ARTICLE IN PRESS

0142-9612/$ - se

doi:10.1016/j.bi

�CorrespondEngineering, N

Tel.: +353 91 4

E-mail addr

sibylle.grad@ao

martin.stoddart

damienohallora

mauro.alini@ao

(A.S. Pandit).1Tel.: +353 92Tel.: +41 813Tel.: +41 814Tel.: +353 95Tel.: +41 81

Biomaterials 29 (2008) 438–447

www.elsevier.com/locate/biomaterials

An injectable cross-linked scaffold for nucleus pulposus regeneration

Damien O. Hallorana,b,1, Sibylle Gradc,2, Martin Stoddartc,3, Peter Dockeryd,4,Mauro Alinic,5, Abhay S. Pandita,�

aNational Centre for Biomedical Engineering Science, National University of Ireland, Galway, IrelandbDepartment of Mechanical and Biomedical Engineering, National University of Ireland, Galway, Ireland

cAO Research Institute, Clavadelerstrasse, 7270 Davos Platz, SwitzerlanddDepartment of Anatomy, National University of Ireland, Galway, Ireland

Received 17 July 2007; accepted 5 October 2007

Available online 23 October 2007

Abstract

Incorporation of scaffolds has long been recognized as a critical element in most tissue engineering strategies. However with regard to

intervertebral disc tissue engineering, the use of a scaffold containing the principal extracellular matrix components of native disc tissue

(i.e. collagen type II, aggrecan and hyaluronan) has not been investigated. In this study the behavior of bovine nucleus pulposus cells that

were seeded within non-cross-linked and enzymatically cross-linked, atelocollagen type II based scaffolds containing varying

concentrations of aggrecan and hyaluronan was investigated. Cross-linking atelocollagen type II based scaffolds did not cause any

negative effects on cell viability or cell proliferation over the 7-day culture period. The cross-linked scaffolds retained the highest

proteoglycan synthesis rate and the lowest elution of sulfated glycosaminoglycan into the surrounding medium. From confined

compression testing and volume reduction measurements, it was seen that the cross-linked scaffolds provided a more stable structure for

the cells compared to the non-cross-linked scaffolds. The results of this study indicate that the enzymatically cross-linked, composite

collagen–hyaluronan scaffold shows the most potential for developing an injectable cell-seeded scaffold for nucleus pulposus treatment in

degenerated intervertebral discs.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Intervertebral disc; Scaffold; Extracellular matrix (ECM); Cell signalling

1. Introduction

Disc degenerative disease (DDD), which may bedefined as an ‘‘aberrant, cell-mediated response to pro-

e front matter r 2007 Elsevier Ltd. All rights reserved.

omaterials.2007.10.009

ing author. Department of Mechanical and Biomedical

ational University of Ireland, Galway, Ireland.

92758; fax: +353 91 563991.

esses: damienohalloran@gmail.com (D.O. Halloran),

foundationn.org (S. Grad),

@aofoundationn.org (M. Stoddart),

n@gmail.com (P. Dockery),

foundationn.org (M. Alini), abhay.pandit@nuigalway.ie

1 495039; fax: +353 91 563991.

414 2390; fax: +353 81 4142288.

414 2448; fax: +353 81 4142288.

1 492784; fax: +353 91 494520.

414 2310; fax: +353 81 4142288.

gressive structural failure’’ [1], is a predominant causeof disability among the 30–50 year old demo-graphic population. The intervertebral disc (IVD) isinherently incapable of adequate self-repair once thedegeneration process has commenced. This may beattributed to the culmination of several factors such as;poor nutritional supply, low oxygen tension, acidic pH,low cell density and viability, and genetic predisposition[2]. Currently the most prevalent treatment moda-lities related to spinal problems associated with IVDsinvolve conservative methods (i.e. physical therapy anddrugs) and interbody fusion surgery [3]. However in2005, the Orthopaedic and Rehabilitation Devices Advi-sory Panel of the US Food and Drug Administration(FDA) proposed that the introduction of less invasivesurgical procedures earlier in the lumbar degenerativedisease cascade could defer the need for fusion or disc

ARTICLE IN PRESSD.O. Halloran et al. / Biomaterials 29 (2008) 438–447 439

replacement [4]. Studies have already indicated thatearly evolution of the degenerative process (i.e. in 18-yearold) is a strong indicator of long-term recurring backpain [5].

Tissue engineering strategies offer many potentialadvantages in the treatment of DDD including: the useof degradable biomaterials, less invasive surgical proce-dures, preservation of native tissue, non-preclusion offuture spinal surgery and multilevel disc treatments.Previous studies in this area have reported severalbiomaterials that can be employed as injectable cell-seededscaffolds (alginate [6–10], types I and II atelocollagen [11],hyaluronan [12,13] and chitosan [14–16]) or preformed cell-seeded scaffolds (type I collagen/GAG [17], type I collagen/hyaluronan [18], gelatin/chondroitin-6-sulfate/hyaluronan[19]).

