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Photobiomodulation of a flowable matrix in a human skin ex vivo model

demonstrates energy-based enhancement of engraftment integration and

remodeling

Lia Mara Grosso Neves1, Nivaldo Antonio Parizotto2, Marcia Regina Cominetti1*,

Ardeshir Bayat3*

1Laboratory of Biology of Aging (LABEN), Department of Gerontology, Federal

University of São Carlos, CEP 13565-905, São Carlos - SP, Brazil

2Physical Therapy Department - Federal University of São Carlos (São Carlos) and

Biotechnology Post-Graduation Program - University of Araraquara (Araraquara), Post-

Graduation in Biomedical Engineering - University of Brasil (São Paulo) – SP, Brazil

3School of Biological Sciences and Health/ Division of Musculoskeletal and

Dermatological Sciences – University of Manchester, Manchester, UK

*Co-senior and corresponding authors: Márcia Regina Cominetti

(mcominetti@ufscar.br) and Ardeshir Bayat (ardeshir.bayat@manchester.ac.uk)

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ABSTRACT

The use of dermal substitutes to treat skin defects such as ulcers has shown promising

results, suggesting a potential role for skin substitutes for treating acute and chronic

wounds. One of the main drawbacks with the use of dermal substitutes is the length of

time from engraftment to graft take, plus the risk of contamination and failure due to

this prolonged integration. Therefore, the use of adjuvant energy-based therapeutic

modalities to augment and accelerate the rate of biointegration by dermal substitute

engraftments is a desirable outcome. The photobiomodulation (PBM) therapy

modulates the repair process, by stimulating cellular proliferation and angiogenesis.

Here, we evaluated the effect of PBM on a collagen-glycosaminoglycan flowable

wound matrix (FWM) in an ex vivo human skin wound model. PBM resulted in

accelerated rate of re-epithelialization and organization of matrix as seen by structural

arrangement of collagen fibers, and a subsequent increased expression of α-SMA and

VEGF-A leading to an overall improved healing process. The use of PBM promoted a

beneficial effect on the rate of integration and healing of FWM. We therefore propose

that the adjuvant use of PBM may have utility in enhancing engraftment and tissue

repair and be of value in clinical practice.

Keywords: Photobiomodulation therapy; low-level laser therapy; wound repair; tissue

remodeling; skin substitute; flowable matrix

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INTRODUCTION

Tissue repair following injury is a well-orchestrated yet complex biological

process involving many local and systemic factors. For this to occur, dynamic

interactions between different cell types and intra and extracellular pathways are

activated to restore integrity and functionality of the tissue [1, 2].

Complete understanding of the role of cellular and molecular mechanisms that

orchestrate skin repair remain poorly understood and thus, current targeted therapies are

limited. The imperfect wound repair process that occurs following chronic wounds

formation is a clinical and therapeutic challenge [3, 4]. Therefore, identifying the

relevant therapeutic modalities to improve cutaneous repair, are a clinical necessity.

Skin tissue engineering through the use of dermal substitutes is an attractive

approach to rebuild lost and damaged tissue [4]. A variety of dermal substitutes are

utilized in the treatment of acute wounds and chronic ulcers. When placed in a wound

bed, these biomaterials often in the form of a gel sheet can stimulate or accelerate

healing by promoting a supportive scaffold for cell migration, revascularization,

epithelialization and tissue remodeling [4]. New generations of skin substitute

biomaterials in form of flowable gels have been developed and considered clinically.

They represent alternatives in place of gel sheet dermal substitutes in view of their form

and pliability to fill defects of any shape and size. Injectable matrices in the form of gels

or fluid pastes have demonstrated great potential as adjuncts in the process of tissue

repair and regeneration [5, 6]. There are a variety of commercially available flowable

matrices including collagen-glycosaminoglycan flowable wound matrix (Integra Life

Sciences, New Jersey, USA) (IFWM), an artificial dermal substitute, which is well

established and approved for use in burns and chronic wounds. IFWM is composed of

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granulated cross-linked bovine tendon collagen and glycosaminoglycan. It provides a

resorbable scaffold for cellular invasion and capillary growth [7].

Physical agents are also employed as adjuncts to facilitate tissue repair including

photobiomodulation (PBM), which is also called low-level laser therapy (LLLT) [8-10],

ultrasound application [11] and microcurrent application [12, 13]. Nowadays, there is an

increasing level of clinical interest in the use of non-invasive therapies that will

optimize the repair process, by reduction of engraftment uptake time period as well as

objectively enhancing the quality of healing. Therefore, the aim of this study was to

investigate the role of PBM in enhancing integration and wound healing following

application of a flowable wound matrix in a human derived ex vivo wound healing

model.

