Studies on the Efficacy of Single and Twice Application of Mesenchymal Stem Cells in Full Thickness Cutaneous Wound Healing

The experimentation on animals were approved by Institutional Animal Ethics Committee.
 
Collection, isolation and expansion of bone marrowderived mesenchymal stem cells
 
Animals were anesthetized using xylazine 6 mg/kg, followed 10 minutes later by ketamine 60 mg/kg, intramuscularly (Amarpal et al., 2010). The bone marrow aspirate was collected using 18 G bone marrow biopsy needle from the lateral aspect of the iliac crest under aseptic conditions. About 2.5 ml of bone marrow was aspirated into a heparinized syringe from each side.
       
Bone marrow aspirate was processed for isolation and ex-vivo expansion of bone marrow stem cells as per the established protocol (Udehiya et al., 2013). Aspirated cell suspension was filtered and mononuclear cells were isolated by centrifugation over Histopaque (10,771; Sigma). Mononuclear cells were recovered and washed twice with phosphate buffered saline (PBS) (70011-044, Gibco Life Technologies). Finally, mononuclear cells were suspended in Dulbecco’s Modified Eagle Medium (DMEM) (D5769; Sigma) containing 15% fetal bovine serum (FBS) and 50 μg/ml gentamycin sulphate (G1272; Sigma), onto tissue culture flasks and cultured in 5% CO₂ at 37°C. Following 48 hours in culture, the non-adherent cells were removed. Adhered cells were cultured up to 80-90% confluence by changing the medium at every 4^th day. The cells were then detached from the culture flasks by 0.25% trypsin solution and washed with PBS, followed by the seeding. Culture medium was changed on every 7^th day and cells were subcultured up to 3^rd passage. The 3^rd passage cells were collected for the further use in the treatment and cell count was adjusted to 1×10⁶ cells/ml of PBS.
 
Characterization of guinea pig MSCs
 
Characterization of in vitro cultured BM MSCs was done by tri-lineage differentiation.
 
Tri-lineage differentiation
 
Bone marrow MSCs isolated from guinea pig were cultured and at third passage the cells were seeded in 4 well culture plates for expansion. At 60-70% confluency, expansion media were replaced with differentiation media for osteogenic lineage using (A10066-01, StemPro, Gibco Life Technologies), chondrogenic (A10064-01, StemPro, Gibco Life Technologies),and adipogenic (A10065-01, StemPro, Gibco Life Technologies) differentiation kits. Differentiation media were changed twice a week for up to 21 days in all three lineages. After 21 days the cells were fixed with 10% formalin and stained with Alizarin Red S stain (A5533, Sigma-Aldrich), Alcian blue 8GX stain (05500, Sigma-Aldrich) and Oil O Red stain (O0625, Sigma-Aldrich) for analyzing the osteogenic, chondrogenic and adipogenic differentiation potential, respectively. Differentiation potential was assessed by the presence of calcium deposits, sulfated and carboxylated mucopolysaccharides and appearance of lipid droplets for osteogenic, chondrogenic and adipogenic differentiation respectively. The wells supplemented with complete expansion media served as negative control (Fig 1).
 

 
Experimental design
 
The wound healing was evaluated using 18 clinically healthy adult Dunkin Hartley guinea pigs of either sex, weighing 450-700g in the age group of 5-6 months. These animals were provided with standard diet and water ad libitum and maintained under uniform managerial conditions. Animals were acclimatized to approaching and handling for 15 days prior to the commencement of the study. One square (2.5×2.5 cm²) full thickness excisional skin wound was created surgically on the dorsum at thoraco-lumbar region of each animal under xylazine-ketamine anaesthesia (Amarpal et al., 2010). The animals were divided into 3 groups having 6 animals in each group. The treatment administered in the animals of different groups is detailed in Table 1.
 

       
Each treatment was evaluated on a total of 6 wounds. Each wound was dressed with hydrogel (10% gel of Pluronic F127) up to day 5 and thereafter, the wounds were cleaned with PBS and dressed with 0.5% povidone iodine daily until complete healing. All the animals were housed in individual cages and administered with broad spectrum antibiotic and anti-inflammatory drugs for initial 5 days.
 