Our tissue engineering approach is based on supplement-ing degenerated IVDs post-nucleotomy/partial nucle-otomy with a cell-seeded atelocollagen based scaffoldthat will ultimately lead to de novo synthesis of replace-ment tissue. Atelocollagen type II was chosen as theprincipal material since collagen type II represents oneof the main extracellular matrix (ECM) components ofthe native nucleus pulposus (NP) tissue, in addition topermitting the use of a minimally invasive, injectablesystem. However, some of the prominent limit-ations associated with using collagen as a biomaterialinclude its weak mechanical and degradative resistanceproperties [20,21]. In previous studies [22,23] we haveshown an increase in the mechanical and physicochemicalproperties of protein based substrates that were cross-linked with microbial transglutaminase (mTGase). Trans-glutaminases (TGases) are a class of natural enzymes thatare involved in catalyzing the acyl-transfer reactionbetween the e-amino group of lysine and the g-carbox-yamide group of glutamine in proteins [24–29]. mTGase,which is derived from the Streptomycete, Streptoverticil-

lium mobaraense [30], (28 kDa) has a high specific activityover a wide range of temperature and pH and is Ca2+

independent. mTGase has been extensively utilized inthe food industry, enhancing the functional propertiesof proteinaceous foods through covalent cross-linking[30–32].

It was hypothesized that re-seeding NP cells (post-expansion in monolayer culture) in an enzymaticallycross-linked scaffold whose composition mimicked theECM of native NP tissue, would provide a suitableenvironment for NP cell phenotype expression. Thespecific objective of this study was to determine the opti-mal scaffold composition for seeding NP cells, by assessingcell proliferation, cell phenotype and viability, sul-fated glycosaminoglycan (sGAG) content and syn-thesis rate, scaffold volume reduction and confinedcompression strength, using non-cross-linked and mTGase(50 mg/ml) cross-linked atelocollagen type II based scaf-folds containing varying concentrations of aggrecan andhyaluronan.

2. Materials and method

2.1. Materials and reagents

All materials and reagents used in this phase of the study were

purchased from Sigma Aldrich Ireland Ltd. (Dublin, Ireland) unless

otherwise stated.

2.2. Scaffold formation

Atelocollagen type II, which was kindly donated by Prof. David Brand

(Veterans’ Affairs Medical Center, and Departments of Medicine and

Molecular Sciences, University of Tennessee Health Science Center

(UTHSC), Memphis, Tennessee, USA), was extracted and purified from

cartilage in fetal bovine fetlock joints by a series of pepsin and salt

precipitation washes [33]. Hyaluronan (mW 300 kDa) isolated from bovine

vitreous humor and aggrecan (mW 2500 kDa) isolated from bovine

articular cartilage were also used in preparation of the scaffolds. mTGase

(ActivasWM, Ajinomoto Co. Inc., Japan) was kindly donated by Prof.

Martin Griffin and Dr. Russell Collighan (School of Life and Health

Sciences, Aston University, Aston Triangle, Birmingham, UK). mTGase

was purified by cation-exchange chromatography and the specific activity

of mTGase was determined to be 27mmol putrescine incorporated/mg/h

[34]. Table 1 identifies the scaffold groups and the concentrations of their

constituents investigated in this study. Lyophilized atelocollagen type II

was reconstituted in 0.05M acetic acid. Hyaluronan and aggrecan were

dissolved in medium comprising complete DMEM (Dulbecco’s Modified

Eagles Medium; Invitrogen), which was supplemented with 0.4M sodium

chloride. mTGase (50mg/ml) was added to the medium, which was then

mixed with the atelocollagen type II solution by gently pipetting up and

down. Sodium hydroxide (1M) was used to neutralize the solutions, after

which gel-like scaffolds were formed after approximately 10min incuba-

tion at 37 1C.

2.3. Rheology

Dynamic rheological measurements were performed on acellular

scaffolds using a stress-controlled AR500 Rheometer (TA Instruments,

AGB Scientific Ltd., Dublin, Ireland) with parallel plate geometry. The

upper aluminum plate (25mm in diameter) was covered with fine

sandpaper to avoid wall slip between the sample and the plate. With the

gap height set at 1mm, 500ml sample volumes were pipetted in solution

state onto the bottom plate, which was held at 15 1C. The temperature was

then ramped to 37 1C. After 5min, the exposed edges were covered with a

low viscosity mineral oil in order to prevent fluid evaporation during the

test and 20min later shear oscillations were carried out at a constant stress

of 0.5 Pa and a frequency of 1Hz (both parameters were found to be

within the linear viscoelastic range of the samples). The evolution of the

storage modulus (G0) was then observed during a test period of 4 h.

