casein was
improved only mildly [417]. A slight improvement in EGCG stability was reported also for
proline, aspartic acid and ovalbumin, whereas glutamic acid, β-cyclodextrin, trehalose, pectin
and alginic acid had no effect on EGCG stabilization [417]. In another study, a GTC mixture (1.09
mM) was added either to a sucrose solution (0.15 g/mL) or to a sucrose-citric acid solution (0.15
g sucrose/mL, 2 mg citric acid/mL), both of which are common additives in tea drinks. The
solution was autoclaved at 120°C for 20 minutes and analyzed monthly. A 6-months
assessment revealed that addition of sucrose had little effect on the stability of GTC, whereas
addition of citric acid destabilized GTC, which could explain the very low content of GTC in the
commercial tea drinks [328]. The stability of EGCG also increased in the presence of Na2EDTA (14
mM), which can scavenge metal ions involved in catalyzing EGCG auto-oxidation [396, 402]
129
Table 2. The effect of antioxidants and stabilizers on the stability of EGCG. The initial concentrations of EGCG, its mean half-life, and its degradation products in respective solvents with additives are listed. GTC = green tea catechins, GCG = (–)-gallocatechin gallate, SOD = superoxide dismutase, AA = ascorbic acid, Pgal = propyl gallate, BHT = butylated dihydroxytoluen, hl = half-life, na = information not available. EGCG, 37 °C, pH 7.4 [396] solvent additive initial concentration mean half-life (t1/2)
% at t = 6 hours (6h) degradation product
Na2HPO4 (50 mM)
- 20 µM t1/2: 30 minutes theasinensin A product 2
Na2HPO4 (50 mM)
saturated nitrogen
20 µM 6h: 100% GCG
Ham’s F12:RPMI (1:1)
- 20 µM t1/2: 20 minutes theasinensin product 2
Ham’s F12:RPMI (1:1)
SOD (5U/mL) 20 µM 6h: 80% GCG
GTC, RT, pH 7.4 [328] solvent additive initial concentration mean half-life (t1/2) degradation product water - 1.09 mM 6 months na water sucrose
(0.15 mg/mL) 1.09 mM 3 months na
water sucrose+citric acid (2 mg/mL)
1.09 mM 1 month na
water sucrose+AA (11 mg/mL)
1.09 mM 0.5 month na
EGCG, RT, pH 5.4 [396] solvent additive initial concentration mean half-life (t1/2) degradation product Na2HPO4 (50 mM)
- 20 µM 30 minutes na
Na2HPO4 (50 mM)
Na2EDTA (14 mM)
20 µM 4 hours na
EGCG 37 °C, pH 6.5 [417] solvent additive initial concentration concentration at t =
5 hours degradation product
PBS (0.05 M) - 218 µM 106.9 µM na PBS (0.05 M) AA (0.1 mg/mL) 218 µM 213.8 µM na PBS (0.05 M) proline (0.1
mg/mL) 218 µM 122.2 µM na
PBS (0.05 M) cysteine (0.1 mg/mL)
218 µM 202.9 µM na
PBS (0.05 M) aspartic acid (0.1 mg/mL)
218 µM 226.5 µM na
PBS (0.05 M) glutamic acid (0.1 mg/mL)
218 µM 220.0 µM na
PBS (0.05 M) casein (0.625 mg/mL)
218 µM 124.3 µM na
PBS (0.05 M) ovalbumin (0.625 mg/mL)
218 µM 133.1 µM na
PBS (0.05 M) b-cyclodextrin (1.25 mg/mL)
218 µM 111.3 µM na
PBS (0.05 M) trehalose (2.0 mg/mL)
218 µM 102.5 µM na
PBS (0.05 M) pectin (0.1 mg/ml) 218 µM 113.4 µM na PBS (0.05 M) alginic acid
(0.5 mg/mL) 218 µM 120.0 µM na
EGCG 37 °C, pH 6.5, t = 10h [417] solvent additive initial concentration concentration at t =
10 hours degradation product
PBS (0.05 M) - 218 µM 19.6 µM na PBS (0.05 M) AA (0.1 mg/mL) 218 µM 43.6 µM na
130
EGCG 37 °C, cell culture conditions [380] solvent additive initial concentration mean half-life (t1/2) degradation product RPMI: Ham’s F12 (1:1)
KYSE 150 cells 20 µM 1 hour theasinensin A H2O2
RPMI: Ham’s F12 (1:1)
KYSE 150 cells SOD (5 U/mL)
20 µM 24 hours GCG dimer
EGCG, 50 °C, dark [406] solvent additive initial concentration t = concentration
decreased to 90% degradation product
Glycerin - 10.9 mM 24.4 days na Glycerin AA (0.1% w/w) 10.9 mM 25.7 days na Glycerin Pgal (0.1% w/w) 10.9 mM 29.3 days na Glycerin BHT (0.1% w/w) 10.9 mM 76.1 days na Glycerin BHT+Pgal
(0.05%+0.05%) 10.9 mM 7.4 days na
Transcutol P - 10.9 mM 22.8 days na Transcutol P AA (0.1% w/w) 10.9 mM 24.7 days na Transcutol P Pgal (0.1% w/w) 10.9 mM 3.5 days na Transcutol P BHT (0.1% w/w) 10.9 mM 8.6 days na Transcutol P BHT+Pgal
(0.05%+0.05%) 10.9 mM 7.0 days na
Oral delivery systems
Oral drug delivery, although not very efficient, is the most convenient and well accepted
route of administration. Encapsulation can protect EGCG from extreme pH changes in the
digestive system as well as increase the GI retention time after oral consumption [420].
Biodegradable and biocompatible cationic polysaccharides, such as chitosan (CS) are suitable
carriers for oral drug delivery. Chitosan adheres to the mucosa through ionic interactions
between its positively charged amine groups and the negatively charged groups present on
intestinal surfaces. Furthermore, interaction between chitosan molecules and polyphenolic
compounds could be a useful strategy to increase their encapsulation efficiency [421]. The
summary of stability and biological activity of encapsulated EGCG compared to free EGCG in
oral delivery formulations is listed in Table 3.
In the absence of food, intestinal mucosa is regularly washed and therefore a relatively
rapid release/absorption of EGCG is required. Diet type and food quantity greatly influence GI
retention time, which should be taken into consideration while developing oral delivery
systems. Significant increases in stability and/or activity of EGCG with sufficient GI retention
time have already been achieved in experimental animals [420]. Stabilization of EGCG and its
optimized release in the GI tract over a period of 20 hours should be adequate for humans.
Oral drug delivery systems demonstrated increased intestinal retention time, stability
and adsorption of EGCG, and consequently enhanced its plasma concentration. However, with
oral administration side effects in non-target tissues can occur. Despite the fact that these
systems are being established in experimental animals, their translation to humans still
requires some effort.
131
Intravenous and local delivery systems
Intravenous administration overcomes the issue of poor intestinal absorption, but can
cause discomfort, rapid particle clearance and can activate host defense mechanisms.
Intravenously administered nanoparticles are passively targeted into tumors due to an
enhanced permeability and retention (EPR) effect. In addition to EPR effect, selective, receptor-
mediated drug delivery of EGCG is another attractive approach to tumor targeting [422].
Biocompatible polymers with a tunable degradation rate and release are often used (e.g. PLA,
PLGA) [423, 424].
The advantage of particles and liposomes – compared to macromolecules – lies in their
reduced kidney clearance, which is related to their larger size. Importantly, surface properties
significantly influence the fate of a particle, e.g. PEG coating protects particles from recognition
by the mononuclear phagocyte system (opsonisation). Properties of particulate systems can be
tuned by integration of ligands, coating, or stimuli-responsive components, to trigger e.g. pH-
catalyzed hydrolysis. The stability and biological effect of encapsulated EGCG compared to free
EGCG in intravenous delivery formulations is listed in Table 4.
Local delivery methods include non-invasive (e.g. topical, ocular) and invasive (e.g. intra-
tumoral) routes of administration. Local delivery avoids rapid kidney clearance of particles and
significantly reduces the risk of systemic side-effects. The stability and biological effect of
encapsulated EGCG compared to free EGCG in local delivery formulations is listed in Table 4.
Modification of (–)-epigallocatechin gallate molecule
Due to high solubility of EGCG in water, it is difficult to obtain effective encapsulation.
One approach to reduce the water solubility of EGCG is to hide hydroxyl groups. Acetylation of
EGCG masked the hydrophilic OH-groups, which increased the antioxidant properties and
solubility of EGCG in fat [425]. In a comparable approach, EGCG was treated with acetic
anhydride and pyridine to create peracetate protection of OH-groups and further encapsulated
into lipid nanoparticles. Despite a partial deacetylation (50% of initially acetylated EGCG), the
amount of EGCG in nanoparticles remained constant over 25 days, suggesting improved
stability [331].
To increase its lipophilicity and bioavailability, the EGCG molecule was esterified with
fatty acids [426]. EGCG lipophilised by docosapentaenoic acid (DPA) exhibited an enhanced
anti-inflammatory effect compared with free EGCG [427].
Cocrystallization can also be used to generate novel forms of EGCG with modulated
dissolution and pharmacokinetic profiles. As demonstrated by Smith et al., cocrystallization can
reduce water solubility of EGCG and slightly increase its bioavailability in mice after oral
administration. Although the in vivo effect compared with free EGCG was very modest in this
study, cocrystallization offers a tool to further modulate its pharmacokinetics [428].
132
Table 3. EGCG stability in oral drug delivery formulations. Physicochemical characteristics [EE = encapsulation efficiency (%), APS = average particle size, ZP = zeta potential (mV), PI = polydispersity index], release properties [IC = initial concentration of EGCG, RM = release media, %(t) =% of EGCG released in time t] as well as biological effect are listed. CS = chitosan, PAA = polyaspartic acid, CPP = caseinophosphopeptide, KBR = Krebs Bicarbonate-buffered Ringers solution, SGF = simulated gastric fluid, SIF = simulated intestinal fluid, * = significant difference in the stability or effect of encapsulated EGCG compared to free EGCG.
