Hydrogels Enhance The Retention Of Bone Marrow Mesenchymal Stem Cells In Mice Allogenic Skin Transplantation And Myocardial Infarction Model

Part I: Combined Transplantation of Human Bone MarrowMesenchymal Stem Cells and Hydrogel in Allogenic Skin TransplantationObjective: To observe the effect of combination of bone marrow mesenchymal stemcells and hydrogel on mice after allogeneic skin transplantation.Methods:(1) The isolation, cultivation and purification of BMSCs: after theinformed consent was obtained,15ml of bone marrow aspirate was obtained from the iliacbone of a donor. The collected sample was cultured in DMEM high-glucose supplementedwith15%FSC for3days until the non-adherent cells were removed, then the cellmorphology and growth were observed. The cells after passage3were used in thisexperiment.(2) Establishment of the animal model:40male C57BL/6mice were used asdonors and40male ICR mice were used as receptors. The whole thickness of the skin wascut out to make a round wound about1cm2. Skin patches from donors were transplantedinto receptors.(3) The ICR mice were randomly divided into four groups, with10mice ineach group, the following procedures were carried out: the control group with PBSinjection; The gelatin group, with hydrogel injection; the cell group with human BMSCsinjection; Mixed group with hydrogel and BMSCs injection.(4) Observation of thesurvival time and conditions of the transplanted flaps.(5) Observation of the distribution ofhuman mesenchymal stem cells in vivo (6) Detection of the inflammatory cytokines in theperipheral blood of the receptor: the inflammatory cytokines: IL–2, IL–4, IL–10, IFN–gamma and TNF-alpha were detected by ELISA.Results:(1) The BMSCs were observed by inverted phase contrast microscope:48hourslater, a few cells were fibroblast-like;3-4days later, the cells began to grow rapidly;12days later, cells reach to90％confluence.(2) Observation of the survival of transplantedskin patches:4days later none of the groups had obvious change. Control group:5days later, the skin turned black and8days later, it had scar necrosis; Gelatin group: small ulcerwas visible8days after the transplantation and on9thdays the scar necrosis appeared; Cellgroup: small ulcer was visible8days after the transplantation and the9thd the skin turnedblack, followed by scar necrosis on the11thday; Mixed group: small ulcer was visible onthe8thday, then on the10thday the skin turned black, and12~13days later the scarnecrosis appeared. The survival time of the transplanted skin: the patches survival time ofthe cell group and the mixed group were longer than the control group (P<0.05); There wasno significant difference between the hydrogel and the cell group (P<0.05); The skin graftof the mixed group had longer survival time compared with the control group, the hydrogelgroup and the cell group (P<0.05).(3) Observation of the remaining subcutaneous humanBMSCs: BMSCs were labeled by CM–DiL and the most BMSCs were remained in themixed group.(4) To observe the distribution of BMSCs in vivo:14days aftersubcutaneous injecting the cells, a large number of fluorescent cells were observed in thelung tissue, and a small amount of fluorescent cells could be observed in the spleen.(5)Detection of the level of peripheral blood cytokines by ELISA (IL-2, IL-4, IL-10, IFN-gamma, TNF-alpha):①The levels of IL-2were significantly higher in the control groupand the hydrogel group than the cell group and the mixed group (P<0.05);②The levelsof IL-4were significantly lower in the control group and the hydrogel group than in the cellgroup and the mixed group (P<0.05);③The levels of IL-10were significantly higher inthe control group and the hydrogel group than in the cell group and the mixed group(P<0.05);④The levels of IFN-gamma were significantly higher in the control groupand the hydrogel group than in the cell group and the mixed group (P<0.05);⑤Thelevels of TNF-alpha were significantly higher in the control group and the hydrogel groupthan in the cell group and the mixed group (P<0.05).Conclusion:(1) Human BMSCs could extend the survival time of allogeneic graft;(2) Thehydrogel can improve the retention of bone marrow mesenchymal stem cells in allergenicskin transplantation model. Part II: Combined Transplantation of Bone Marrow Mesenchymal StemCells and Hydrogel in Acute Myocardial Infarction Model Objective: To verify the effect of combination of bone marrow mesenchymal stem cellsand hydrogel on myocardial infarction.Methods:(1) Isolation, culture and purification of SD-BMSC by density gradientcentrifugation;(2) Establishment of myocardial infarction model:20male SD rats wererandomly divided into myocardial infarction group and control group. MI model wasestablished by left descending artery (LAD) ligation.Results:(1) The BMSCs were observed by inverted phase contrast microscope:48hourslater, a few cells were fibroblast-like;3-4days later, the cells began to grow rapidly,after10-14days cultured primary MSCs achieved80%-90%fusion, the results showed thatalmost all of SD rat bone marrow mesenchymal stem cells expressed CD29(cell integrinmolecules), CD90(adhesion molecule); but did not express the hematopoietic progenitorcell surface markers like CD34,CD45,CD106.（2）To observe the cardiac function: thecardiac functions of the rats in each groups were damaged after the surgery, then theyrecovered gradually, especially in the combined treatment group.Conclusion: The hydrogel can improve the retention of bone marrow mesenchymal stemcells on rat myocardial infarction model thus contributing to the ebhance cardiac functionin the mixed group.