Enhancement of Therapeutic Benefits of Split Thickness Skin Grafts using Pre-vascularized Human Mesenchymal Stem Cells
Lei Chen, MD, PhD1, Qi Xing, PhD2, Qiyi Zhai, MS1, Mitchell Tahtinen, BS2, Fei Zhou, MD1, Shaohai Qi, MS1, Mohamed Magdy Ibrahim, MD3, Feng Zhao, PhD2.
1First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, 2Michigan Technological University, Houghton, MI, USA, 3Duke University Medical Center, Durham, NC, USA.
PURPOSE: Split thickness skin graft (STSG) implantation is one of the standard therapies for full thickness wound repair when autologous skin full thickness graft (FTG) or skin flap transplants are inapplicable. However, STSG are more fragile than FTG and can contract significantly during the healing process. Human mesenchymal stem cells (hMSCs) are capable of accelerating the wound healing process. So we use pre-vascularized hMSC sheets (PHCS) to study if they would further improve the repair quality of STSG.
METHODS: In vitro cultured control hMSC cell sheets (HCS) were obtained after four weeks culture in complete culture medium (CCM), then endothelial cells (ECs) were cocultured on top of the hMSC sheets for 1 week to get PHCS. Immunofluorescence staining was used to characterize both cell sheets. The progenitor population and multi-lineage differentiation ability of hMSCs inside the cell sheets were tested. The angiogenic growth factor amount present in the cell sheets was also analyzed using an enzyme-linked immunosorbent assay (ELISA). The cell sheets were applied in combination with an autologous STSG in rats with full thickness skin wound. Graft survival and contraction was evaluated and the implants were harvested at predetermined time points for analysis (n=6 animals per group). Histology and immunohistochemistry were performed to test if PHCS can enhance the therapeutic effects of STSG.
RESULTS: The hMSCs in HCS still maintain self-renewal and differentiation capacity after extended periods of culture. The TGF-β1, ANG1 and ANG2 amount in PHCS was higher than HCS (p<0.05) whereas the VEGF amount in PHCS was significantly lower than that in HCS (p<0.05). The HCS and the PHCS implantation significantly reduced skin contraction and improved cosmetic appearance relative to the STSG control group. The PHCS group experienced the least hemorrhage and necrosis, and lowest inflammatory cell infiltration. It also induced the highest neovascularization in early stages, which established a robust blood micro-circulation to support grafts survival and tissue regeneration. Moreover, the PHCS grafts preserved the largest amount of skin appendages (including hair follicles and sebaceous glands) and developed the smallest epidermal thickness. The collagen deposition and fibril morphology in PHCS grafts were also similar to normal skin, indicating a lower degree of skin fibrosis. The superior therapeutic effect seen in PHCS groups was attributed to the elevated presence of growth factors and cytokines in the pre-vascularized cell sheet, which exerted a beneficial paracrine signaling during the early stage of wound repair.
CONCLUSION: The strategy of combining autologous STSG with PHCS implantation appears to be a promising approach in regenerative treatment of full thickness skin wounds.
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