Chitosan (CS) microparticles aim: stability increase [421] delivery route
physicochemical characteristics release properties significance of encapsulation*
biological effect
na EE: 47.12±2.01% APS: 1.79±0.38 µm
ZP: na PI: na
IC: 218 mM catechin extract RM: PBS pH 5.5 PBS pH 7.4
%(t): 80% in 4 hours (60% in 1st hour)
na na
Nanoparticles CS aim: oral delivery [330, 429]
delivery route
physicochemical characteristics release properties significance of encapsulation*
biological effect
oral EE: na LE: 3.7±0.1 µg/mg APS: 440±37 nm
ZP: +25±1 PI: na
IC: 1.09 mM RM: KBR, pH 6.0
%(t): 60% in 2 hours
p < 0.05 in vitro (organ culture) in vivo
Nanoparticles CS-PAA aim: oral delivery [420]
delivery route
physicochemical characteristics release properties significance of encapsulation*
biological effect
oral EE: 25.0±2.1% APS: 102.4±5.6 nm
ZP: 33.3±0.2 PI: 0.224
IC: 4.4 mM RM: SGF, SIF
%(t): 100% in 5 hours
p < 0.05 in vivo (rabbit)
Nanoparticles CS-CPP aim: chemoprevention [430]
delivery route
physicochemical characteristics release properties significance of encapsulation*
biological effect
not specified
EE: 81.7±4.1% APS: 143.7±6.7 nm
ZP: 30.8±4.6 PI: 0.08-0.13
IC: 5.5 mM RM: PBS pH 6.2
%(t): 48% in 20 hours
p < 0.01 in vitro
Nanoliposomes aim: tumor delivery [431]
delivery route
physicochemical characteristics release properties significance of encapsulation*
biological effect
not specified
EE: 85.79±1.65% APS: 180±4 nm
ZP: na PI: na
IC: 10.6 mM RM: SGF, SIF
%(t): SGF: 21% in 4 hours SIF: 35% in 4 hours
p < 0.05 in higher concentrations
in vitro
Nanoparticles Gum Arabic-Maltodextrin aim: chemoprevention [432]
delivery route
physicochemical characteristics release properties significance of encapsulation*
biological effect
not specified
EE: > 80% APS: 120±28 nm
ZP: -12.3±0.8 PI: 0.45±0.23
IC: 109 mM RM: PBS pH 7.4
%(t): >40% in 10 minutes
p < 0.05 in lower concentrations
in vitro
133
Table 4. EGCG stability in intravenous and local drug delivery formulations. Physicochemical characteristics [EE = encapsulation efficiency, APS = average particle size, ZP = zeta potential (mV), PI = polydispersity index], release properties [IC = initial concentration of EGCG, RM = release media, %(t) =% of EGCG released in time t] as well as biological effect are listed. PLA = polylactic acid, PEG = polyethylene glycol, EtOH = ethanol, CTAB-LN = cetyltrimethylammonium bromide lipid nanoparticles, DDAB-LN = dimethyldioctadecylammonium bromide lipid nanoparticles, PLGA = poly(lactic-co-glycolic acid), CP = citrate-phosphate, * = significant difference in the stability or effect of encapsulated EGCG compared to free EGCG.
Nanoparticles PLA-PEG aim: chemoprevention [423]
delivery route
physicochemical characteristics release properties significance of encapsulation*
biological effect
systemic injection
EE: na APS: 260 nm
ZP: -7.92 PI: 0.07
IC: 218 mM RM: blood
%(t): 22-65 µM during 4 hours
p < 0.05 in vitro in vivo
Niosomes aim: tumor targeting [422]
delivery route
physicochemical characteristics release properties significance of encapsulation*
biological effect
systemic injection
EE: 30.3±2.8% APS: 294 nm
ZP: -36 PI: 0.414
IC: 2.2 mM RM: PBS
%(t): 80% in 24 hours
p < 0.05 in vitro in vivo
Lipid nanocapsules aim: stability increase [331]
delivery route
physicochemical characteristics release properties significance of encapsulation*
biological effect
not specified
EE: 80% APS: 57 nm
ZP: na PI: < 0.15
IC: 32.8 mM acetyated EGCG RM: water
%(t): 60% in 8 days
- -
Liposomes (-EtOH) aim: intra-tumor retention [433]
delivery route
physicochemical characteristics release properties significance of encapsulation*
biological effect
intra-tumor injection, topical
EE: 36.3-89.7% APS: na
ZP: na PI: na
IC: 10.9 mM RM: na
%(t): na in most tests not found
in vivo
Liposomes (+EtOH) aim: intra-tumor retention [424]
delivery route
physicochemical characteristics release properties significance of encapsulation*
biological effect
intra-tumor injection
EE: >98% APS: 378.2±10.9
ZP: -26±0.1 PI: na
IC: 17.2 mM RM: CP buffer pH 7.4
%(t): 0% in 12 hours
p < 0.05 for concentrations below 50 µM
in vitro in vivo
Lipid nanoparticles CTAB-LN, DDAB-LN
aim: ocular delivery [434]
delivery route
physicochemical characteristics release properties significance of encapsulation*
biological effect
not specified
EE: 98.99±0.19% 96.86±1.88% APS: 149.1±1.78 143.7±0.45
ZP: 20.8±0.89 25.7±1.42 PI: 0.240±0.008 0.160±0.015
IC: 26.2 mM RM: na
%(t): na na na
Nanoparticles PLGA aim: chemoprevention [411]
delivery route
physicochemical characteristics release properties significance of encapsulation*
biological effect
topical EE: 26% LE: 5.76% APS: 127.2±12 nm
ZP: -24.5 ± 1.89 PI: 0.189
IC: 87 mM RM: PBS, pH 7.4
%(t): 50% in 24 hours
p < 0.05 in vivo
134
6.6 Conclusions
Diverse beneficial activities of EGCG were demonstrated in cell culture models, among
which its antioxidant, anti-inflammatory and anti-cancer effects are most pronounced. This
indicates that EGCG is indeed a promising compound for clinical translation. However, EGCG is
less effective in corresponding animal experiments and moreover, only limited effects are
observed in human epidemiological studies.
The inconsistency between the activity of EGCG in the cell cultures and in the whole
organism can be attributed to the insufficient stability of the EGCG molecule: uncontrolled
EGCG degradation does not only lead to inaccurate conclusions of in vitro studies, but can also
limit EGCG’s bioavailability in vivo, hence decreasing its beneficial effects. For this reason,
accurate translation of in vitro and preclinical research to humans has not yet been successful.
Various encapsulation techniques for plant polyphenols have been developed, however,
they have several disadvantages. Classical methods include the use of extreme conditions (pH,
temperature), which causes a low encapsulation efficiency of EGCG, and the use of toxic
solvents with the need for time-consuming post-treatments. Moreover, the hydrophilic nature
of EGCG further reduces its encapsulation efficiency, as EGCG tends to leak between the
organic and aqueous phases during the standard encapsulation process.
Bearing in mind the clinical translation, stabilization method should reflect the final
EGCG application: targeted delivery with rapid release of a toxic dose is desired in cancer
treatment, whereas slow release of low EGCG doses over longer periods of time may be needed
to treat age-related diseases like atherosclerosis or neurodegeneration. Reducing agents and
appropriate encapsulation strategies, which sufficiently improve the stability of EGCG during
the delivery process, in combination with in vivo stabilizing mechanisms such as antioxidant
enzymes and lower oxygen partial pressure, will allow for full manifestation of EGCG’s
beneficial activities in vivo.
6.7 Acknowledgement
The authors have no conflict of interest. The authors would like to thank the Mäxi
Foundation for financial support and Prof. Dr. Paola Luciani and Prof. Dr. Bruno Gander for their
valuable advice.
135
6.8 Supplement Supplementary Table 1. The effect of concentration on the stability of EGCG. The initial concentrations of EGCG, its mean half-life, and its degradation products in respective solvents are listed. GCG = (–)-gallocatechin gallate, RT = room temperature, AA = ascorbic acid, hl = half-life, na = information not available.
EGCG, RT, pH 5.4 [396] solvent additive Initial concentration % at t = 7 days (7d)
mean half-life (t1/2) degradation product
water - 0.51 mM 7d: 80% GCG water - 2.06 mM 7d: 80% GCG water - 6.99 mM 7d: 100% GCG water - 96 µM t1/2: 150 minutes theasinensin A water - 20 µM t1/2: 30 minutes theasinensin A EGCG, RT, pH 7.4 [409] solvent additive initial concentration % at t = 2 days
degradation product
HEPES - 0.55 µM 0% na HEPES - 1.09 µM 0% na HEPES - 54.5 µM 0% na HEPES - 218 µM 18% na HEPES - 654 µM 58% na HEPES - 1964 µM 80% na HEPES AA 0.25% (w/v) 0.55 µM 0% na HEPES AA 0.25% (w/v) 1.09 µM 0% na HEPES AA 0.25% (w/v) 54.5 µM 90% na HEPES AA 0.25% (w/v) 218 µM 98% na HEPES AA 0.25% (w/v) 654 µM 98% na HEPES AA 0.25% (w/v) 1964 µM 100% na EGCG, 37 °C, pH 7.4 [400] solvent additive initial concentration mean half-life (t1/2)
degradation product
K2HPO4 buffer (50 mM)
- 3.27 µM 40 minutes na
K2HPO4 buffer (50 mM)
- 164 µM 40 minutes na
K2HPO4 buffer (50 mM)
- 545 µM 50 minutes na
EGCG, various temperatures (-20 °C to +25 °C) [409] solvent additive initial concentration % at t = 7 days
degradation product
Transcutol P (20%, v/v)
AA (0.25%, w/v) ethanol (24%, v/v)
1964 µM ±100% na
Transcutol P (20%, v/v)
AA (0.25%, w/v) ethanol (24%, v/v)
982 µM ±100% na
Transcutol P (20%, v/v)
AA (0.25%, w/v) ethanol (24%, v/v)
654 µM ±100% na
Transcutol P (20%, v/v)
AA (0.25%, w/v) ethanol (24%, v/v)
218 µM ±100% na
Transcutol P (20%, v/v)
AA (0.25%, w/v) ethanol (24%, v/v)
54.5 µM ±100% na
136
Supplementary Table 2. The effect of pH on the stability of EGCG. The initial concentrations of EGCG, its mean half-life, and its degradation products in respective pH are listed. GTC = green tea catechins, GCG = (–)-gallocatechin gallate, hl = half-life, na = information not available.