METHODS

Ex vivo wound healing model assay design and insertion of IFWM

Normal unscarred skin samples were obtained from five Caucasian subjects

undergoing elective cosmetic abdominoplasty surgery with appropriate ethical

committee and human tissue authority approval (16/NW/0736 IRAS 214160). Biopsy

samples of tissue were removed from harvested tissue using a 6 mm punch biopsy

device. A further wound was created in the centre of each of these skin samples in the

shape of a donut using a smaller 3 mm punch biopsy kit.

Subsequently, the biopsy samples were washed with sterile PBS containing 1%

(v/v) penicillin/streptomycin and inserted into the 24-well plate inserts (Corning, USA).

The Integra® flowable wound matrix (IFWM) was prepared and inserted into the wound

area. The wells were supplemented with 500 µL of complete DMEM (10% FBS) so that

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the epidermis of the ex vivo tissue was air exposed. The skin samples were cultured for

7 and 14 days with medium changed every 2 days (Supplementary Fig. 1).

Division of experimental groups and photobiomodulation (PBM) of wound models

Donut wound models were divided into six experimental groups (Supplementary

Fig. 2). For the PBM treatment of donut wound models; a continuous-wavelength of

InGaAlP laser (660 nm), was chosen due to its known depth of penetration to reach

epidermis and deeper dermis, with a power output of 30 mW (Therapy XT – DMC,

Brazil) for light irradiation. Initially, three different energy doses per point were tested:

0.9 J (30 seconds); 2.7 J (90 seconds) and 4.5 J (150 seconds). The irradiations were

performed at a single point in the center of the wounds three times a week for two

weeks (Supplementary Fig. 3).

Histology

Ex vivo wound models (n = 2) were removed from culture at 7 and 14 days for

detailed analysis through histology of wound cross-sections. Tissue was fixed in 4%

(v/v) formalin and processed to inclusion on paraffin. For wound closure analysis,

sequential wound cross-sections (5 μm) were taken containing both wound area tissue

and adjacent non-injured tissue. Sections were de-paraffinised, stained with

hematoxylin-eosin for the structure analysis and Sirius red (fast green) for the analysis

of collagen fibers density. The sections stained were analyzed using Case Viewer and

ImageJ software, respectively.

Viability Assay

To assess the viability of ex vivo tissue during the culture period, the

colorimetric LDH (lactate dehydrogenase) assay was used (Thermo Scientific, UK),

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according to the manufacturer’s instructions. The tissue culture medium of days 3, 5, 7,

10, 12, 14 were removed from ex vivo cultures. Twenty µL of the removed medium was

aliquoted into new 384-well plates and 20 µL reaction mix (LDH) was added to aliquots

of medium. The reaction was incubated under gentle shaking, in the dark at 20ºC for 30

min. After incubation, 20 µL of stop solution was added to stop the reaction. The

absorbance was obtained by a spectrophotometer at OD 490 nm (corrected for OD 680

nm). The results are represented by means of triplicate of the three independent assay

reactions, for each ex vivo sample.

Evaluation of the optimal energy dose

The skin samples were removed from culture for analysis of application of

different levels of energy doses of PBM in different time points (data not presented),

however energy dose at 4.5J in 14 days, produced optimal results compared with other

doses and time points (Supplementary Fig. 4).

Histology and Immunohistochemistry (optimal dose 4.5J - 14 days)

Ex vivo wound models (n = 3) were removed from culture at 14 days for analysis

of wound healing through histology of wound cross-sections. Tissue was fixed in 4%

(v/v) formalin and processed to inclusion on paraffin. For wound closure analysis,

sequential wound cross-sections (5 μm) were taken containing both wound area tissue

and adjacent non-injured tissue. Sections were deparaffinised, stained with

hematoxylin-eosin for the structure analysis. For immunohistochemical staining,

paraffin embedded sections were cut and mount sections on slides coated with a suitable

tissue adhesive. After this, the sections were deparaffinized in xylene and rehydrated

through graded alcohols. The antigen retrieval was performed with citrate buffer (0.01

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M, pH 6.0) in microwave for 20 minutes at low power. After that a Novolink ™

Polymer Detection System (Sigma-Aldrich) was used for IHC technique. The

manufacturer's instructions were followed, and the markers were performed for primary

antibodies anti-Collagen I (1:1000) – Ab34710 (Abcam), anti-Collagen III (1:1000) –

Ab6310 (Abcam), anti-VEGFA (1:100) – Ab1316 (Abcam) and anti-Actin Alpha

Smooth Muscle (1:40) – A5691 (Sigma-Aldrich). Then the sections were dehydrated

and diaphanized. The slides were mounted and scanned in automated equipment.