2.4. Cell isolation and seeding within scaffolds

Fresh 3–4 month old bovine calf tails were used as a source for NP

cells. During dissection only the innermost central region of the IVD tissue

was used for NP cell isolation. Tissues were minced and washed with

Tyrode’s balanced salt solution (TBSS) containing 10% Penicillin/

Streptomycin (Pen/Strep) (Invitrogen) in spinner flasks. DMEM contain-

ing 0.2% pronase (Roche Applied Science), 0.004% Deoxyribonuclease II

(DNase II, Sigma) was then added to each flask and further stirred in an

incubator (37 1C, 5% CO2) for 90min. Fresh DMEM containing 0.05%

collagenase II (BioConcept), 0.01% hyaluronidase and 0.004% DNase II

was added and left stirring overnight in the incubator. After filtering the

digested solution through 70mm cell strainers (Millipore) the cell

suspension was centrifuged at 1400 rpm at 4 1C. The viability of the cells

was confirmed using trypan blue exclusion dye. The cells were ex-

panded for 1 passage only in monolayer culture in complete DMEM

ARTICLE IN PRESS

Table 1

Scaffold Constituent Final concentration

(mg/ml)aPercentage set covariance

(mean7SEM)bFluoresence (� 1000)

(mean7SEM)c

C Atelocollagen type II 5 33.972.1 53.274.0

CH Atelocollagen type II 5 32.173.7 43.670.9

Hyaluronan 0.55

xCH Atelocollagen type II 5 36.273.1 48.876.7

Hyaluronan 0.55

mTGase 0.05

CAH Atelocollagen type II 5 29.671.6 40.671.0

Aggrecan 1

Hyaluronan 0.55

xCAH Atelocollagen type II 5 25.273.6 51.677.0

Aggrecan 1

Hyaluronan 0.55

mTGase 0.05

Data represent mean7SEM (n ¼ 3).aScaffold groups and composition.bPercentage set covariance of NP cell distribution throughout the scaffolds at day 2.cAlamarBlueTM analysis of the viability of NP cells seeded within scaffolds at day 7 of culture. An average fluorescence value (� 1000) of 29.570.4 was

found at day 0 of culture.

D.O. Halloran et al. / Biomaterials 29 (2008) 438–447440

(supplemented with 10% heat inactivated fetal bovine serum (Invitrogen)

and 1% Pen/Strep. Confluent cells were detached with trypsin–ethylene

diamine tetraacetic acid (EDTA) (Invitrogen). Cells (5� 105) were

suspended in 30ml medium and were pipetted up and down gently in

approximately 500ml neutralized scaffold solution, which was maintained

at room temperature. The cell-seeded scaffolds were then pipetted through

1ml tips into well-inserts of 24-well plates (United Drug) and incubated at

37 1C, 5% CO2. The medium was changed every 2 days throughout the

experiment, which was completed after 7 days for analysis.

2.5. Stereology

At day 2 of culture, cell-seeded scaffolds were fixed in 4% neutral

buffered formaldehyde solution at 4 1C. After dehydration through a

series of ethanol and xylene washes, the samples were embedded in

paraffin blocks. Five micrometer thick sections were cut with a microtome

and after de-paraffinization, the sections were stained with safranin 0 and

fast green FCF. Finally the sections were mounted on coverslips and

viewed under a light microscope (Olympus BX51, Olympus UK Ltd.,

London, UK) and analyzed using an image analysis software (Image Pro

Pluss, Media Cybernetics Inc., MD, USA). Stereological methods

employed by our research group [35] were used to determine the

distribution pattern (i.e. percentage set covariance) of cells seeded within

the scaffolds. Briefly, this involved measuring the area fraction of cells as a

percentage of the total area of the scaffold, at regular cross-section

intervals throughout the samples [36]. From an initial pilot study, it was

determined that the minimum number of sections required for a

representative analysis of each cell-seeded scaffold was 8.

2.6. DNA content

At day 2 and day 7 of culture, cell-seeded scaffolds were digested

in 0.5mg/ml of proteinase-K (Roche Applied Science) solution. The

HOECHST 33258 fluorescence dye assay (Polyscience Inc.) was used

with a plate reader (Perkin Elmers HTS 7000, Monza, Italy) at

360 and 465nm2 excitation and emission wavelengths, respectively. The

DNA content of the samples was quantified by interpolating values

from a linear standard curve generated from calf thymus DNA

(Invitrogen) [37].

2.7. Viability

After 7 days in culture, cell viability was assessed using the AlamarBlue

assayTM (BioSource). The cell-seeded scaffolds were removed from the

original wells and placed in fresh wells containing 1ml of AlamarBlueTM

solution. After 3 h incubation, the fluorescence was measured using a plate

reader (Perkin Elmers HTS 7000) at 520 and 590nm2 excitation and

emission wavelengths, respectively.