EGCG, 4 °C [406] pH solvent Initial concentration % at t = 28 days (28d)
mean half-life (t1/2) degradation product
3 citrate buffer (0.05 M)
109 mM 28d: 90% na
5 citrate buffer (0.05 M)
109 mM 28d: 90% na
7 phosphate buffer (0.05 M)
109 mM t1/2: 7 days na
9 boric acid-borax buffer (0.05 M)
109 mM t1/2: 5 days na
EGCG, 21 °C [406] pH solvent initial concentration % at t = 28 days (28d)
mean half-life degradation product
3 citrate buffer (0.05 M)
109 mM 28d: 90% na
5 citrate buffer (0.05 M)
109 mM 28d: 70% na
7 phosphate buffer (0.05 M)
109 mM t1/2: 2 days na
9 boric acid-borax buffer (0.05 M)
109 mM t1/2: 1 day na
EGCG, 50 °C [406] pH solvent initial concentration mean half-life (t1/2) degradation
product 3 citrate buffer
(0.05 M) 109 mM 28 days na
5 citrate buffer (0.05 M)
109 mM 4.5 days na
7 phosphate buffer (0.05 M)
109 mM < 10 hours na
9 boric acid-borax buffer (0.05 M)
109 mM < 10 hours na
EGCG, RT [409] pH solvent Initial concentration % at t = 2 days degradation
product 3.5 HEPES 54.5 µM 40% na 7.4 HEPES 54.5 µM 0% na 3.5 HEPES 1.09 µM 0% na 7.4 HEPES 1.09 µM 0% na GTC mix [328] pH solvent initial concentration % at t = 18 hours (18h),
6 months (6m) degradation product
- water 1.09 mM 6m: 50% na 4 sodium
phosphate buffer (60 mM)
1.09 mM 6m: 30% na
5 sodium phosphate buffer (60 mM)
1.09 mM 6m: 10% na
5 sodium phosphate buffer (60 mM)
1.09 mM 18h: 100% na
6 sodium phosphate buffer (60 mM)
1.09 mM 18h: 80% na
137
Supplementary Table 3. The effect of temperature on the stability of EGCG. The initial concentrations of EGCG, its mean half-life, and its degradation products in respective temperatures are listed. GTC = green tea catechins, GCG = (–)-gallocatechin gallate, na = information not available. EGCG, pH 7, PBS [406] temperature solvent initial concentration mean half-life (t1/2) degradation product 4 °C PBS 109 mM 7 days na 21 °C PBS 109 mM 2 days na 55 °C PBS 109 mM less than 10 hours na EGCG, pH 7.4 [409] temperature solvent initial concentration % at t = 2 days degradation product 4 °C HEPES 54.5 µM 60% na 25 °C HEPES 54.5 µM 0% na 4 °C HEPES 1.09 µM 43% na 25 °C HEPES 1.09 µM 0% na GTC mix [328] temperature solvent Initial concentration % at t = 3 hours degradation product 100 °C deionized water 1.09 mM 75% na 70 °C deionized water 1.09 mM 80% na 24 °C deionized water 1.09 mM 100% na GTC mix [327] temperature solvent initial concentration % at t = 7 hours degradation product 98 °C distilled water 10.9 mM 80% GCG 37 °C distilled water 10.9 mM 100% na
6.5 sodium phosphate buffer (60 mM)
1.09 mM 18h: 50% na
7 sodium phosphate buffer (60 mM)
1.09 mM 18h: 30% na
7.4 sodium phosphate buffer (60 mM)
1.09 mM 18h: 17% na
EGCG [328] pH solvent initial concentration % at t = 6 days degradation
product 7.4 PBS (60 mM) 1.09 mM 0% na EGCG, 120 °C [327] pH solvent initial concentration % at t = 20 minutes degradation
product 3 citric acid-sodium
citrate buffer (0.1 M)
1.09 mM 95% GCG 1%
4 citric acid-sodium citrate buffer (0.1 M)
1.09 mM 90% GCG 5%
5 NaH2PO4-Na2HPO4 buffer (0.1 M)
1.09 mM 55% GCG 19%
6 NaH2PO4-Na2HPO4 buffer (0.1 M)
1.09 mM 20% GCG 8%
- distilled water 1.09 mM 75% GCG 13%
138
Supplementary Table 4. The effect of ionic strength on the stability of EGCG. The initial concentrations of EGCG, its mean half-life, and its degradation products in respective ionic strengths are listed. na = information not available. EGCG, pH 5, t=50 °C, acetate buffer (0.05 M) + NaCl to adjust ionic strength
[406]
ionic strength
solvent initial concentration t = concentration decreased to 90%
degradation product
0.1 M acetate buffer 109 mM 7.35 hours na 0.2 M acetate buffer 109 mM 5.51 hours na 0.3 M acetate buffer 109 mM 5.16 hours na 0.4 M acetate buffer 109 mM 4.52 hours na 0.5 M acetate buffer 109 mM 3.21 hours na
139
Chapter 7
Towards controlled delivery of epigallocatechin
3-gallate as an anti-inflammatory treatment for
degenerative disc disease
Olga Krupkova, Aikaterini Zafeiroupoulou, Oddny Björgvinsdóttir, David
Eglin, Stephen J Ferguson, Karin Wuertz-Kozak
Report of work in progress
140
141
7.1 Abstract
We have previously identified the polyphenol epigallocatechin 3-gallate (EGCG) as a
promising therapeutic candidate for the treatment of degenerative disc disease. In human
intervertebral disc (IVD) cells, EGCG not only inhibits inflammatory responses, but also reduces
the detrimental effects of oxidative stress. In addition, EGCG ameliorates IVD-related
radiculopathy in experimental animals, which underlines its clinical significance. However, the
EGCG molecule is prone to degradation, which complicates clinical translation of EGCG-based
treatments. Therefore, a delivery system that protects EGCG and provides its stable release has
to be developed to ensure efficient long-lasting treatment of degenerative disc disease.
The overall goal of this project is to develop and characterize an intradiscal delivery
system, stabilizing EGCG and providing sustained release. The delivery system consists of
EGCG-containing gelatin microparticles dispersed in injectable thermo-reversible hydrogel (HA-
pNIPAM). Microparticles were created by electrospraying and their biocompatibility was
verified on human IVD cells in vitro using MTT assay. The release test was designed and the
release of free EGCG from HA-pNIPAM was measured, to establish a baseline release. This
report outlines methodology, current progress, and future steps of the project.
142
7.2 Introduction
Degeneration and aging of the intervertebral disc (IVD) includes morphological and
functional changes such as calcification of endplates, disruption of the annulus fibrosus (AF)
and loss of extracellular matrix (ECM) in the nucleus pulposus (NP). The degenerative process
involves a combination of multiple events including inflammation, auto-immune reactions,
senescence and cell death [43, 240]. Although IVD degeneration is not symptomatic in all cases,
it is known to be one of the major factors in the development of low back pain [128]. Despite
the high prevalence of discogenic low back pain and the consequently high economic burden,
only symptomatic treatments (e.g. analgesics or spinal surgeries) are available [435-437]. Novel
therapeutic strategies such as molecular and cell therapies could restore the disc function in a
biologically relevant manner. Injected or implanted cells can support the old disc population,
produce ECM and “rejuvenate” the aging disc. However, as degenerative IVDs are often
catabolic, inflammatory, and acidic, the implanted cells are at risk of apoptosis [59, 177].
Inflammation and catabolism in the IVD can be regulated by molecular therapeutics, which can
prevent premature aging, reduce cell death, enhance ECM turnover and inhibit expression of
pain-mediators, thus enhancing survival and functionality of the resident cells while
counteracting nociception. Several pharmacological molecules promoting IVD regeneration
have been tested to date, with encouraging results [16, 176, 438].
Recently our group has shown that phytochemicals can serve as promising compounds
to reduce inflammation, catabolism and pain arising from the IVD [18, 170, 171, 173].
Epigallocatechin gallate (EGCG), a polyphenol found in tea (Camelia sinensis), interferes with
the IL-1β-induced inflammatory cascade, inhibiting the key inflammatory mediators NF-kB, JNK
and p38 and reducing the expression of inflammatory and catabolic genes/proteins (IL-6, IL-8,
MMP1, MMP3, MMP13, TLR2, COX-2, NGF, iNOS). Moreover, local application of EGCG in a rat
model of IVD herniation attenuates radiculopathy, hence supporting the clinical relevance of
this compound. In addition to its anti-inflammatory effects, EGCG also possesses the ability to
inhibit free radical-mediated damage [439]. We have shown that EGCG does not only
ameliorate inflammation and pain, but also reduces oxidative stress-induced cell death of IVD
cells, via up-regulation of PI3K/Akt pathway [311]. This suggests that EGCG constitutes a
promising therapeutic option, as it targets multiple deregulated processes in the degenerative
IVD.
Oral drug delivery of pharmaceutical compounds has the highest compliance rates.
However, after the oral ingestion of green tea or EGCG supplements, only 0.1-1.1% of the orally
administered dose reaches the systemic circulation [329]. On the other hand, when high doses
of EGCG-containing preparations (10-29 mg/kg/day) are ingested, cases of hepatotoxicity and
nephrotoxicity can occur [391]. Importantly, the avascular disc has limited blood supply.
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Therefore, EGCG delivered orally is most likely not appropriate for IVD applications, as rapid
degradation or side effects in non-target tissues can occur. Local delivery (e.g. intradiscal
injection) overcomes the poor intestinal absorption and reduced blood supply to the IVD and
significantly lowers the risk of systemic side-effects [31]. To promote the successful clinical
translation of safe and therapeutically active EGCG doses, delivery system with the ability to
enhance EGCG’s stability, to provide sustained delivery, and to account for specific local tissue
requirements is necessary.
Although various encapsulation techniques for plant polyphenols have been developed,
these techniques are not suitable for the encapsulation of EGCG [51]. Unlike most of other used
compounds, EGCG is hydrophilic and thus tends to leak out during encapsulation, causing low
encapsulation efficiency. Moreover, the EGCG molecule is prone to degradation in extreme
conditions (high temperature and pH), common to traditional encapsulation methods [31, 439].
