Bone marrow-derived mesenchymal originate cell (BM-MSC) therapy is a novel, therapeutic option for the particular acceleration of corneal healing in the horse. In our within vitro study, at 72 h after scratch initiation, we documented a significant percentage decrease in the scratch region remaining between the BM-MSC and MSC-Sp groups compared to the manage group. Additionally , the BM-MSC group showed improved recovery compared to the MSC-Sp group as there was a significant percentage reduction in the scratch area remaining between the BM-MSC and MSC-Sp groups at 36 h post-scratch and all time factors thereafter. The cytokine TGF-β 1 was found in considerably higher concentrations in the BM-MSC group compared to the control team, but not when compared to the MCS-Sp group.
It is expected that both BM-MSC and MSC-Sp would have a positive influence on wound recovery, as demonstrated in this study. It is known that MSCs secrete soluble factors to exert influence over around tissue [ 31 ] and the beneficial effects of BM-MSCs, therefore , are believed to be due in large part to the potent paracrine factors that the cells secrete, which are both anti-inflammatory plus immunomodulatory [ 26 , 27 , 32 ]. These interactions likely also be involved in paracrine signaling to host cells, for example within the cornea, to promote repair of local tissue. It has been earlier shown that MSC-conditioned media has a therapeutically positive impact on tissue repair without the use of whole cells [ 33 ]. This likely explains why a beneficial effect in the MSC-Sp group was demonstrated. Although this effect was not because significant as when there was direct biological feedback among cell types, this result strengthens the argument to be used of a stem cell-derived but cell-free product clinically, which might prove beneficial due to its lack of immunogenicity, ease of storage, plus preferred routes for administration.
Despite their potent immunomodulatory effects, BM-MSCs can still be immunogenic due to their constitutive surface expression associated with major histocompatibility complex (MHC) class I and adjustable surface expression of MHC class II [ 34 ]. MHC class II proteins are potent alloantigens resulting in recognition by alloreactive CD4 + T cells and promotion of T-cell proliferation [ 35 ], and it has been shown that MHC class II healthy proteins are upregulated by inflammatory mediators including interferon (IFN)-γ [ 34 ]. Recent studies in the horse have shown that will allogeneic donor BM-MSCs that are MHC-mismatched to the recipient trigger both cellular and humoral immune responses in the receiver [ 28 , 34 ] whether they are MHC class II negative or positive, suggesting that recipient recognition associated with foreign MHC class I alone is capable of leading to a significant immune response. Furthermore, antibodies present in the receiver antisera following donor BM-MSC injection are capable of killing subscriber BM-MSCs in vitro through the complement cascade suggesting that will allogeneic BM-MSCs may have a limited therapeutic effect [ 29 ].
It is important to remember that the normal cornea is considered an immune privileged site because of lack of corneal vasculature and lymphatics, low numbers of nearby antigen-presenting cells, high concentrations of immunomodulatory cytokines, plus low constitutive expression of MHC class I upon corneal cells [ 36 ]. Therefore , it is unclear exactly what effect, if any, allogeneic BM-MSCs would have on immune system responses in this predominantly immune-tolerant environment. Damage to the surface epithelium can result in impairment of normal immune tolerance, however , resulting in recruitment of antigen-presenting cells to the limbus, production associated with inflammatory cytokines, antigenic stimulation, and upregulation of MHC class II [ 37 ]. For these reasons, it is reasonable in order to still recommend the use of an autologous as opposed to an allogeneic source in a wounded corneal environment, as used in this particular study. This, unfortunately, is not ideal for acute corneal injuries as autologous BM-MSCs can take weeks to culture plus achieve numbers for use. Ideally, a supernatant product or maybe the use of an allogeneic source that is MHC class We low and MHC class II negative would be favored. Further studies are needed to evaluate the safety and effectiveness of various allogeneic BM-MSCs donors.