Qualitative and quantitative evaluations were done from sections through the central

region of wounds in order to obtain the maximum wound and border area for

evaluation. The sections stained were analyzed using Case Viewer software and

evaluation morphometric for positive immunostaining intensity was performed with

NIH ImageJ software; all the analyses were run as triplicates.

Statistical analysis

Experimental data were presented as the mean ± SD of three independent

experiments. Statistical differences were determined using One-way ANOVA with

Bonferroni’s post-hoc test, with significant difference of the p-value < 0.05.

RESULTS

Histology (ex vivo organ culture – 7 and 14 days)

In this study, we investigated the effects of photobiomodulation (PMB) on a

collagen-glycosaminoglycan flowable wound matrix (IFWM) using an ex vivo human

skin wound healing model assay. Structural analysis of skin samples removed from

tissue culture after 7 and 14 days were performed in representative histological sections

stained with hematoxylin and eosin (Fig. 1A and B). The samples from all groups were

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composed of stratified, keratinized and squamous epithelial tissue. Epidermis and

dermis without histological changes in the unwounded skin group maintained in culture

for 7 and 14 days were observed (Fig. 1A and B). In the wounded skin group and in the

treated groups the formation of granulation tissue in the wound bed is evident at 7 days

post-wound induction. It was also possible to observe epithelium in the treated groups

when compared to the untreated wound group. There were no significant histological

changes between the untreated group and the groups treated for 7 days in culture (Fig.

1A), indicating that the treatments did not produce significant effects on the early stages

of tissue repair in ex vivo human skin culture.

Figure 1A - Histological photomicrographs (7 days). Samples of ex vivo cutaneous wounds (3mm) treated with IFWM and PBM at 7 days of culture. Hematoxylin and eosin staining. X5 magnifications. ED: epidermis; PD: papillary dermis; RD: reticular dermis. Scale bar = 200µm.

On day 14 post-wound induction, there was an increased level of cellular

density, evaluated by a qualitative analysis, with the formation of new small blood

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vessels, and evidence of re-epithelialization in the treated groups compared to the

untreated group. Furthermore, there was an intense re-epithelization in the IFWM +

PBM (4.5J) group, showing a better efficacy at this laser dose (4.5J) when compared

0.9J and 2.7J (Fig. 1B).

Figure 1B - Histological photomicrographs (14 days). Samples of ex vivo cutaneous wounds (3mm) treated with IFWM and PBM at 14 days of culture. Hematoxylin and eosin staining. X5 magnifications. ED: epidermis; PD: papillary dermis; RD: reticular dermis. Scale bar = 200µm.

In the structural analysis of the organization and density of collagen fibers of

skin samples with induced wounds maintained in culture for 14 days, a more organized

network of collagen fibers in the reticular dermis was observed. In addition, a parallel

structure to the epidermis and longer fibers in the samples of the groups treated

following application of IFWM and PBM (0.9, 2.7, 4.5J) was observed, when compared

with the wounded skin and IFWM groups. In the last two groups, it was possible to

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observe a thin, short and disorganized collagen fibers structure and bundle arrangement

(Fig. 1C).

Figure 1C - Organization and density of collagen fibers. Qualitative analysis of birefringent collagen fibers in samples collected of tissue culture on the 14 th day, after the induction of wound and evaluated under polarized light. X20 Magnifications. Picrossirius red staining. ED: epidermis; PD: papillary dermis; RD: reticular dermis. Scale bar = 50µm.

Viability of ex vivo tissue

The viability of the tested skin samples over the culture period was confirmed

through lactate dehydrogenase (LDH) assays. There was no significant increase in cell

death and the survival of the tested samples was maintained during the culture period.

The level of cell death of the culture after 14 days indicated that the skin samples were

still viable in all groups (Fig. 2).

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Figure 2 - LDH analysis of model viability during ex vivo culture. The assays were performed from the culture media extracted on days 3, 5, 7, 10, 12 and 14 of the unwounded skin, wounded skin, IFWM and IFWM + PBM (4.5J) groups.