Bearing in mind future clinical application, the use of toxic solvents and encapsulation-
enhancing additives is not appropriate.
Electrospraying is an electrohydrodynamic encapsulation method that overcomes these
limits. Electrospraying can be used to generate solid particles in a one-step process without the
need of employing high temperatures or toxic solvents. During electrospraying, the solution jet
breaks down into fine droplets, which acquire spherical shapes due to the surface tension,
subsequently producing solid microparticles upon solvent evaporation [54]. EGCG-loaded
particles, created by electrospraying, exhibit high encapsulation efficiency [54]. However, once
injected, particles can escape the IVD space, or be damaged by mechanical loading.
To reduce particle displacement and damage, microparticles can be dispersed in in situ-
forming biomaterials, which provide protection and in the ideal case also improve the load-
bearing function of the NP. Environmentally-responsive hydrogels solidify after their injection
into the IVD tissue. Thermo-reversible poly(N-isopropylacrylamide) (pNIPAM) solidifies via intra-
and inter-molecular hydrophobic interactions between isopropyl groups of pNIPAM at body
temperature (gel point ~32°C). High molecular weight (Mw) pNIPAM is non-biodegradable, but
shorter pNIPAM chains (up to Mw 10.000) can undergo renal excretion [200]. A modified
pNIPAM has been recently used as a vehicle for siRNA and celecoxib in IVD degeneration
studies in vitro and in vivo [191, 197]. As hyaluronic acid (HA) is the most abundant water-
absorbing molecule in the NP tissue, HA-based biomaterials are suitable for the use in IVD
repair and regeneration [155]. HA-grafted pNIPAM (HA-pNIPAM) has been successfully tested
for delivery of cells and growth factors into the degenerative IVD [198, 199, 440, 441].
The overall goal of this project is to achieve controlled delivery of EGCG as an anti-
inflammatory therapeutic method in the treatment of degenerative disc disease. We
hypothesize that electrosprayed EGCG microparticles dispersed in HA-pNIPAM can serve as an
intradiscal delivery system with enhanced stability and sustained release of EGCG, compared to
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direct incorporation of EGCG in HA-pNIPAM or compared to pure EGCG microparticles without
a carrier. The specific aims of the project are (1) to prepare stable biocompatible EGCG
microparticles for dispersion in HA-pNIPAM and (2) to characterize the release of EGCG from
this delivery system.
7.3 Methods
Electrospraying of gelatin microparticles
The biocompatible natural material gelatin was used for the encapsulation of EGCG.
Gelatin A (G1890, Sigma) was dissolved in 20% (v/v) acetic acid (A6283, Sigma), stirred at 40°C
for 6 hours and cooled down to a room temperature for electrospraying [54, 442]. As gelatin
tends to swell and dissolve rapidly at 37°C, the cross-linking agent glutaraldehyde (GA, 5% w/v),
added at different concentrations (37.5, 62.5 and 87.5 μg/mL), was tested to extend the lifetime
of the gelatin microparticles [443]. 6% (w/v) gelatin was used for preparation of non-cross-
linked microparticles, whereas 4% (w/v) gelatin was used for crosslinking [54, 442].
Electrospraying was conducted using a previously described custom-made setup
connected to high voltage supply and situated within a climate chamber for environmental
control, with the temperature and humidity being fixed to 24°C and 40%, respectively [444].
Spherical particles can be formed by varying electrospraying parameters, namely flow rate,
voltage and concentration [445]. Gelatin was introduced in a 5 mL plastic syringe, pumped at a
steady flow-rate and voltage (2 and 4 mL/min, 20-22 kV) through a 20G stainless steel needle
and microparticles were collected on an aluminum sheet placed at a distance of 10 cm from the
spray needle [443, 446]. Samples of particles were sputter-coated with gold under vacuum (8
µm) and analyzed by scanning electron microscopy (SEM, FEI Quanta 200F) at an accelerating
voltage of 10 kV and a working distance of 10 mm. Particle shape and size distribution was
calculated from 3 independent images analyzed by Image J (>100 particles in total).
Biocompatibility of gelatin microparticles
Biocompatibility of gelatin microparticles without EGCG was tested by MTT assay, to
exclude toxicity of the solvent acetic acid and of the crosslinking agent glutaraldehyde. Human
NP tissue (grade III-V), isolated from donors undergoing spinal surgeries for degenerative disc
disease or disc herniation (approved by Kantonale Ethikkommission Zürich EK-16/2005), was
used. The tissue was enzymatically digested and primary cells were isolated [18]. Primary cells
were seeded in Dulbecco’s Modified Eagle’s Medium (DMEM/F12, D8437, Sigma, St. Louis, MO,
USA), supplemented with 10 % fetal calf serum (FCS, F7524, Sigma), penicillin (50 units/mL),
streptomycin (50 μg/mL) and ampicillin (125 ng/mL, 15240-062, Gibco, Carlsbad, CA USA) and
sub-cultured using 1.5% trypsin (15090- 046, Gibco). Microparticles were sterilized by UV-C (3
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hours) and placed in the upper part of a trans-well system (ThinCert™ Tissue Culture Inserts,
82051-570, VWR), with human primary IVD cells seeded on the bottom. On day 4, fresh MTT (3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, M5655, Sigma) solution in media
(0.5 mg/mL) were added and maintained for 3 hours at 37°C. MTT was discarded, cells were
lysed in DMSO (D8418, Sigma) and the absorbance was measured at 565 nm [311]. The result
was expressed as percentage of control cells, without exposure to microparticles.
The release of EGCG from HA-pNIPAM
The release test of free EGCG from HA-pNIPAM was performed at 37°C in 10 mL of 0.9%
NaCl as release media. 10 mL of the release media was collected and exchanged at 1h, 24h and
on days 3, 5 and 7. The release of EGCG was tested by ferrous tartarate assay, specific for
catechins. EGCG dyeing solution [1 g ferrous sulfate (F8633, Sigma) + 5 g potassium sodium
tartrate tetrahydrate (S2377, Sigma) in 1000 mL of distilled water] was mixed with 0.067 M
potassium phosphate buffer (pH 7.5) as 1:4. This mixture was used to dilute EGCG samples for
colorimetric spectroscopy (540 nm) in a 1:1 ratio [410]. UV-VIS spectroscopy (275 nm) was
employed as a complementary method to ensure accuracy of values obtained by ferrous
tartarate assay [54].
7.4 Results
Electrospraying of gelatin microparticles
Well-shaped microparticles with an average diameter of 351.52 ± 62.56 nm can be
produced by electrospraying of 6% non-cross-linked gelatin in 20% acetic acid under 21 kV and 2
µL/min. 5 mM EGCG (E4143, Sigma) was mixed with 6% non-cross-linked gelatin in 20% acetic
acid and sprayed under the same conditions. As a result, spherical microparticles with diameter
496.67 ± 151.66 nm were formed (Figure 1).
As non-cross-linked gelatin exhibits quick degradation at 37°C, crosslinking is required.
Glutaraldehyde (GA) crosslinking increases the stability of gelatin, reduces its solubility, and
provides slower release of encapsulated compounds [443]. Therefore, 37.5 µg/mL, 62.5 µg/mL,
and 87.5 µg/mL of GA was added into plain 6% gelatin and sprayed under the flow rate of 2 and
4 µL/min, based on the initial results described above. However, the addition of glutaraldehyde
resulted in formation of fibers between particles, especially at the higher flow rate (Figure 2).
To reduce the fiber formation, the concentration of gelatin for GA cross-linking was
lowered to 4%, while the flow rate was unchanged. As a result, smaller fibers were formed. 2
µL/min flow rate further reduced the fiber formation, especially in the group cross-linked with
37.5 µg/mL GA (Figure 3). Gelatin cross-linked with 37.5 µg/mL GA and sprayed under the 2
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µL/min yielded best results, with an average particle size of 907.5 ± 241.49 nm and was thus
selected for further experiments. EGCG encapsulation tests are currently ongoing.
Figure 1. SEM images of electrosprayed non-crosslinked 6% gelatin and gelatin-EGCG. The most suitable parameters for electrospraying of plain gelatin have been determined as 6 % (w/v), 2 µl/min and 21 kV. EGCG was encapsulated and sprayed under the same conditions, forming spherical particles with diameter 496.67 ± 151.66 nm.
Figure 2. Scanning electron microscopy images of electrosprayed cross-linked 6 % gelatin. Glutaraldehyde (GA) was used as a crosslinking agent. 37.5 µg/mL, 62.5 µg/mL, and 87.5 µg/mL of GA in 6% gelatin solution caused fiber formation, especially at flow rate of 4 µL/min.
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Figure 3. Scanning electron microscopy images of electrosprayed cross-linked 4 % gelatin. Glutaraldehyde (GA) was used as a crosslinking agent. 37.5 µg/mL, 62.5 µg/mL, and 87.5 µg/mL of GA in 4% gelatin solution reduced fiber formation, especially at the flow rate of 2 µL/min.
Biocompatibility of gelatin microparticles
While the non-toxicity of HA-pNIPAM has previously been demonstrated [441], the
biocompatibility of the cross-linked gelatin microparticles was examined within this study.
Biocompatibility of cross-linked gelatin microparticles was tested on human IVD cells (n = 3
independent repeats of one donor). 15, 30, 60 mg of particles were tested to span the amount
planned to be used in the proposed delivery system (= 42.3 mg). Particles were added into 2 mL
of complete media and kept at 37°C for 4 days. No major changes in cell viability were detected
in the groups treated with 15 and 30 mg of particles cross-linked with 62.5 µg/mL GA compared
to untreated IVD cells. However, 60 mg of particles reduced cell viability, indicating toxicity of
higher quantities of microparticles, most likely due to the increased concentration of GA (Figure
4). With the lower GA concentration, the designed delivery system is thus expected to be non-
toxic for IVD cells. Experiments with pure gelatin (without crosslinking) as well as with lower
GA concentrations (37.5 µg/mL GA ) are currently conducted to confirm this notion.
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Figure 4. Biocompatibility test of electrosprayed cross-linked gelatin particles. 15 and 30 mg of gelatin particles, cross-linked with 62.5 µg/mL glutaraldehyde, did not activate cell death in IVD cells during 4 days in culture, while a drop in viability was observed at the highest concentration. Graph shows mean ± SD (n = 3).