Determining the specific soluble factors that BM- MSCs secrete can help us to further clarify and specify their role in tissue repair. Therefore , we examined the media from each treatment group at the end of the research for peptide growth factors typically found and important in healing of the injured equine corneal environment, which includes EGF, PDGF-BB, and TGF-β 1 . Previous studies have examined the efficacy of certain exogenous growth factors on the acceleration of wound healing. A study by Haber ou al . documented good proliferative effects of exogenous EGF and PDGF-BB on both mount corneal epithelial cells and keratocytes in vitro [ 38 ]. EGF has been shown to increase cell proliferation and chemotactic migration [ 39 , 40 ], and PDGF-BB increases matrix manufacturing and chemotaxis and enhances inflammatory reactions to speed up tissue repair [ 38 , 41 ]. EGF was a component of the particular naive fibroblast media and serum-free media and, consequently , its detection in all groups in this study was anticipated. EGF was, however , not found in a higher concentration within our treatment groups compared to the control population, indicating it was not really upregulated by BM-MSCs during wound healing within the framework of this assay. PDGF-BB was not found in any group within our study.
TGF-β 1 was not expressed in the control group; however , improved concentrations of TGF-β 1 were noted in both the particular BM-MSC and MSC-Sp groups. This is supported by prior studies which demonstrated higher TGF-β 1 expression associated with MSCs compared to limbal stem cells [ 42 ] and higher expression of TGF-β 1 in verweis corneas treated with MSC therapy [ 43 ]. TGF-β one has been shown to induce connective tissue growth factor through fibroblasts [ 44 , 45 ] which is important for fibroblast expansion and extracellular matrix component production, including collagen. TGF-β 1 has also been shown to stimulate integrin expression which is involved with acceleration of wound repair [ 46 ]. However , within the study by Haber et al. exogenous TGF-β one had a negative effect on proliferation of corneal epithelial tissues and keratocytes [ 38 ]. Therefore , its effects upon wound healing are variable and warrant further analysis. Additionally , the increased concentration of TGF-β 1 within our study was only significant for the BM-MSC group when compared to control group. We suspect the MSC-Sp group could have gained significance with an increased population of horses examined.
An additional objective of our study was to determine the phenotype of the corneal cellular material cultured, as we strived to create an in vitro atmosphere as similar to the wounded equine cornea as possible, and to evaluate and differentiate them from a BM-MSC phenotype. Through stream cytometry, our corneal cells were determined to be of fibroblastic morphology. Fibroblasts are an appropriate model for this project since keratocytes undergo fibroblastic transformation after injury and are and so the primary cell type in wound repair [ 47 ]. Our own primary culture media was supplemented with 5% FBS, which likely explains the fibroblastic phenotype. It has been earlier shown that equine primary cultures generated from stromal explants plated in media supplemented with 10% FBS selectively induce fibroblasts instead of keratocytes [ 48 ] which is in agreement with our study.
Limitations of this study include the few horses. It is possible additional horses may strengthen the findings of this study. Additionally , there was variability in the number of pathways fibroblasts underwent prior to the scratch assay as this was dependent upon how quickly each horse’ s BM-MSCs took to achieve confluence. Variability among horses was also noted in the proliferative capabilities of fibroblasts which may have influenced our outcomes. Lastly, we do not yet know what concentration of BM-MSCs is optimal for having the best effect on stromal cells.
Future goals of the research would include in vivo studies to prove effectiveness of topical products for clinically affected epithelial plus stromal lesions. Prior to these studies, it would be important to enhance our stem cell product by determining the focus of cells that maintain the most positive therapeutic impact. Additionally , it has been previously shown that MSCs of passing 4 have an increased wound healing potential compared to tissue only undergoing two passages [ 49 ]. The BM-MSCs in our study underwent two passages prior to placement within the transwell inserts and, therefore , it would be beneficial to know when continued passages of these cells would result in faster injury healing.