2.8. Percentage of original volume

The dimensions of the cell-seeded scaffolds were measured using vernier

callipers on day 2 and day 7 of culture. The volume of the constructs at

day 2 and day 7 were calculated as a percentage of the original volume,

which was measured on day 0 using acellular scaffolds.

2.9. Sulfated glycosaminoglycan (sGAG) content

At day 2 and day 7 of culture, cell-seeded scaffolds were digested in

0.5mg/ml of proteinase-K solution and mixed with dimethyl methylene

blue (DMMB) buffer solution before measurement with a plate reader

(Perkin Elmers HTS 7000) at 535 nm absorbance [38]. A standard curve

was generated using chondroitin-4-sulfate dissolved in phosphate buffered

saline (PBS) and complete DMEM, for analysis of the sGAG retained in

the scaffolds and eluted into the medium, respectively.

2.10. 35S sulfate incorporation

The rate of sGAG synthesis by cells seeded in the scaffolds was

measured using 35S sulfate radiolabeling (GE Healthcare Biosciences AB,

Amersham, Uppsala, Sweden). About 24 h before the day 2 and day 7

culture time-point, the cell-seeded scaffolds were cultured for 24 h in 35S

sulfate radiolabeled medium (2.5mCi/ml). The cell-seeded scaffolds were

then digested in 0.5mg/ml of proteinase-K solution. Five hundred

microliters of both the radiolabeled digests and medium were loaded

onto a PD-10 desalting column (GE Healthcare Biosciences AB) and

washed with an eluent solution (pH 7.5) comprising; 4M guanidine

hydrochloride, 0.1M anhydrous sodium sulfate, 0.05M Tris/ hydrochloride

and 0.5% triton X-100. Fractions of 500ml were collected in 5ml vials

ARTICLE IN PRESSD.O. Halloran et al. / Biomaterials 29 (2008) 438–447 441

(Simports, Quebec, Canada), combined with 3.5ml of scintillation fluid

(OptiPhase HiSafeTM 3, PerkinElmers Inc.) and the activity was counted

using a liquid scintillation counter (Wallac 1414 Liquid Scintillation

Counter, PerkinElmers Inc., Regensburg, Switzerland).

2.11. Confined compression

The compressive strength of acellular scaffolds (i.e. day 0) and day 2

and day 7 cell-seeded scaffolds was measured using a custom built

confined compression apparatus. This apparatus was composed of a

10mm diameter confining chamber (polytetrafluorethylene (PTFE), Dawn

Lough), with a rigid porous polyethylene base plate (pore size ranging

between 20 and 90 mm; Bel Arts Products). The upper plate (PTFE) was

attached to a Dynamic Mechanical Thermal AnalyserTM 2980 (TA

Instruments, AGB Scientific Ltd.). A tare load of 0.001N was used and

the scaffolds were loaded at a constant rate of 0.5N/min at room

temperature (approximately 23 1C).

2.12. Statistical analysis

Statistical analysis was performed using statistical software (MINI-

TABTM version 13.32, Minitab Inc., Coventry, UK). Data were compared

by a one-way analysis of variance check (ANOVA) and multiple pairwise

comparisons were carried out using the Bonferroni post-hoc t-test.

Statistical significance was set at po0.05.

0

250

500

750

1000

1250

1500

0 1 2 3 4

Time (hours)

Sto

rag

e M

od

ulu

s (

Pa

)

C CH xCH CAH xCAH

*

Fig. 1. Storage modulus (G0) (Pascals) of non-cross-linked and cross-

linked acellular scaffold groups at 37 1C. Data represent mean7SEM.

*Denotes statistical difference at 4 h (po0.05) (n ¼ 3).

C CH

CAH xCAH

Fig. 2. Safranin 0/fast green FCF stained sections of NP cell-se

3. Results

Table 1a identifies the scaffold groups that wereproduced in this study and the final concentration of theconstituents present in each of the scaffolds.After 4 h rheological testing at 37 1C, it was seen that

both the cross-linked scaffold groups (i.e. xCH and xCAH)had statistically higher storage moduli (G0), comparedto the non-cross-linked C, CH and CAH scaffold groups(Fig. 1).In order to avoid major cross-contamination with

intermediate zones, only the innermost central regions ofIVD tissue were used to procure NP cells. From fresh 3–4-month-old bovine calf tails, typically 6.8� 10672.2�106 cells/g wet weight of tissue were isolated. Cell viability(trypan blue) from this method of isolation was determinedto be 98.571.4%.Sections of the NP cell-seeded scaffolds were stained