Design of EGCG release tests
To determine the release of EGCG from the delivery system, appropriate control systems
are included (Table 1). The delivery system is composed of EGCG, encapsulated in 4% gelatin
particles cross-linked with 37.5 µg/mL glutaraldehyde, and thermo-sensitive injectable HA-
pNIPAM, provided by the AO Foundation Davos (System 1). Control systems contain free 5 mM
EGCG in HA-pNIPAM (System 2) and pure EGCG-microparticles without any carrier (System 3).
Additional controls include free 5 mM EGCG (System 4), and HA-pNIPAM without particles
(System 5).
The release of EGCG from HA-pNIPAM
The data on the release of free EGCG from HA-pNIPAM (System 2) is presented in this
report. 5 mM EGCG was mixed with 1 mL of 10% and 15% HA-pNIPAM to establish a baseline
release of the free compound over a period of 7 days (n = 5) (Figure 5A). Cumulative release was
calculated from the obtained data (Figure 5B). Free EGCG, incorporated in HA-pNIPAM, showed
high-dose burst release during the first 24 hours. EGCG was completely released before the day
5. This demonstrated that improvement of EGCG retention in the HA-pNIPAM through
encapsulation is needed. No major difference between EGCG release from 10% and 15% HA-
pNIPAM was detected.
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Table 1. Experimental design of the release tests: EGCG delivery system and control systems. EGCG particles, formed by electrospraying of 4% gelatin, will be mixed with 1 mL of HA-pNIPAM for the release tests (System 1). Appropriate control systems will be used (System 2 - 5). A total concentration of 5 mM (2.293 mg/mL) EGCG was chosen, aiming for long-term sustained release of 5 - 15 µM EGCG. 100% encapsulation efficiency of EGCG in gelatin particles is assumed. Incorporation of 5 mM EGCG into 1 mL of HA-pNIPAM for the release tests
Free EGCG
(mg) % of EGCG in particles
EGCG particles (mg)
HA-pNIPAM 10% / 15% (mg)
1 EGCG particles in HA-pNIPAM
- 5.42% 42.3 100 / 150
2 Free EGCG in HA-pNIPAM
2.293 - - 100 / 150
3 EGCG particles - 5.42% 42.3 -
4 Free EGCG 2.293 - - -
5 HA-pNIPAM - - - 100 / 150
Figure 5. EGCG release (A) and cumulative EGCG release (B) from 10% and 15% HA-pNIPAM measured by the ferrous tartarate assay. Burst release of free EGCG was detected during first 24 hours. EGCG was completely released before the day 5. Graph shows mean ± SD (n=5).
7.5 Discussion
The molecular structure of EGCG is prone to rapid degradation, which causes issues in
clinical translation of EGCG-based therapeutics, despite their high potential. Therefore, a
suitable delivery system, including an effective means to protect EGCG from degradation, has
to be developed for each specific application. To stabilize EGCG and to ensure its sustained
release in the degenerative IVD, EGCG was incorporated into gelatin microparticles, which will
be then combined with the injectable carrier HA-pNIPAM. The anticipated benefits from this
system are: (1) Minimal-invasive applicability, (2) tissue retention, (3) sustained release, (4)
degradation protection and consequently (5) prolonged activity of EGCG.
Biocompatible polymers such as PLGA, pNIPAM, chitosan, or gelatin, have been tested as
drug carriers for the IVD [155, 447-449]. Gelatin is a collagen derivate, hence closely resembles
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one of the main extracellular matrix components of the IVD. Gelatin was previously shown to
be appropriate for intradiscal delivery: The injection of pure gelatin-based microparticles
without addition of cells or drugs already positively affected the NP microenvironment and
decreased the rate of apoptosis in a rabbit IVD degeneration model [150, 450]. Gelatin
microparticles were successfully used to deliver platelet rich plasma (PRP) into the NP in rabbits,
with sustained release through diffusion via the pores of the slowly degrading gelatin network
[192, 193].
Aside from gelatin-EGCG-microparticles, a critical component of the designed delivery
system is the carrier. HA-pNIPAM hydrogel was selected as the carrier, as it has previously been
shown to be suitable for intradiscal delivery [440]. A major challenge of injectable hydrogel-
based therapies in degenerative disc disease is to replicate the mechanical properties of the
native NP. Although hydrogel-based materials are presumed suitable for mimicking the NP
environment [451], their mechanical properties such as the Poisson’s ratio, bulk and shear
modulus, can differ from the native tissue. Poisson’s ratio of the NP determines the change of
lateral expansion or shrinkage under compressive or tensile loading. The bulk modulus
indicates the stiffness of the NP under compression or tension and the shear modulus
determines the torsional stiffness of the NP. For instance, the complex shear modulus (G*) of a
human NP is in the range of 7–21 kPa [451, 452], while G* of HA-pNIPAM (at 37°C) has been
determined as 4.6 kPa [441], suggesting lower mechanical stability of the HA-pNIPAM. The
influence of the hydrogel on the mechanical properties of the NP can be investigated by
measuring the change in the aforementioned parameters, before and after injection, using e.g.
whole organ cultures in suitable bioreactors. Based on the results, the mechanical properties of
HA-pNIPAM can be tuned by varying the concentration and ratio of hydrogel constituents.
It has been suggested that slowly biodegradable materials are favorable for IVD repair.
Although high Mw pNIPAM is not biodegradable and does not undergo renal clearance [155], it
has been shown that short pNIPAM chains (Mw < 100.000) undergo renal excretion in vivo
[453]. Moreover, HA-pNIPAM can be broken down in vivo due to the degradation of HA. A
modified HA-pNIPAM with hydrolytically degradable linkers is currently under development
(AO Foundation, Davos). The linkers will enable generation of small Mw degradation products
for improved renal excretion, hence increasing clinical applicability of HA-pNIPAM [200].
To date, we have optimized the conditions for electrospraying of EGCG-gelatin
microparticles and verified biocompatibility of particles following crosslinking with
glutaraldehyde. We observed that formation of beaded fibers, which occurred specifically under
increased flow rate and with gelatin crosslinking, could be compensated by lowering the
gelatin concentration. We have shown that electrospraying of 4 % gelatin, cross-linked with
37.5 µg/mL glutaraldehyde, resulted in formation of well-shaped and biocompatible
microparticles. We have also shown that free EGCG mixed with HA-pNIPAM (System 2)
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exhibited an unfavorable high-dose release, indicating the need to include an EGCG
encapsulation in the system.
The aim 1 was achieved, while the aim 2 will be completed in the future. The release of
EGCG from the delivery system (System 1) will be measured in solution and compared with
control systems (System 2, 3, 4 and 5). The release of EGCG will be determined by ferrous
tartarate assay, as described above. In addition, gelatin degradation in the delivery system will
be measured, as the release of EGCG from the microparticles is expected to occur due to the
gelatin degradation. Gelatin degradation will be measured by the presence of hydroxyproline
(HYP) in the release media, using the HYP assay kit (MAK008, Sigma) [30]. Gelatin degradation
will be expressed relative to control HA-pNIPAM without particles.
To verify whether encapsulation influenced the functionality of the EGCG molecule, the
antioxidant activity of EGCG in release media will be measured using 2, 2’-diphyenyl-1-
picrylhydrazyl (DPPH) radical scavenging assay (D9132, Sigma) at 517 nm (n = 5). 100 μl of EGCG
solution will be incubated with 500 μl of DPPH (250 μM in ethanol; D9132 Sigma) for 1 hour at
RT. DPPH radical scavenging, measured at 517 nm, manifests as a decrease in absorbance. L-
Ascorbic acid (A4403, Sigma) and ethanol (O2860, Sigma) in equal amounts will be used as
assay controls [454]. In the future, the long-term anti-inflammatory activity of optimized EGCG
delivery system will be characterized in IVD cell and organ cultures treated with inflammatory
cytokines. The anti-inflammatory activity will be measured by RT-qPCR, as described before [18].
It has been reported that 1-3 mL of material can be injected into the center of human
lumbar degenerative NP [455, 456]. Therefore, we incorporate EGCG microparticles in 1 ml of
HA-pNIPAM. Our aim is to achieve a stable release of 5-15 µM EGCG over a period of one month
[457]. Given the fact that diurnal fluid loss in the human NP is 10-20 % [458, 459] and burst
release always occurs [31], our currently ongoing release tests are designed to encapsulate a
high-dose (5 mM) of EGCG. As the relationship between the concentration of encapsulated
EGCG, the quantity of HA-pNIPAM, and the EGCG diffusion rate is not yet known, the amounts
of these components may need to be adjusted, to achieve optimum EGCG activity. Once
optimized and fully characterized, the HA-pNIPAM-based delivery system can be compatible
also with other small-molecular therapeutics such as kinase inhibitors and other
phytochemicals, hence offering great clinical versatility/applicability.
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153
Chapter 8
Discussion
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155
8.1 Summary
Molecules targeting deregulated signaling pathways involved in painful degenerative
disc disease (DDD) are promising therapeutic candidates. Natural compounds can exhibit
antioxidant, anti-inflammatory and pro-survival activities via modulation of multiple signal
transduction and apoptotic cascades, with minimal adverse effects. The overall goal of this
thesis was to evaluate polyphenol epigallocatechin gallate (EGCG) as therapeutic candidate
against DDD. In this thesis, the anti-inflammatory (Aim 1) and the antioxidant (Aim 2)
properties of EGCG were investigated, mainly in primary cell cultures. Subsequently, a
degenerative nucleus pulposus (NP) organ culture model was developed in order to assess the
effects of EGCG in the NP tissue (Aim 3). An intradiscal sustained release system for EGCG was
designed and is currently under development (Aim 4).
