1
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
([email protected]) and Ardeshir Bayat ([email protected])
2
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
3
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
4
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
5
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),
6
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
7
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
8
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
9
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
10
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).
11
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
12
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
13
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.
14
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
15
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
16
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,
17
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
18
This work was supported by São Paulo Research Foundation - FAPESP (grants
# 2013/27021-8, 2015/24940-5 and 2016/24907-3).
REFERENCES
1. Stadelmann, W.K., A.G. Digenis, and G.R. Tobin, Impediments to wound healing. Am J Surg, 1998. 176(2A Suppl): p. 39S-47S.
2. Ojeh, N., et al., Stem Cells in Skin Regeneration, Wound Healing, and Their Clinical Applications. Int J Mol Sci, 2015. 16(10): p. 25476-501.
3. Eming, S.A., P. Martin, and M. Tomic-Canic, Wound repair and regeneration: mechanisms, signaling, and translation. Sci Transl Med, 2014. 6(265): p. 265sr6.
4. Greaves, N.S., et al., The role of skin substitutes in the management of chronic cutaneous wounds. Wound Repair Regen, 2013. 21(2): p. 194-210.
5. Shilo, S., et al., Cutaneous wound healing after treatment with plant-derived human recombinant collagen flowable gel. Tissue Eng Part A, 2013. 19(13-14): p. 1519-26.
6. Brigido, S.A., et al., Use of an acellular flowable dermal replacement scaffold on lower extremity sinus tract wounds: a retrospective series. Foot Ankle Spec, 2009. 2(2): p. 67-72.
7. Hodgkinson, T. and A. Bayat, Ex vivo evaluation of acellular and cellular collagen-glycosaminoglycan flowable matrices. Biomed Mater, 2015. 10(4): p. 041001.
8. Tatmatsu-Rocha, J.C., et al., Low-level laser therapy (904nm) can increase collagen and reduce oxidative and nitrosative stress in diabetic wounded mouse skin. J Photochem Photobiol B, 2016. 164: p. 96-102.
9. de Medeiros, M.L., et al., Effect of low-level laser therapy on angiogenesis and matrix metalloproteinase-2 immunoexpression in wound repair. Lasers Med Sci, 2016.
10. Ferraresi, C., Y.Y. Huang, and M.R. Hamblin, Photobiomodulation in human muscle tissue: an advantage in sports performance? J Biophotonics, 2016. 9(11-12): p. 1273-1299.
11. Guimarães, G.N., et al., Effect of ultrasound and dexpanthenol on collagen organization in tegumentary lesions. Rev Bras Fisioter, 2011. 15(3): p. 227-32.
12. de G de Gaspi, F.O., et al., Effects of the Topical Application of Hydroalcoholic Leaf Extract of Oncidium flexuosum Sims. (Orchidaceae) and Microcurrent on the Healing of Wounds Surgically Induced in Wistar Rats. Evid Based Complement Alternat Med, 2011. 2011: p. 950347.
13. Castro, F.C., et al., Effects of microcurrent application alone or in combination with topical Hypericum perforatum L. and Arnica montana L. on surgically induced wound healing in Wistar rats. Homeopathy, 2012. 101(3): p. 147-53.
14. Xu, W., et al., Application of a partial-thickness human ex vivo skin culture model in cutaneous wound healing study. Lab Invest, 2012. 92(4): p. 584-99.
15. Zhang, Y., et al., cDNA microarray analysis of gene expression profiles in human fibroblast cells irradiated with red light. J Invest Dermatol, 2003. 120(5): p. 849-57.
19
16. Abergel, R.P., et al., Biostimulation of wound healing by lasers: experimental approaches in animal models and in fibroblast cultures. J Dermatol Surg Oncol, 1987. 13(2): p. 127-33.
17. Darby, I.A., et al., Fibroblasts and myofibroblasts in wound healing. Clin Cosmet Investig Dermatol, 2014. 7: p. 301-11.
18. Esmaeelinejad, M., et al., The effects of low-level laser irradiation on cellular viability and proliferation of human skin fibroblasts cultured in high glucose mediums. Lasers Med Sci, 2014. 29(1): p. 121-9.
19. Tomasek, J.J., et al., Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol, 2002. 3(5): p. 349-63.
20. Kondo, T. and Y. Ishida, Molecular pathology of wound healing. Forensic Sci Int, 2010. 203(1-3): p. 93-8.
21. Cury, V., et al., Low level laser therapy increases angiogenesis in a model of ischemic skin flap in rats mediated by VEGF, HIF-1α and MMP-2. J Photochem Photobiol B, 2013. 125: p. 164-70.
22. Park, I.S., P.S. Chung, and J.C. Ahn, Adipose-derived stromal cell cluster with light therapy enhance angiogenesis and skin wound healing in mice. Biochem Biophys Res Commun, 2015. 462(3): p. 171-7.