A Study Of Bone Marrow Mesenchymal Stem Cells Accelerate The Angiogenesis In Full-thickness Skin Wound In Pig

Objective:To investigate the clinical value of BMSCs treatment in full-thickness skin defect wounds, the expression of vascular factors and microvessel density were detected in this research.Method:2 one-year-old pigs were employed for BMSCs isolation.15-20 ml bone marrow were harvested from ileac bone using a ileac needle and mononuclear cells were isolated using density gradient centrifugation and cultured in F-12 with 15% fetal bovine serum. After 48 hours of cell isolation medium was replaced and after then cell medium was replaced every 2 days. Culture cells were daily observed under phasecontrast microscope and passaged when cells almost 80% confluent.24 3cm×3cm full-thickness skin wounds were made on the back of each pig after anesthesia. Every wound was divided into different group. Group A:1×107 cultured BMSCs in 1 ml PBS were administrated to each wound followed by autologous microskin grafting, and covered by acellular dermal matrix (ADM); Group B:After BMSCs administration like Group A, auto-split skin were transplanted to each wound and covered with ADM; Group C:After BMSCs administration like Group A, wounds were covered with ADM. As control groups, Group a, Group b, and Group c, all steps were similar to above but BMSCs administration was replaced by PBS injection. At 7,14 days post operations, wound tissues were harvested for histology and total RNA abstraction. Actin was detected by immunohistochemistry staining, and microvessel density (MVD) was counted under microscope. VEGF, VCAM, and ANG mRNA expression were detected by real time-PCR.Result:Combined with cultured BMSCs administration, full-thickness skin wounds closure were accelerated.①Among three experimental groups, wounds of Group B were complete healed at 14 days after operation, but small open wounds were still exist in Group A, and Group C. Compared with Group C, at day 14, opening wounds of Group A were smaller.②MVD analysis expressed that counting of Actin-positive cell clusters in wound beds and leading edges in Group B were higher than other groups (P< 0.05).③Relative quantitative analysis by real time PCR shows, in all groups, VEGF mRNA expression at day 7 post operation were higher than its expression at day 14. But in three experimental groups combined with BMSCs administration, VEGF mRNA were higher than control groups. Among three experimental groups, its expression in Group C was highest, and its expression in Group B was higher than Group A. VCAM, ANG mRNA expression pattern were similar to VEGF mRNA.Conclusion:BMSCs could accelerate full-thickness skin wound healing by improving angiogenesis, and the main mechanism may promote the expression of VEGF, VCAM, and ANG.. But only local BMSCs administration could not replace auto-split skin grafting.