The model systems used in our studies were chosen to adequately represent
inflammation and oxidative stress that occur in diseases. During DDD, regulation and function
of the IL-1 pathway are altered. Synthesis of IL-1α, IL-1β and IL-1R increases, whereas the
expression of IL-1R antagonist remains unchanged [82]. Cytokine-stimulated cell cultures
appropriately reproduce deregulated inflammatory signaling, representing a good system for
the evaluation of anti-inflammatory treatments. Similarly, hydroxyl radical, released from H2O2,
induces oxidative stress in a biologically relevant manner, allowing to test antioxidant
molecules in cell cultures [460]. However, IL-1R antagonists and natural antioxidant enzymes
are commonly extracellular (e.g. diffused in the ECM). Therefore, organ cultures are essential
for investigating the complex effects of the therapeutic compounds, as organ cultures contain
both, the cells and the native ECM. In this thesis, cell cultures were used to reveal the
mechanisms involved in EGCG action, whereas organ cultures were employed to investigate
the effects of EGCG on the whole NP. Importantly, clear evidence that EGCG interferes with the
inflammatory and the oxidative stress responses in the IVD and exhibits analgesic effects in
vivo could be provided.
8.1.1 Clinical relevance
Low back pain, often caused by complex interactions of biological and psychological
factors, is comprised of both nociceptive and neuropathic component [461].
Molecules that play a major role in the neuronal sensitisation are prostaglandins.
Biosynthesis of prostaglandin E2, mediated by cyclooxygenase-2 (COX-2), significantly increases
in degenerative and injured tissues, magnifying nociceptive and inflammatory responses [462].
The acute nociceptive IVD-related pain can be reduced by analgesics such as non-steroidal anti-
inflammatory drugs (NSAIDs) that target the production of prostaglandins. However,
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prolonged PGE2-mediated sensitization of the nociceptor neurons of dorsal root ganglion (DRG)
contributes to the transition from acute to chronic pain [463]. NSAIDs are not effective in
chronic pain conditions, hence being unable to prevent the DDD progression. As EGCG does not
only reduce the expression of COX-2, but also inhibits other pain-related mediators and
inflammation, it is considered a good therapeutic candidate for the management of chronic
pain.
Aside from these effects, EGCG was furthermore able to reduce nerve growth factor
(NGF) expression. NGF has been recognized as an important mediator of chronic pain and as a
marker of pathological inflammation [464, 465]. NGF binds to two different receptors on the
nociceptor neurons: (1) To the low-affinity 75 kDa neurotrophin receptor (p75NTR), which binds
all neurotrophins, and (2) to the tropomyosin-related tyrosin kinase A (TrkA), which binds NGF
more selectively than the other neurotrophins. NGF-activated p75NTR regulates a wide range of
functions, including axonal growth, apoptosis and myelination [466]. NGF signaling through
TrkA mediates the nociceptive functions of the sensory neurons through enhanced expression
of substance P, calcitonin gene-related peptide (CGRP) and transient receptor potential
vanilloid 1 (TRPV1) [464, 467]. Therefore, EGCG-mediated inhibition of NGF expression in the IVD
may reduce both, nerve ingrowth and nerve sensitization.
Neuropathic pain, arising from neuronal damage, often fails to respond to ordinary
medication. Therefore, therapeutics with the ability to attenuate the IVD-related radicular pain,
are desired. In order to determine the effectiveness of EGCG against neuropathic pain, in vivo
testing is required. As currently no model of DDD neuropathy exists, a well-established rat
model of IVD-induced radicular pain, coupled with the von Frey test, was employed in our
study. The von Frey test is widely used to investigate pain-related behavior that is caused by
radiculopathy with limb symptoms, typical for IVD herniations [170, 230, 232]. In our 28-day
animal experiment, EGCG significantly improved the hind paw withdrawal threshold,
compared to the NP-only groups. We hypothesize that EGCG attenuated disc-related radicular
pain by inhibiting the inflammatory responses of the DRG, induced through the herniated NP
material. Recently, it has been reported that EGCG and its derivatives can reduce the intensity
of neuropathic pain after chronic nerve constriction injury in mice through an interaction with
NF-κB and the fatty acid synthase (FASN) in the dorsal horn of the spinal cord [468].
Furthermore, nerve damage and resulting neuropathic pain are closely linked with the
increased levels of the reactive oxygen or the nitrogen species [469]. As nitric oxide synthase
(iNOS in IVD cells, nNOS in nervous tissue) is involved in the generation of reactive nitric oxide
(NO) [470], elevated presence of NOS in DRGs and IVDs can intensify the pain sensation [471,
472]. Although we did not specifically test the DRG tissue, we demonstrated that EGCG reduced
the expression of iNOS in IVD cells. This possibly contributes to the amelioration of pain in vivo.
Moreover, EGCG enhanced survival of the IVD cells in the presence of the oxidative stress.
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Recently, EGCG was shown to exhibit significant anti-apoptotic effects in skeletal muscles after
induction of sciatic nerve crush injury in mice [473], supporting our hypothesis on the protective
role of EGCG against oxidative stress-induced neuronal damage.
Interestingly, the improvement in the pain behavior was not immediate. This could be
caused by interaction between the extruded NP tissue and the immune system. It is well
known that macrophages are recruited to herniated NP material, where they can initiate
resorption through the release of MMPs and stimulate the IVD cells to express e.g. MMP1 and
MMP3 [474, 475]. On the other hand, macrophages also contribute to the increased cytokine
levels, hence promoting DRG damage [476]. Based on literature and our data, we suggest that
although EGCG reduced radiculopathy by inhibiting inflammation, it may have also affected
clearance of the displaced NP tissue in this model. Additional tests on the effects of EGCG on
macrophage-induced expression of MMPs will be necessary to determine whether resorption
of the herniated material may be slowed down upon treatment. If so, EGCG may not be
suitable for treating pain in patients with NP extrusions. To closely investigate the effects of
EGCG on radicular pain, an animal model where NP is not directly exposed to the DRG (e.g.
nerve constriction injury model) should be used.
We showed that EGCG did not only inhibit the expression of molecules involved in
nociception (cytokines, COX-2, NGF), but also reduced neuropathic pain markers (oxidative
stress, iNOS) and limb symptoms in experimental animals. Therefore, EGCG may be superior to
current clinical strategies, as it can attenuate both, the inflammation and pain, providing a
possible solution for the management of chronic and radicular pain in affected patients.
As 10 µM EGCG exhibited promising effects in the cell and organ cultures, we believe
that it possesses the ability to balance the inflammatory and catabolic changes also in human
IVDs in vivo. However, the exact dose and the duration of an EGCG intervention for DDD
therapy are yet to be established. The low stability of the EGCG molecule reduces its
bioavailability and limits its activity in biological systems. Upon oral ingestion, only 0.01 - 0.1%
of EGCG is absorbed, leading to maximum plasma concentration of 1 µM with a half-life of no
more than 12 hours. Oral intake of higher doses of EGCG can cause side-effects, affecting the
liver and the kidney [477, 478]. Therefore, encapsulation of EGCG into microparticles is desired
to increase its stability, bioavailability, and at the same time to protect the patient from
unspecific toxicity. Nevertheless, oral delivery is not practical for targeting the human IVD, due
to its avascular nature. Moreover, DDD is further limiting the diffusion of the solutes from
blood into the center of IVD. Therefore, encapsulated EGCG should be delivered locally in a
minimally-invasive manner.
An ideal NP delivery system would be composed of a biocompatible material that is
liquid before application and thus injectable through a needle, but rapidly solidifies after the
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injection [200]. A biomaterial with similar mechanical properties as the native NP is needed for
normal IVD function. However, the mechanical properties of the NP are complex and vary with
the stage of degeneration, thus difficult to replicate [452]. We are exploring and option of using
an injectable thermo-reversible hydrogel HA-pNIPAM to deliver EGCG encapsulated in gelatin
microparticles. Previously, HA-pNIPAM was proven suitable for stable drug release in the IVD,
exhibiting rapid gelation and good mechanical stability [200]. Although our delivery system has
not yet been fully tested, we provide evidence that electrosprayed gelatin microparticles are
biocompatible for IVD cells. We believe that once established, this treatment option can
constitute a clear therapeutic advantage over the current treatments of DDD.
8.1.2 Clinical applicability
The therapeutic activity of EGCG in the IVD is yet to be established in clinical trials.
Nevertheless, several phase I-III clinical trials have been performed on the effects of EGCG in
neurodegenerative diseases [479, 480], cancer chemoprevention [481-483] and metabolic
disorders such as insulin resistance and obesity [484-486], with promising results. In addition, it
has been proposed that the stabilization of EGCG via encapsulation can facilitate the clinical
translation of EGCG. Hydrogel-based injectable systems for the delivery of the therapeutic
agents and cells into the IVD are currently being tested in phase I-III clinical trials [487]. In our
approach, an injectable in-situ forming hydrogel was chosen to deliver EGCG-loaded
microparticles into the IVD. The ultimate goal is to reduce inflammation, catabolism, and pain
occurring due to the degenerated IVD.
Once the preclinical phase is successfully completed, clinical trials will determine to
which extend our experimental results reflect the treatment outcomes in clinical situation.
Careful patient selection is needed to evaluate treatment suitability. We believe that our
strategy can be useful in the treatment of early and moderate painful DDD with no major
defects in the annulus fibrosus (AF) structure. In early and moderate DDD, homeostasis in the
NP has shifted towards the catabolism. In such condition, cytokines and pain-mediating
chemicals are released from IVD cells, irritating existing and newly formed nerve fibers in the
AF. At the same time, the proteoglycan content in the NP is still sufficient and the disc height is
preserved. According to the diagnostics using T2-weighted MRI images, our strategy is intended
for homogeneous (grade I) or inhomogeneous (grade II) painful discs with bright hyperintense
white signal intensity and normal disc height as well as for inhomogeneous discs with
intermittent gray MRI signal intensity, without clear distinction between the NP and the AF,
and normal or slightly decreased height (grade III) [488]. Therapeutic interventions using anti-
inflammatory and anti-catabolic compounds are thought to restore homeostasis and
functionality of the resident disc cells. On the other hand, the proposed therapeutic strategy
may not be suitable as a single treatment for the advanced stages of DDD, characterized by
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hypointense dark gray MRI signal and decreased disc height (grade IV), or by hypointense black
MRI signal with collapsed disc space (grade V) [488]. In advanced DDD, proteoglycans of the NP
are degraded, disc cells are dysfunctional or disappearing, and the AF is affected by non-
physiological load distribution. The total disc implants or the tissue-engineered materials are
more appropriate for the treatment of advanced DDD. Whether a combination of tissue-
engineered materials with EGCG microparticles could provide any benefits will have to be
tested in future experiments.