Gross wound evaluation
 
Grossly all the wounds of different groups were observed for quantity and type of exudate, peripheral swelling and complete healing of the wounds.
 
Wound contraction analysis
 
The rate of wound healing was evaluated on the basis of reduction in the wound area at subsequent intervals. Wound size was measured on 3^rd, 7^th, 14^th, 21^st, 28^th, 35^th and 42^nd postoperative days. Total contraction, percentage contraction and the mean percentage of wound contraction for each interval were calculated for each group.
 
Photographic evaluation
 
Wounds were photographed on days 0, 3, 7, 14, 21, 28, 35 and 42 post-surgery and evaluated by independent observers to assess the quality of wound healing and cosmetic outcome.
 
Histopathological analysis
 
Full-thickness skin tissue samples from 3 healing wounds of each group of animals were collected from centre of the healing tissue on 21^st and 42^nd postoperative days and fixed in 10% buffered formalin. After fixation, the tissues were processed by paraffin embedding technique and 4-5 µm thick tissue sections were cut and stained with Haematoxylin and Eosin (H&E) stain. The H&E stained sections were evaluated microscopically by using histological scoring system as per the method described by Smith et al., 2008. Sections from regenerated tissue were observed under light microscope for presence and type of epithelialization, inflammation (cellular infiltration), fibroblast, collagen and neovascularization. The scoring system used to grade the healing in histological evaluation is presented in Table 2.
 

 
Histochemical analysis
 
Duplicate sections from each treatment group were stained using special staining techniques namely Masson’s Trichrome stain (Masson, 1929; Lillie, 1948) and Gomori’s Aldehyde Fuchsin method (Mallory, 1942) for detection and grading of collagen and elastin fibres, respectively, in the healing tissue.
 
Statistical analysis
 
The means of parametric observations were compared by one way Analysis of Variance (ANOVA) and DMRT (Duncan’s new multiple range test) using SPSS software. Non-parametric observations were compared by Kruskal-Wallis test (Siegel and Castellan, 1998). The significance level was set to *p<0.05 and **p<0.01 for statistically significant and highly statistically significant differences, respectively.

Bone marrow derived mesenchymal stem cells (MSCs) have a good potential as therapeutic agents for promoting tissue regeneration and repair in response to injury, as these cells have shown to influence each and every step of wound healing. They contribute to wound healing either by differentiation or by paracrine signalling, out of which paracrine signalling is the primary mechanism responsible for MSCs response to injury such as inflammation, angiogenesis and fibroproliferation (Gnecchi et al., 2008; Lau et al., 2009).
 
Gross wound evaluation
 
In group III there was significant improvement in the quantity of exudate on day 3 onwards as compared to day 5 in group II. Exudation observed was of serous type in group I whereas it was dried in groups II and III on day 3. Group II showed maximal reduction in peripheral swelling by day 3. Gross observations like quantity and type of exudate, peripheral swelling around wound margin showed better score in group where MSCs were used as compared to the control group. This  could be due to the beneficial paracrine action of these MSCs in the wound bed as they produce and secrete a variety of cytokines and chemokines specifically vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF), keratinocyte growth factor (KGF) and TGFα which positively affects wound repair (Gnecchi and Melo, 2009). MSCs are also known to secrete a variety of angiogenic, mitogenic and anti-apoptotic factors such as, hepatocyte growth factor (HGF), angiopoietin-1, adreno medullin (AM) and IGF-1, in vitro (Kinnaird et al., 2004). Repeated application of MSCs over the wounds positively affected the gross appearance of the wound as the wounds showed earlier appearance of granulation tissue, epithelization and faster wound contraction as compared to the single application and control group.
       