Histology (optimal dose 4.5J - 14 days)

After determining the best efficacy laser dose (4.5J), three new independent

cultures were performed: unwounded skin, wounded skin, IFWM and IFWM + PBM

(4.5J) during a period of 14 days. In the histological analysis post hematoxylin and

eosin staining, the same pattern as previously described in the analyses carried out on

the sample size model of 6mm and wound size of 3mm was observed (Fig. 3).

Immunohistochemical analysis of Collagen I, III, α-SMA and VEGF-A

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An immunohistochemical analysis was performed to quantify the expression

levels of collagen I, III, α-SMA and VEGF-A (Fig. 4A). The analysis of collagen I

demonstrated a smaller amount in the wounded skin in IFWM and IFWM + PBM (4.5J)

groups compared to the unwounded skin group, indicating that the treatments had no

effect on increasing levels of collagen I synthesis.

Figure 3 - Histological photomicrographs (optimal dose 4.5J – 14 days). Samples of ex vivo cutaneous wounds (4mm) treated with IFWM and PBM (4.5J) at 14 days of culture. Hematoxylin and eosin staining. X3 magnifications. ED: epidermis; PD: papillary dermis; RD: reticular dermis. Scale bar = 500µm.

In the analysis of collagen III, only the IFWM group presented a smaller amount

of protein when compared with the other groups, and there was no significant

differences between the unwounded skin, wounded skin and IFWM + PBM (4.5J)

groups (Fig. 4B). On the other hand, there was a significant increase in α-SMA levels in

IFWM (11.39%) and IFWM + PBM (14.05%) treated wounds compared to the control

wounded skin group (8.44%). The quantification of VEGF-A growth factor expression

by immunohistochemical analysis revealed a significant difference between the IFWM

+ PBM and the wounded skin group (p<0.01), which represents an increase of 5.09% in

VEGF-A expression after the combined treatment with IFWM and PBM. The IFWM

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group (16.95%) presented lower levels of this angiogenic growth factor when compared

to the wounded skin group (19.15%) (Fig. 4C).

Figure 4A - Immunohistochemical analysis. Collagen I, collagen III, VEGF-A and α-SMA expression in skin samples of unwounded skin, wounded skin, IFWM and IFWM+PBM (4.5J) groups, removed from tissue culture at 14 days after the wounds induction. X3 magnifications. ED: epidermis; PD: papillary dermis; RD: reticular dermis. Scale bar = 500µm.

Figure 4B - Immunohistochemical analysis of Collagen I and Collagen III. Data are expressed as mean±SD. *P<0.05, tested by one-way ANOVA with Bonferroni’s post-hoc test.

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Figure 4C - Immunohistochemical analysis of α-SMA and VEGF-A. Data are expressed as mean±SD. **P<0.01 and ***P<0.001, tested by one-way ANOVA with Bonferroni’s post-hoc test.

DISCUSSION

The application of phototobiomodulation (PBM) with different energy doses

with a dermal substitute (Integra® flowable wound matrix, IFWM) was investigated in

this present study using an ex vivo human skin wound healing model. In order to test the

viability of the ex vivo wounded skin samples that were maintained in culture for 14

days, we performed the LDH assay, in which it was possible to confirm a stable

maintenance of the viability of all assayed samples. The viability test of ex vivo

wounded skin over the same culture time (14 days) was originally performed by

Hodgkinson and Bayat [7] (senior author) using the same approach. Our findings

corroborate with the authors’ previous results, showing that the samples remained viable

until the end of the culture period.

In our study, the purpose of evaluating different energy doses of PBM was to

find an optimal dose that would produce biomodulatory effects on tissue repair

processes with a focus on the rate and level of enhanced engraftment integration in a

simple validated ex vivo human skin wound healing model. Our findings indicate a

possible beneficial action of the different tested doses of PBM associated with IFWM in

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the preservation of epithelial thickness, maintaining morphological characteristics found

in an in vivo human skin. In addition, our findings revealed an increase in epidermal

thickness in all ex vivo human skin culture samples in relation to normal human skin in

vivo. In agreement with our experimental work, Xu et al. [14] had previously described

a significant increase in epidermal thickness of ex vivo skin samples removed from the

culture at 4 and 10 days when compared to fresh skin samples processed within 4 hours

after patient collection, the authors attributed a larger number of keratinocytes layers.