The majority of novel treatment strategies for DDD, including our approach, involve
injection of therapeutic agents or cells into the IVD. However, it has been reported that the
needle puncture of the AF can cause micromechanical alterations and accelerate disc
degeneration [489]. It has been reported that the provocative discography (using
predominantly 21G and 26G needles [490]) can result in higher incidence of disc degeneration
over a 10-year period in some patients [127, 491]. Nevertheless, it was shown that most discs,
subjected to discography, d0 not degenerate [127]. Importantly, the puncture outcome is likely
determined by the needle size, as demonstrated in experiments in rabbits [127].
Despite the concerns, intradiscal injections are being used in clinical practice not only
for provocative discography, but also to deliver conventional analgesics. Serious complications
of the current spinal injection therapy (e.g. cauda equina syndrome, septic facet joint arthritis,
puncture-related disc damage, or paraplegia) have been reported, but only in rare cases, hence
making it an overall low-risk intervention compared to classical surgical treatments.
Consequently, there has been no strong evidence for or against the use of any type of injection
therapy in individuals with chronic low back pain [492]. Based on the available data, we
conclude that employing intradiscal injections in our therapeutic approach can be safe. We aim
to use needle size, no bigger than 22G, with which the delivery of cells in the HA-pNIPAM has
been previously validated [441].
Apart from the intradiscal injections, free or encapsulated EGCG can be injected into the
vicinity of the painful IVD, to avoid further damage to the IVD as a result of needle puncture. As
an example of similar approach, a sustained release curcumin depot was recently implanted
near the sciatic nerve in mice and tested for the treatment of IVD-related neuroinflammation
[493]. Topical delivery of EGCG, e.g. via lumbar patches, may also be possible. However,
transdermal delivery systems of EGCG are not yet well developed, due to the hydrophilic nature
of EGCG molecule and the need for using potentially toxic skin penetration enhancers [494].
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8.2 Limitations
The main limitation of primary IVD cell cultures is their tendency to dedifferentiate in
vitro, thus losing the IVD phenotype. Dedifferentiation can be partially avoided using primary
cells in low passages and limiting cell expansion. However, the main disadvantage of this
approach is the low amount of primary cells available for the experiments. As primary IVD cells
are isolated from multiple donors with different genetic background and medical condition,
inter-donor variability occurs, leading to high variations in the cellular responses.
All our in vitro experiments have been performed in normoxia (21 % O2). However, O2
concentration in the center of the healthy human IVD can decrease below 4 % [58]. Low oxygen
concentration in the NP is considered physiological, as it is involved in maintaining the
phenotype of healthy NP cells and ECM. Therefore, our experimental conditions may not
exactly replicate the in vivo situation. On the other hand, it has been reported that normoxia in
vitro correlates with the non-physiological oxidative stress in the degenerative IVD [40, 92].
Therefore, normoxia most likely does not interfere with either of our hypotheses.
Although our studies provided evidence that EGCG functions as an IL-1 pathway
antagonist and PI3K/Akt activator, these studies did not address the exact mechanisms of
action. Previously, EGCG has been shown to interact with surface receptors such as TLR4,
laminin receptor or RTK [378, 495, 496]. Although we did not test this alternative, we
hypothesize that the interaction between EGCG and the surface receptors may be involved in
the effects observed in our studies. Mechanism of EGCG-mediated activation of PI3K/Akt and
its link to protection of mitochondria should be investigated in more detail.
The natural level of the inflammation-related gene expression and ROS was measured
neither in obtained biopsy tissue, nor in healthy donors. Therefore, the anti-inflammatory and
anti-oxidant effects of EGCG on non-stimulated IVD tissue could not be tested. In the animal
study, neither inflammatory responses of the DRG were evaluated, nor were the effects of
EGCG on the resorption of herniated NP tissue. Future studies should address these points.
A clear limitation of our approach is the low stability of the EGCG molecule, which may
impair the drug development process. If this will be the case, using EGCG derivatives with
chemically increased stability such as lipophilic EGCG [426] instead of natural EGCG may solve
this problem.
161
As our proposed therapeutic strategy involves an injection, it is not completely non-
invasive. In order to increase patient compliance, the EGCG sustained release system must
exhibit no side effects and possess high efficacy with therapeutic advantage over current
injection strategies. These aspects can be thoroughly evaluated in animal studies. Single
injection approach is preferred.
8.3 Future steps
Although we showed the promising effects of 10 µM EGCG in vitro, the appropriate dose
and duration off its application in the treatment of DDD in vivo is not known. In DDD, the
expression of MMPs significantly increases, shifting the metabolism towards catabolism and
reducing ECM turnover. Similarly, regulation and function of the IL-1 pathway and ROS is
altered, activating an inflammatory cascade [82]. On the other hand, it is well documented that
balanced levels of cytokines, MMPs, and ROS play an important role in IVD homeostasis, [209,
497], which can be disturbed by common therapeutics. As an example, corticosteroids, which
are classical anti-inflammatory agents often used in clinical practice, directly inhibit the
synthesis and release of TNF-α, IL-1, arachidonic acid, and its metabolites [498], and can thus
significantly reduce the activity of cytokine-regulated pathways in vivo. Consequent complete
cytokine inhibition, as well as the negative effects of corticosteroids on proliferation
morphology, exocytosis, phagocytosis, or cell motility [499, 500], suggest potential damaging
consequences of their use in the IVD. Therefore, molecules that are able to preserve naturally
low levels of cytokines, MMPs, and ROS in vivo, such as the herein tested substance EGCG, can
be better therapeutic candidates. Our studies suggest that EGCG does not pose a risk to the
non-degenerated IVD. However, the mechanisms of EGCG action in healthy and degenerative
NP in vivo are unknown and thus should be carefully investigated in the future.
We hypothesize that the increased EGCG stability and its sustained release will be
linked to more profound effects in the biological systems. However, the release properties of
EGCG from our delivery system are yet to be evaluated. It has been shown that the release of
EGCG in fully controlled in vitro experiments can differ from its release in the tissues. We
expect that the release properties in vivo will depend on the absolute amount of the material
injected, the EGCG dose, the stage of NP degeneration and the applied mechanical loading.
To answer some of these questions, long-term study on the activity of free and
encapsulated EGCG will be performed in the near future, using the degenerative NP culture
model, developed within this thesis. The NP culture model will allow us to evaluate the
therapeutic potential of EGCG and to fine-tune the dose before designing animal studies.
162
In the future, thorough release-activity studies performed in vitro, in organ cultures,
under mechanical loading, and in experimental animals are needed to fully evaluate the
behavior of our delivery system. In addition, the effects of EGCG on adjacent tissues (e.g.
vertebrae and endplates) must also be determined in vivo.
The evidence that phytochemicals can possess beneficial effects in the treatment of the
painful DDD has been provided. Phytochemicals are promising candidates to inhibit
inflammation, catabolism and oxidative stress in the degenerative tissues, while restoring
homeostasis. As different molecular therapeutics function via different mechanisms,
appropriate combinations of these molecules can produce additive and synergistic effects. In
the future, EGCG will be combined with other phytochemicals, to screen for additive and
synergistic effects. In addition, our EGCG-based approach will be tested together with
molecular therapeutics such as growth factors or LinkN, and cell therapies, to investigate full
potential of EGCG in the regeneration of IVD. Our NP culture model will serve for high
throughput preclinical tests of these treatment combinations.
8.4 Conclusion
Management of chronic pain conditions that arise from the degenerative IVD is
challenging due to the multifactorial nature of DDD. Degradation of the ECM, inflammation
and oxidative stress are common hallmarks of painful DDD. However, these hallmarks are not
yet being therapeutically targeted in a specific way. Unspecific analgesics and invasive
surgeries are used nowadays as standard treatments, with limited success. We believe that
minimally invasive procedures, delivering regenerative and analgesic substances and/or cells
will be superior to current standard treatments. Although some progress in the use of ECM
anabolics has been made in preclinical research, anti-inflammatory and antioxidant
approaches have not yet been thoroughly explored. The goal of this thesis was to identify and
test natural compounds which can interfere with the multiple aspects of the DDD, and to
evaluate their potential therapeutic use. In this thesis, evidence could be provided that EGCG is
a promising therapeutic candidate, as it reduces inflammation and oxidative stress in vitro as
well as pain intensity in vivo. To increase the clinical applicability of our findings, a delivery
system for EGCG has been designed and is currently under development. The question whether
our strategy is optimal for the clinical use will hence be answered in the future, after additional
preclinical and possibly also clinical testing.
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Appendix
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193
List of abbreviations
A/PI Annexin V-FITC/Propidium Iodide
AA Ascorbic acid
ADAMTs A disintegrin and metalloproteinases with thrombospondin motifs
AF Annulus fibrosus
Akt Protein kinase B
AP-1 Activator protein 1
APS Average particle size
BHT Butylated dihydroxytoluen
BDNF Brain-derived neurotrophic factor
BMP Bone morfogenic protein
COMT Catechol-O-methyltransferase
COX Clooxygenase
CPP Caseinophosphopeptide
CS Chitosan
CTAB Cetyltrimethylammonium bromide
DA Deoxycholic acid
DDAB Dimethyldioctadecylammonium bromide
DDD Degenerative disc disease
DMBA Dimethylbenz(a)anthracene
DMEM Dulbecco’s Modified Eagle’s Medium
DMMB Dimethylmethylene blue
DP Docosapentaenoic acid
DRG Dorsal root ganglion
EE Encapsulation efficiency
EGCG Epigallocatechin 3-gallate
ELISA Enzyme-linked immunosorbent assay
EPR Electron paramagnetic resonance
FGF Fibroblast growth factor
GA Glutaraldehyde
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GAG Glycosaminoglycan
GCG (–)-gallocatechin gallate
GI Gastrointestinal
GTC Green tea catechins
HMGB1 High-mobility group protein B1
HPLC High-performance liquid chromatography
HYP Hydroxyproline
IGF Insuline growth factor
IKK Inhibitor of κB kinase
IL Interleukin
ILEX Isolated lumbar extension
IP Intraperitoneal
IRAK-1 Interleukin1 receptor-associated kinase 1
IVD Intervertebral disc
JC-1 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide
JNK c-Jun N-terminal protein kinase
LBP Low back pain
LE Loading efficiency
LinkN N-terminus of Link protein
LNs Lipid nanoparticles
LY LY294002
MAPK Mitogen-activated protein kinase
MLL Mixed-lineage leukemia
MMP Matrix metalloproteinase
MRP Multidrug resistance-associated protein
MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide
NF‑κB Nuclear factor kappa-light-chain-enhancer of activated B cells
NGF Nerve growth factor
NO Nitric oxide
iNOS Inducible nitric oxide synthase
NP Nucleus pulposus
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OA Osteoarthritis
P-gp P-glycoprotein
PAA Polyaspartic acid
PCL Polycaprolactone
PEG Polyethylene glycol
Pgal Propyl gallate
PGES Prostaglandin E synthase (COX-2)
PI Polydispersity index
PLA Polylactic acid (PEG)
PLGA Poly(lactide-co-glycolic acid)
PTGS2 Prostaglandin endoperoxide synthase 2 (COX-2)
ROS Reactive oxygen species
RT Room temperature
SA β-gal Senescence-associated β-galactosidase
SASP Senescence-activated secretory phenotype
SEM Scanning electron microscopy
SGF Simulated gastric fluid
SIF Simulated intestinal fluid
SOD Superoxide dismutase
SULT Phenolsulfotransferases
TBP Tata box-binding protein
TIMP Tissue inhibitor of matrix metalloproteinase
TLRs Toll-like receptors
TNF-α Tumor necrosis factor alpha
UGT UDP-glucuronosyltransferases
ZP zeta potential
196
Acknowledgement
First and foremost, I would like to thank my doctoral supervisors, Prof. Dr. Karin Wuertz-Kozak
and Prof. Dr. Stephen Ferguson for the opportunity to work under their guidance. I would like to
thank them for their understanding, support, and encouragement throughout the project.