with safranin-O/fast green FCF after 48 h in culture. Fromstereological analysis of the stained sections, the percentageset covariance (i.e. variation in the global spatial arrange-ment of cells within the scaffolds) for each cell-seededscaffold group was relatively low (Table 1b). There was nostatistical difference in the homogeneity of the celldistribution between the scaffold groups and the averagevalues ranged from 25.273.6% to 36.273.1%. Thesefindings were indicative of a homogeneous distributionpattern of cells throughout the scaffolds, which can also beobserved in the histological stained sections presented inFig. 2.The DNA content remained stable throughout the

culture period with an average value of 15.370.3 mgDNA/scaffold. The viability of NP cells seeded within thescaffolds was measured using AlamarBlueTM assay. Therewas no statistical difference observed between the scaffoldgroups over the 7 day culture period (Table 1c).The volume of the cell-seeded scaffolds at day 2

and day 7 of culture were compared to acellular scaffolds

xCH

eded scaffolds at day 2 of culture. Scale bar equals 100mm.

ARTICLE IN PRESS

C CH xCH CAH xCAH

Day 0

Day 7

Fig. 3. Acellular scaffolds (i.e. day 0) and NP cell-seeded scaffolds on culture day 7, on the top and bottom row, respectively. Progressing from left to right

are the scaffold groups: collagen (C), collagen/hyaluronan (CH), mTGase cross-linked collagen/hyaluronan (xCH), collagen/aggrecan/hyaluronan (CAH),

mTGase cross-linked collagen/aggrecan/hyaluronan (xCAH).

50

60

70

80

90

100

0 2 7

Culture Day

Pe

rce

nta

ge

Orig

ina

l V

olu

me

C CH xCH CAH xCAH*

*

Fig. 4. Percent reduction in volume of day 0 (acellular) scaffolds and day

2 and day 7 cell-seeded scaffolds. 100% value is given to scaffold groups at

day 0. Data represent mean7SEM. *Denotes statistical difference

between scaffold groups at day 7 compared to day 0) (po0.05) (n ¼ 3).

D.O. Halloran et al. / Biomaterials 29 (2008) 438–447442

(i.e. day 0), and the values were expressed as a percentageof the original day 0 volume. At day 2 no distinct changeswere observed for all the scaffold groups. By day 7however, all scaffold groups exhibited a statistically lowerpercentage of the volume measured at day 0 (Figs. 3 and 4).The C scaffold group had the lowest percentage of the day0 volume (59.873.7%), which was statistically lower thanthe CH, xCH and CAH scaffold groups at day 7. The xCHscaffold group showed the highest resistance to volumereduction by day 7, maintaining 85.470.5% of the originalscaffold volume measured at day 0.

In measuring the amount of sGAG retained by thescaffolds, the scaffold groups without aggrecan (i.e. C, CHand xCH) are reported separately to the scaffold groupsalready containing aggrecan (i.e. CAH and xCAH) inTables 2a and b, respectively. The amount of sGAGretained by the scaffolds was expressed as a percentage ofthe total sGAG in the system (i.e. sGAG that was retainedby the scaffold plus the amount eluted into the medium) atday 2 and day 7 of culture. For all scaffold groups, therewas a significant drop in the percentage sGAG retained by

the scaffolds on day 7 in comparison to day 2. On bothday 2 and day 7, the xCH scaffold group retained a higherpercentage of sGAG compared to the C and CH scaffoldgroups, which was found to be statistically significant atday 2. Also, a higher percentage of sGAG was retained bythe xCAH scaffold group in comparison to the CAHscaffold group at day 2 and day 7.Although NP cells in both the xCH and xCAH scaffold

groups synthesized more sGAG over a 24 h culture periodcompared to the remaining scaffold groups (i.e. C, CH andCAH) at day 2 and day 7 (Fig. 5), this increase was notfound to be statistically significant.From the compressive stress results shown in Fig. 6, the

effect of the mTGase cross-linker is seen from the increasein strength of the acellular (i.e. day 0) xCH and xCAHscaffold groups compared to the C, CH and CAH scaffoldgroups. The compressive stresses increased with time inculture; with the CH, xCH and xCAH scaffold groups atday 7 showing statistically higher values compared to theC and CH scaffold groups at day 0.