Granulation tissue consists of fibroblasts, growing capillaries and infiltered inflammatory cells. It is formed on the surface of wounds to protect and provide nutrition to the wound. Among all the groups, groups II and III showed better granulation tissue level on day 3 as compared to group I. Group III showed decreased level of granulation tissue on day 35 as compared to other groups suggesting early maturation of granulation tissue than rest of the groups. Wan et al., 2013 reported transplantation of MSCs promoted granulation tissue proliferation by enhancing cell multiplication, which in turn facilitated the wound healing process and led to better tissue regeneration. Time of epithelization was shortest in group III followed by group II because MSCs secrete mitogens that stimulate proliferation of keratinocytes, dermal fibroblasts in vitro (Smith et al., 2010), or have an accelerating effect on migration of keratinocytes and epithelialization (Gurtner et al., 2008). Nakagawa et al., 2005 also showed that MSCs transdifferentiated into the epithelium in rat skin defect model.
       
Though the original wound created in each animal was of 2.5 cm × 2.5 cm dimensions and the original size should have been 6.25 cm² in all the groups, but almost all the wounds were expanded to certain extents. Thus, immediately after the surgery all the wounds had an area greater than 8 cm². The wound area decreased significantly (P<0.01) from day 14 onwards in all the groups as compared to their base values. On day 21, the wound area was significantly (P<0.05) lesser in the animals of group III as compared to that in groups II and I (Fig 2). Per cent healing of the wounds was fastest in the animals of group III where 99.74% wound healing was recorded by day 35 and 100% healing on day 42, followed by group II (94.69%) and then group I (91.51%). (Table 2).
 

 
Histopathological evaluation
 
As histomorphological assessment of healing in open wounds allows more precision than clinical examination of wounds therefore wounds were also evaluated using following parameters (Table 3). On day 21^st postoperatively, surface epithelium was only in initialized stage in all the groups and was thicker than adjacent normal skin. But by day 42, group III showed complete epithelialization comparable to that of normal skin, which was followed by group II (Fig 3). Application of MSCs might have led to epithelialization as studies showed transdifferentiation of human MSCs into epithelial cells (Gurtner et al., 2008). On day 42, groups II and III showed thickness of epithelium resembling to the normal skin indicating the MSCs results in early maturation of the epithelium but in group I, it was still moderately thick. Inflammatory reaction was recorded as mild to nil in group III probably because of anti-inflammatory effect of MSCs. MSCs causes a decrease in the secretion of the pro-inflammatory cytokines TNFα and Interferon-γ (IFNγ) while simultaneously increase the production of anti-inflammatory cytokines interleukin-10 (IL-10) and IL-4 (Aggarwal and Pittenger, 2005). Granulation tissue width subsequently decreased in all the groups on day 42 as compared to day 21 but group III showed appreciably narrow width, early maturation and well organised granulation tissue in the form of collagen fiber bundles. Fibroblast proliferation was moderate (groups III) to severe (groups I and II) on day 21 in different groups which indicated rapid increase in the concentration of fibroblast over the wound bed in initial phase of healing. The formation of new blood vessels is necessary to uphold the newly formed connective tissue and the survival of dermal cells (Pratheesh et al., 2017). Groups in which MSCs were applied showed greater number of newly formed capillaries on day 21 which positively influenced wound healing. While on 42^nd postoperative day the score for neovascularization was lower in group II and III as compared to group I indicating early maturation of granulation tissue with decreased number of newly formed blood vessels eventually. Nuschke (2014) reviewed the role of MSCs in chronic wound healing concluding that these cells have the ability to suppress excessive inflammation and reduce scarring while stimulating de novo angiogenesis in the wound bed, all leading to promising outcome in wound repair.
 

 


Collagen, elastic fiber density and thickness showed all over better score in group III compared to rest of the groups. Thicker collagen fibers were present in group III as compared to rest of the groups on day 21 and 42. The arrangement of collagen fibers was best in animals of group III on day 42. Studies have shown that dermal fibroblasts secrete increased amounts of collagen type I and alter gene expression in response to either MSCs or MSC conditioned medium (Smith et al., 2010).
 
Group III depicted better regeneration of wounds, followed closely by group II as compared to the control group. From this study it was concluded that double application of MSCs by injection around the wound periphery leads to relatively faster and qualitatively better healing than single application of stem cells.