My acknowledgement also belongs to Dr. Junichi Handa and Dr. Marian Hlavna for their work
and discussions that contributed to this thesis. I also would like to thank my collaborators Dr.
Juergen Klasen (Spital Zollikerberg), Dr. Miho Sekiguchi (Fukushima Medical University), Prof.
Keita Ito (Technical University Eindhoven), and Dr. David Eglin (AO Research Institute Davos), for
their inputs to the studies, revisions of manuscripts, and provided materials. I would like to
thank Helen Greutert for technical support.
I appreciate the students Sophie Chabloz, Aikaterini Zafeiroupoulpu, Julie Amir Tahmasseb,
Sonia Rossi, Seraina Domenig, Joel Zvick and Dominik Kunz, for their work within the
framework of this thesis. Their thoughtful remarks were very helpful, and some results of their
work are adopted in this thesis. In addition, I would like to thank Dr .Karin Wuertz-Kozak, Dr.
Ilsoo Koh and Dr. Hadi Seyed Hosseini for proofreading my thesis.
A special acknowledgement extends to the funding agencies: European Union/Marie Curie
action (FPT7-PITN-GA-2009-238690-SPINEFX), the Herzog-Egli foundation, and the Mäxi
Foundation, for providing the financial support of the studies.
I would like to sincerely thank my friends Dominika Ignasiak, Ilsoo Koh, Patrick Vogt, and Yabin
Wu for listening, advices, and valuable discussions about work and daily life throughout the
years of my PhD.
Last but not least, I would like to thank Hadi and my family for their patience, understanding
and endless support.
197
Curriculum Vitae
Olga Krupkova
born 1.9. 1986 in Brno, Czech Republic.
EDUCATION
2013-2016 PhD candidate, Institute for Biomechanics, ETH Zurich, Switzerland
Supervisors: Prof. Dr. Stephen John Ferguson, Prof. Dr. Karin Wuertz-Kozak
Thesis: Activity and controlled delivery of epigallocatechin 3-gallate in the
treatment of degenerative disc disease
2012-2013 SpineFX Marie Curie training fellow, Institute for Biomechanics, ETH Zurich,
Switzerland
Supervisors: Prof. Dr. Stephen John Ferguson, Prof. Dr. Karin Wuertz-Kozak
Project: Testing the anti-inflammatory effects of epigallocatechin 3-gallate in
intervertebral disc
2010-2011 Research assistant, Faculty of Medicine, Masaryk University, Brno, Czech
Republic
Supervisor: Dr. Stjepan Uldrijan
Project: Therapeutic potential of p38 MAPK inhibition in melanoma
2005-2010 BSc and MSc in Molecular Biology and Genetics, Faculty of Science, Masaryk
University, Brno, Czech Republic
Supervisor: Prof. Dr. Renata Veselská
Thesis: Expression and subcellular localization of nestin in tumor cells
HONORS AND AWARDS
2015 Travel award for participation at winter school of Electrospin paramagnetic
resonance, Ascona, Switzerland.
2014 2nd best student oral presentation award at the 15th eCM conference: Cartilage &
Disc: Repair and Regeneration, Davos, Switzerland.
2010 Special Rector´s research award for the best diploma thesis in the study
program Molecular Biology and Genetics, Masaryk University, Brno.
2010 Dean´s Award for excellent organization of The Student Scientific Conference on
Genetically Modified Organisms, Masaryk University, Brno.
198
PUBLICATIONS
Published
2016 Krupkova O, Ferguson SJ, Wuertz-Kozak K. Stability of (–)-epigallocatechin gallate
and its activity in liquid formulations and delivery systems – a review. J Nutr
Biochem. 2016 Nov(37):1–12.
2016 Handa J, Sekiguchi M, Krupkova O, Konno S. The effect of serotonin noradrenaline
reuptake inhibitor duloxetine on the intervertebral disc-related radiculopathy in
rats. Eur Spine J. 2016 Mar(3):877-87.
2016 Krupkova O, Handa J, Hlavna M, Klasen J, Ospelt C, Ferguson SJ, Wuertz-Kozak K.
The natural polyphenol epigallocatechin gallate protects intervertebral disc cells
from oxidative stress. Oxid Med Cell Longev. 2016 Jan (9):1-17.
2014 Krupkova O, Sekiguchi M, Klasen J, Hausmann O, Konno S, Ferguson SJ, Wuertz
K. Epigallocatechin 3-gallate supresses Interleukin-1β-induced inflammatory
responses of intervertebral disc cells in vitro and reduces radiculopathic pain in
rats. Eur Cell Materials. 2014. 28: 372-86.
2014 Klawitter M, Hakozaki M, Kobayashi H Krupkova O et al., Expression and
regulation of toll-like receptors (TLRs) in human intervertebral disc cells. Eur Spine
J. 2014. 23 (9) 1878-91.
2011 Krupkova O, Loja T, Redova M, Neradil J, Zitterbart K, Sterba J, Veselska R. Analysis
of nuclear nestin localization in cell lines derived from neurogenic tumors. Tumour
Biol. 2011. Aug(4):631-9.
2010 Krupkova O, Loja T, Zambo I, Veselska R. Nestin expression in human tumors and
tumor cell lines. Neoplasma. 2010. 57(4): 291-8.
Under review
2016 Krupkova O, Hlavna M, Amir Tahmasseb J, Zvick J, Kunz D, Ferguson SJ, Wuertz-
Kozak K. An inflammatory nucleus pulposus tissue culture model to test molecular
regenerative therapies: Validation with epigallocatechin 3-gallate.
PRESENTATIONS
Conferences
2015 Krupkova O, Sekiguchi M, Handa J, Klasen J, Konno S, Wuertz-Kozak K, Ferguson
SJ. Polyphenol Epigallocatechin 3-gallate Significantly Decreases Inflammation
and Oxidative Stress in Human Intervertebral Disc Cells. Podium presentation at
the 3rd International ORS Spine Symposium, Philadelphia, USA.
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2015 Krupkova O, Handa J, Hlavna M, Klasen J, Wuertz-Kozak K, Ferguson SJ.
Combating oxidative stress-induced premature senescence in the intervertebral
disc. Podium presentation at the Eurospine, Copenhagen, Denmark.
2014 Krupkova O, Sekiguchi M, Klasen J, Konno S, Wuertz-Kozak K, Ferguson SJ.
Epigallocatechin 3-gallate suppresses interleukin-1β-induced inflammatory
responses in intervertebral disc cells in vitro and reduces radiculopathic pain in
vivo. Podium presentation at the 15th Cartilage & Disc: Repair and Regeneration,
Davos, Switzerland.
2013 Krupkova O, Sekiguchi M, Klasen J, Konno S, Wuertz-Kozak K, Ferguson SJ. The
potential of EGCG for the treatment of painful disc degeneration. Podium
presentation at the Swiss-Japanese Symposium on Musculoskeletal Research,
ETH Zurich, Switzerland.
2013 Krupkova O, Klasen J, Wuertz-Kozak K, Ferguson SJ. The potential of
Epigallocatechin 3-gallate for the treatment of painful intervertebral disc
degeneration. Podium presentation at the Eurospine, Liverpool, UK.
Guest lectures
2016 Targeting degenerative disc disease with bio-active compounds. Center for
Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich
STUDENT SUPERVISION
Master Theses
2016 Aikaterini Zafeiroupoulou (TU Delft/ETH Zurich). Controlled delivery of anti-
inflammatory compounds as a therapeutic strategy for degenerative disc disease.
2015 Sophie Chabloz (ETH Zurich). Investigating the anti-aging effects of resveratrol on
intervertebral disc cells.
Semester Projects
2016 Julie Amir Tahmasseb (ETH Zurich). Anti-inflammatory activity of
epigallocatechin gallate in bovine disc organ culture.
2013 Ion Tripa (ETH Zurich). Anti-oxidant and DNA-protective activity of resveratrol in
human intervertebral disc cells.
Internships
2016 Joel Zvick (ETH Zurich). Analysis of glycosaminoglycan composition in
degenerative nucleus pulposus culture in artificial annulus fibrosus.
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2016 Dominik Kunz (Zurich University of Applied Sciences). Analysis of collagens and
water content in degenerative nucleus pulposus culture in artificial annulus
fibrosus 2016)
2016 Seraina Domenig (ETH Zurich). Effect of curcumin on apoptotic and survival
pathways in intervertebral disc cells.
2014 Sonia Rossi (Kungsholmen gymnasium Stockholm, Sweden). The effects of
natural compounds on intervertebral disc degeneration.
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