4. Discussion

It has been demonstrated that the inclusion of cells inscaffold-based IVD regeneration strategies is necessary foreffective tissue regeneration, as the injection of acellularscaffolds alone gave poor results in a rabbit disc degenera-tion model (aspiration of NP tissue) [20]. Hence we haveinvestigated the behavior of NP cells within 5 cell-seededbiologically relevant scaffolds. Much work has been carriedout with various biomaterials in IVD tissue engineering,but this is the first study to develop an mTGase cross-linked composite scaffold comprising hyaluronan andaggrecan together with atelocollagen type II. The scaffoldsformed in this study were all atelocollagen type II-basedsince collagen type II is one of the main ECM componentsfound in native NP tissue and it also facilitates the use ofan in situ curable, injectable-type scaffold. The removalof the telopeptides during pepsin digestion removes

ARTICLE IN PRESS

Table 2a

Percentage sGAG retained by the scaffolds (without aggrecan) at day 2 and day 7 of culture

Scaffold (%) Day 2 (%) Day 7

C 59.174.3 a0

b

375 21.671.9

b0

375CH 58.574.3 a0 20.272.0

xCH 86.172.8 a 27.175.9

Data represents mean7SEM. Similar letter denotes statistical difference to letter0 (a-a0, b-b0) (po0.05) (n ¼ 6).

Table 2b

Percentage sGAG retained by the scaffolds already containing aggrecan at day 2 and day 7 of culture

Scaffold (containing aggrecan) (%) Day 2 (%) Day 7

CAH 77.074.6

a

#11.271.7

a0

#xCAH 82.571.5 15.771.9

Data represents mean7SEM. Similar letter denotes statistical difference to letter0 (a-a0) (po0.05) (n ¼ 6).

0

200

400

600

800

1000

C CH xCH CAH xCAH

CP

M/µ

g D

NA

/24

hrs

Day 2 Day 7

Fig. 5. Rate of synthesis of total sGAG in the system (i.e. sGAG that was

retained by the scaffold plus the amount eluted into the medium) per mgDNA, over a 24 h culture period at day 2 and day 7 of culture. Data

represent mean7SEM (n ¼ 3).

0

5

10

15

20

25

C CH xCH CAH xCAH

Co

mp

ressiv

e S

tre

ss (

kP

a)

Day 0 Day 2 Day 7

a

a', b' a', b' a', b'

b

Fig. 6. Compressive stress (kPa) of the day 0 (acellular scaffolds) and day

2 and day 7 cell-seeded scaffolds measured at 99% strain. Data represent

mean7SEM. Similar letter denotes statistical difference to letter0 (a-a0,

b-b0, c-c0) (po0.05) (n ¼ 3).

D.O. Halloran et al. / Biomaterials 29 (2008) 438–447 443

a significant portion of the antigenic sites on the collagenmolecule [39]. However, it remains to be proven if thisprocess results in bovine atelocollagen type II being

completely non-immunogenic since the degree of antigeni-city is said to be related to the donor/recipient pair [40] andno study to date has been carried out using the bovine/human pair. However, due to the NP implantation siteremaining isolated and avascular, it has been described as‘‘immuno-privileged’’ [41].The remaining constituents of the matrix (i.e. aggrecan

and hyaluronan) were chosen as they represent the majorECM macromolecules present in native IVD and are ofimportance for the functionality of NP tissue. Although innative NP tissue the ratio of collagen to aggrecan isapproximately 2:5 [42], from preliminary scaffold-forma-tion studies, the optimal ratio of atelocollagen type II toaggrecan was found to be 3:1. The ratio of collagen tohyaluronan (9:1) used in the scaffolds mimicked thecollagen to hyaluronan ratio, which is found in nativeNP tissue [18].Preliminary work carried out using 3mg/ml atelocolla-

gen scaffolds showed significant problems associated withrapid volume reduction and degradation. Hence, for thisstudy, the atelocollagen concentration was increased to5mg/ml, in addition to incorporation of the mTGase cross-linking enzyme. In choosing cross-linking agents in orderto enhance biomaterial properties it is imperative that thefunctionality of the biomaterial is not changed to such anextent that its clinical application becomes reduced. SincemTGase displays a wide pH activity at an optimaltemperature of 37 1C, the use of mTGase as a cross-linkerwith composite atelocollagen type II based scaffolds, stillmaintains the favorable in situ gelling (i.e. injectable)property.Rheological analysis of acellular scaffolds under con-

stant shear oscillation loading showed that the incorpora-tion of mTGase in the xCH and xCAH scaffold groupsresulted in a statistical increase in the storage modulus (G0)compared to the non-cross-linked C, CH and CAHscaffold groups. G0 of the xCH and xCAH scaffold groups

ARTICLE IN PRESSD.O. Halloran et al. / Biomaterials 29 (2008) 438–447444

was on average 2.2 and 1.7 times higher than the CH andCAH scaffold groups, respectively. This increase in G0 maybe linked to the formation of stronger, covalently cross-linked collagen fibrils by the catalytic cross-linking reactionof mTGase [23]. Importantly, all the scaffold groupsdemonstrated their potential for use as an injectabletreatment, since the scaffolds recovered their gelatinous-structural integrity after being passed through a tip of1mm diameter.