It has been previously shown that the PBM generated by the red laser (628nm -

0.88J/cm²) promotes moderate stimulation of human fibroblast culture proliferation.

Two signaling pathways have been identified, p38 MAPK and PDGF, as playing an

important role in mediating the effects of red laser irradiation on human fibroblast

proliferation [15]. In addition to its effects on cell proliferation, red laser irradiation can

also regulate gene expression in relevant cells in relation to microcirculation, anti-

apoptosis, anti-oxidation, and DNA repair [15]. A possible effect on cellular

proliferation may be due to re-epithelialization found most evidently in the IFWM +

PBM group (4.5J), that may be attributed to, by the amount of energy that was

dispensed to the tissue undergoing the repair process generating an effective

biomodulatory effect. These findings point to the importance of correct choice of the

parameters that involve PBM in order to obtain an adequate biological response in terms

of skin wound healing. Interestingly, we found an increased level of density and

organization of collagen fibers in the samples treated with IFWM and PBM (0.9, 2.7,

4.5J) compared with PBM alone, since the untreated groups showed a lower density and

organization of the collagen fibers.

We also evaluated the expression of collagens I or III and did not find

differences among the experimental groups. In contrast to our findings, qRT-PCR

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analysis of gene expression in models treated with IFWM with and without cell

incorporation after 2 weeks of ex vivo culture showed significantly increased levels of

collagen I and III expression (p<0.05 and p<0.0001) in the groups treated with cell

incorporated scaffolds [7]. Based on these data, we demonstrated that the addition of the

same dermal substitute with PBM was not able to increase levels of collagen I and III

expression, even if known that PBM produced an increase in collagen gene expression

in different experimental models [16]. We therefore suggest that it may be necessary to

use higher doses of PBM in order to increase the number of days subjected to treatment

to produce an effective response in increasing the expression of collagen I and III.

Fibroblasts are the most common cell type found in the dermis as the main

source of collagen synthesis. In tissue repair, more specifically in granulation tissue,

these cells are activated and acquire α-SMA, changing their phenotype to

myofibroblasts involving synthesis and deposition of extracellular matrix components

that replace the provisional matrix [17]. Of note, fibroblasts irradiated with 632.8 nm

light show an increase in their proliferation and viability, demonstrating the stimulatory

effect of PBM and the potential use of this therapy in the wound repair process [18].

Remarkably, our findings demonstrated an increase in α-SMA (a reliable marker of the

myofibroblast-like phenotype) expression in PMB + IFWM group, when compared to

wounded skin alone. It is therefore possible to increase α-SMA expression post-PMB

resulting in a greater effect on contraction and maturation of the granulation tissue,

progressively leading to induction of remodeling of dermal tissue, with increased

turnover and replacement of collagen type III by collagen type I [19].

In the tissue repair process, angiogenesis is considered as an important event, as

part of new granulation tissue formation in the proliferative phase [20]. Molecular

markers such as VEGF-A and bFGF are potent angiogenic growth factors [20]. Notably,

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we showed an increase in the expression of VEGF-A in the PMB + IFWM group.

Indeed, we corroborate similar findings by Cury et al. [21] who observed a significant

increase in the number of vessels in the skin flap of animals treated with two lasers of

different wavelengths, 660 nm and 780 nm with concomitant increase in the expression

of VEGF mRNA. In addition, Park et al. [22] observed an enhanced differentiation and

secretion of FGF and VEGF growth factors in spheroids composed of hASCs (adipose-

derived stromal cell) post-PMB application. To the best of our knowledge, there are no

reports in the literature of the use of PBM and IFWM in an ex vivo skin culture to

corroborate or compare to our results.

CONCLUSION

Taken together, our results demonstrate that the use of PBM at an optimal dose

of 4.5 J of energy with IFWM served as adjuvant and contributed to accelerated

engraftment, and tissue repair process, as evident by an increase in the expression of α-

SMA and VEGF-A, and enhanced organization in the disposition of the collagen fibers

in the dermis and increase in re-epithelialization in our unique wound model. In

conclusion, the adjuvant use of PBM and IFWM may have utility in enhancing dermal

substitute biointegration and tissue repair with potential future relevance in clinical

practice.

CONFLICT OF INTEREST

Authors declare no conflict of interest.

FUNDING

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This work was supported by São Paulo Research Foundation - FAPESP (grants

# 2013/27021-8, 2015/24940-5 and 2016/24907-3).

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