Wound healing requires a synchronized interplay among cells, growth factors, and extracellular matrix proteins. Several studies have demonstrated that mesenchymal stem cells coordinate the repair response by recruiting other host cells and secreting growth factors and matrix proteins.
This study was performed to evaluate the role of allogeneic ASCs in acute and chronic wound healing therapies.
Considering this aim, there are essential characteristics that must be met to allow these cells to be candidates for cell-based therapy.
As we have previously mentioned, these cells can differentiate into multiple tissue-forming cell lineages. During differentiation, upregulation or suppression of transcription factors occurs via specific signaling pathways. In osteogenic differentiation, Runx2 is a key transcription factor that elevates osteoblast differentiation. Chondrogenic differentiation is driven by Sox9, and adipogenic differentiation is mainly controlled by PPARγ. We reported the expression of these transcription factor markers involved in early differentiation of osteoblasts, adipocytes, and chondrocytes, and we presented images of differentiated cells. These data are in agreement with the study performed by Almalki et al. [6]. (Fig. 1.).
As discussed in the Materials and Methods, we utilized TBP gene for normalization of qRT-PCR data, according to Rangi et al [11], who found that TBP has the top position for both geNorm and NormFinder analysis in MSCs, and Vandesompele et al found that TBP has the top position for geNorm analysis in skin wound [12].
Particular attention should be given to the homing capability of ASC, allowing the cells to find the way of injury and inflammation. Figure 2 shows the capacity of these cells under normoxic conditions when stimulated by conditioned medium as well as by the inflammatory cytokine GM-CSF to migrate and mediate regenerative effects at sites of tissue damage. It is note that the role of conditioned medium in increasing the transmigratory effect of ASCs, which implies that in the process of proliferation, ASCs release factors and cytokines with chemoattractant activity. Enciso et al [1] demonstrated the expression of MMP-2 and MMP-9 in ASCs enables the breakdown of the endothelial basement membrane.
Our pilot approach, reported by Enciso et al [49], demonstrated a higher regenerative capacity with earlier and faster closure in wounds treated with ASCs in comparison to other forms of treatment.
In this paper, we extend this previous study to 24 dogs and evaluated the clinical value and safety of the application of cultured adipose allogenic ASCs for treating acute and chronic skin wound healing in the canine model described above.
Damage of the skin certainly induces local inflammation; this process involves multiple mediators, including chemokines, pro- and anti-inflammatory cytokines and growth factors. To analytically evaluate the etiological role of inflammatory processes in systemic compartments, it is necessary to quantify the concentrations of relevant biomarkers in fluids, such as serum.
As shown in Fig. 3, of the cytokines studied, we did not observe increased levels in the serum of dogs treated with ASCs for 7 or 30 days. Although we observed a significant increase in IL8 cytokine levels in wounds treated with ASCs at both 7 and 30 days, the levels of IL-8 (3858.64 pg/ml) in the serum of animals at day 30 posttreatment were similar to the median value (3329 pg/ml) of healthy dogs, which is data obtained with the Multiplex assay by Safra, O’Neill and Kjegaard-Hansen [46, 47]. These results ensure the safety of our protocol.
These data, together with our studies related to the immunomodulatory capacity of ASCs (49) [49], allow us to guarantee the welfare and safety of our treatment.
Figure 4A and B shows the wound contraction and percent of re-epithelization in acute and chronic wounds as well as in non-treated wounds. The percentages of wound contraction and re-epithelialization were significant (p < 0.0001) between wounds treated with ASCs and control wounds both in acute and chronic conditions at 7, 30 and 90 days posttreatment. At 90 days, re-epithelization of acute and chronic wounds reached more than 97%. On the other hand, we have analyzed data of acute wounds that have received one or two doses, obtaining the same significant difference p < 0.0001.
These data indicate the success of ASCs in wound healing therapy, providing faster wound healing and re-epithelization than that of other treatments.
With respect to the histopathological study, Fig. 5A and 5B data support the active regenerative process, which revealed better organization of re-epithelialization, reduced inflammatory infiltrate, marked collagen fibers and the presence of multiple hair follicles in different stages of activity on day 7 after treatment with ASCs by promoting epidermal and dermal regeneration.
In this study, we analyzed the gene expression of IL10, MMP-2, GM-CSF and VEGFA in the process of cutaneous wound healing in a canine model treated with ASCs.
It is well known that the process of wound healing is an organized event resulting in the restoration of the skin. It involves the interactions of many different cell types, matrix components, and biochemical factors. In this context, the GM-CSF gene is a possible candidate for the regulation of wound healing because it is synthesized by a number of cells involved in the repair process [50–54] .
Consistent with these comments, we found an upregulation of GM-CSF at the gene level in our canine model treated with ASCs. GM-CSF has been shown to exert beneficial effects on wound healing in patients suffering from poorly healing wounds and chronic skin ulcers with diverse etiology [55, 56] .
Considering the role of GM-CSF in transmigration (Fig. 2), the increased gene expression levels of GM-CSF may contribute to the recruitment of cells that participate in skin repair to the site of injury.
However, for IL10 and MMP-2 genes, there were no significant differences between treatments. With respect to VEGFA gene, we expected an increase in gene expression, taking into consideration the histological findings, which revealed better organization of re-epithelialization; on the contrary, a significant decrease was found. However, other authors showed that increased vascularization is associated with GM-CSF [55] .
In view of the data we present in this study, we suggest that the beneficial effects observed in canine wounds after allogenic ASC therapy are due not only to direct ASC action but also to indirect paracrine processes through the induction of secondary factors involved in wound repair.