There was no significant change in the DNA contentbetween the scaffolds from day 2 to day 7 in culture,indicating the presence of a low-proliferating but stable cellpopulation. This low proliferation tendency has beenobserved in other studies, where IVD cells and chondro-cytes were re-introduced from monolayer conditions into athree-dimensional environment [6,43,44]. It has also beenshown that the molecular weight and concentration ofexogenous hyaluronan has a strong influence on theproliferation response of porcine NP cells [43] andchondrocytes [45,46] seeded in three-dimensional con-structs after 7 days in culture. However, in our study nosuch relationship between the level of cell proliferation andthe presence of hyaluronan in the scaffold was observed.This may be due to the low molecular weight (i.e.3�105Da) hyaluronan that was used in this study, which maynot be as influential as the higher molecular weighthyaluronan used in other studies (i.e. 8� 105–3� 106Da).

From preliminary investigations it was established thatmTGase concentrations above 50 mg/ml had a negativeeffect on NP cell viability. Thus the scaffolds in this study(i.e. xCH and xCAH) were cross-linked with 50 mg/mlmTGase. Results from the AlamarBlue

TM

assay after 7 daysin culture indicated no difference in viability for the cross-linked scaffold groups in comparison to the non-cross-linked scaffold groups.

Measuring the percentage of original volume thescaffolds maintained over the culture period is importantsince in vivo, any reduction in volume could potentially leadto a loss of contact between the cell-seeded scaffold and thehost-tissue, in this case the inner annulus fibrosus (AF). Noreduction in volume was noted for the acellular scaffoldsthroughout the entire culture period. The cell-seededscaffold volumes remained relatively stable at day 2, butmeasurements taken on day 7 showed the C scaffold grouphad the lowest percentage of original volume, while thexCH scaffold group showed the highest resistance tovolume reduction at day 7. The observed volume reductionis not referred to as ‘‘scaffold contraction’’, due to the factit is likely there were two forces at work in this instance:(1) some catabolic proteins may have been synthesised bythe NP cells [2], which would have led to increaseddegradation of the scaffolds; and (2) the mechanicalstiffness of the scaffolds was not sufficient enough to fullyresist cell-mediated contraction. Therefore, it is feasiblethat the xCH scaffold group was more resistant todegradation and cell-mediated contraction than the otherscaffold groups.

In another study [47], where chondrocyte-seeded col-lagen–GAG scaffolds were cross-linked with dehydrother-mal treatment, ultraviolet irradiation, glutaraldehyde and1-ethyl-3-(3-dimethylamino-propyl) carbodiimide, after 4weeks, some association was found between the compres-sive modulus of the scaffolds and the level of cell mediatedcontraction. However, no correlation between the percen-tage volume reduction and the confined compressivestrength (R2

¼ 0.37) or storage modulus (G0) (R2¼ 0.29)

was found in this study. It is possible that besides theinherent biomechanical property of the scaffold itself, theextent of adherence between the cells and the ECM, whichis regulated by the ECM macromolecules presented to theintegrins of the cells, plays a significant role in how the cellsinteract and contract the surrounding ECM [48]. It wasalso shown [49] that when chondrocytes were seeded in acollagen type I scaffold, significantly more scaffoldcontraction was observed in scaffolds that were detached(i.e. free floating) from the surrounding walls of the wells incomparison to scaffolds that remained attached. In ourstudy, most of the cell-seeded scaffolds had detached fromthe wells by day 4 in culture, which may explain thesignificant reduction in the volume of the cell-seededscaffolds observed by day 7. The xCH scaffold groupretained statistically higher amounts of sGAG compared toits respective non-cross-linked CH scaffold group. It washypothesized that the cross-linked collagen fibrillar net-work led to the physical entrapping of more sGAG withinthe scaffold. Although not statistically higher, there wasmore sGAG retained by the xCAH scaffold group incomparison to the CAH scaffold group at day 2 of culture.By day 7 however, the percentage retention values werestatistically lower compared to day 2, for all scaffoldgroups. In another phase of our study [50], IVD tissue wasisolated from 3–4 month bovine calf tails and sectionedinto AF, NP and intact IVD (with end-plates removed)tissue. These explant-tissues were cultured at 37 1C incomplete DMEM and the percentage sGAG eluted fromthe tissues was quantified over a similar culture period.Statistically lower amounts of sGAG were retained by theNP tissue compared to the AF tissue and the intact IVDtissue. Additionally, if the endplates were preserved it islikely that there would have been more sGAG retained bythe intact IVD tissue. This finding highlights the impor-tance of maintaining cell-seeded scaffolds in confinedculture conditions, which would inevitably lead to highersGAG retention values.Since the CAH and xCAH scaffold groups already

contained sGAG, it was necessary to measure the rate ofsGAG synthesis by cells seeded in the scaffolds using 35Ssulfate radiolabeling. No statistical difference in sGAGsynthesis levels were found between the values at day 2compared to day 7 for all scaffold groups. However, linearregression analysis between day 2 sGAG synthesis valuesand day 0 and day 2 compressive strength values gave astrong indication that levels of sGAG synthesis weredependent on the compressive strength of the scaffolds

ARTICLE IN PRESS

Table 3

Assessment of NP cell seeded scaffold groups after 7 days in culture

C CH xCH CAH xCAH

Storage modulus + + ++a + ++a

Cell distribution + + + + +

DNA Content + + + + +

Viability + + + + +

Percentage of original volume �� + ++a + �

sGAG retention by matrix � � + � �

sGAG synthesis � � + + +

Compressive stress � + + � +

aDenotes statistically higher values compared to all other scaffold groups.

D.O. Halloran et al. / Biomaterials 29 (2008) 438–447 445

(R2¼ 0.89 and 0.80, respectively). These data are consis-

tent with findings from another study [47] that showedhigher ECM synthesis levels associated with collagen–GAG scaffolds having higher compressive moduli. It ispossible that the stronger scaffolds provided more resis-tance to the NP cells which placed them in a stressed ratherthan relaxed environment [49]. Integrins, which are presenton the surface of the mechanosensitive NP cells [51], maytranslate these biomechanical differences from the sur-rounding ECM through its association with the actincytoskeleton of the cell [48,52]. Signaling pathways maythen be activated, leading to upregulation of ECMsynthesis levels. On day 7 however, no correlation wasfound between sGAG synthesis levels and compressivestrength values. This suggests other factors may beinfluencing the NP cells in synthesizing sGAG, such asthe endogenous production of catabolic or anabolicfactors. Alternatively, once the cells start to produce theirown matrix, the external matrix becomes less important.

Due to differences in the volume of the cell-seededscaffolds, compressive stress measurements were reportedat 99% strain, which ensured that the force from the upperplate of the confined apparatus was applied over an equalarea of the samples. Acellular scaffolds (i.e. day 0), showedthat there was an increase in compressive stress in thescaffold groups due to the presence of the mTGase cross-linker. As observed from rheological findings, the increasein strength may be attributed to the formation ofcovalently cross-linked collagen fibrils within the scaffold[23]. The strength of the cell-seeded scaffolds increasedwith time in culture, but the enhanced effect of mTGasewas observed between the CAH and xCAH cell seededscaffold groups only. The observed increase in compressivestress with time in culture may be attributed to forcesexerted by the cells on the surrounding ECM [47–49]. Theforce that was generated from the cells appears to be moreinfluential in the case of the CH and xCH scaffold groups,where no difference between the compressive stresses ofthese scaffolds was observed at day 2 and day 7 of culture.This may be due to the presence of different cell–ECMinteractions present within the scaffolds that may affectother modulators of cell signaling events [53].

The overall goal of this study was not to produce NPtissue in vitro, but to investigate non-cross-linked andmTGase cross-linked composite scaffolds, based on thestability of the scaffolds and the behavior of seeded NPcells. Based on properties relevant to NP tissue engineering,a summary of the results obtained from this study ispresented in Table 3.

5. Conclusion

In conclusion, the present study demonstrated thatin situ gelling non-cross-linked and mTGase cross-linkedatelocollagen type II-based composite scaffolds werecapable of supporting NP cell viability and normalphenotype expression. The mTGase cross-linked atelocol-lagen/hyaluronan composite scaffold group (i.e. xCH)displayed more favorable properties compared to the otherscaffold groups. The most outstanding property main-tained by this scaffold group was its ability to significantlyresist scaffold volume reduction after 7 days in culture. ThexCH scaffold group also retained the highest amount ofsGAG and along with the CAH and xCAH scaffoldgroups, the xCH scaffold group displayed the strongestconfined compressive strength.The xCH scaffold could potentially be used in future

studies as a scaffold for seeding mesenchymal stem cells(MSCs). It is conceivable that the temporary physical andbiomechanical environment provided by the ECM of thescaffold could present important cues that would stimulateMSCs to differentiate into and maintain the NP cellphenotype, thus leading to the synthesis of replacement NPtissue.

Acknowledgments

The authors would like to extend their thanks to Mr.Robert Peter (AO Foundation, Davos, Switzerland) andMr. John McCabe and Mr. John Street (orthopaedicsurgeons, Merlin Park Hospital, Galway, Ireland). Theauthors would also like to acknowledge the Irish Councilfor Science, Engineering and Technology; Funded by theNational Development Plan and Science Foundation

ARTICLE IN PRESSD.O. Halloran et al. / Biomaterials 29 (2008) 438–447446

Ireland; Research Frontiers Program 2007, for researchfunding.

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