Prelimitary Study Of Bone Marrow Mesenchymal Stem Cells Combined With Porcine Vaginal Acellular Matrix To Construct Tissue Engineering Vagina

Objective: To explore whether the application of bone marrow mesenchymal stem cells(BMSCs)combined with porcine vaginal acellular matrix AVM)to construct tissue engineered vagina in rats is superior to that of use AVM alone.Methods: The BMSCs were isolated from bilateral femur and tibia from 20 days old Sprague-Dawley male rats,the whole bone marrow adherence method was used to culture BMSCs.The third passage of BMSCs was combined with AVM and cocultured for 24 h.Scanning electron microscope was used to observe the growth of the cells on the scaffold.The third passage cells were marked by CM.dil.Staining effect and label efficiency were observed with fluorescence microscope.The animal model was established by resection of vaginal in rats,and divided into three groups according to different construction methods :(1)The AVM combined with BMSCs labled by CM.dil,namely composite BMSCs group.(2)The rat tissue engineered vagina was constructed by simple AVM,namely simple AVM group.(3)The normal rats’ vaginas were used as control group.Tissue engineering vagina were collected at 1,2,3,4,8weeks after surgery.The gross morphology was observed and microscopic morphological were evaluated by HE and Masson staining.Immunofluorescence staining was used to detect the expression of epithelial,smooth muscle,nerve and blood vessels of BMSCs and the new vaginal tissues.Functional evaluation of new vagina was tested by tissue bath method.High concentration K+ solution was used to evaluate the activity of the new vagina.Electrical field stimulation was used to access contractile effects of the new vginas.At an amplitude of 30 V and varying frequencies(10~100Hz).Phenylephrine(10-6~2*10-4M),Phentolamine mesilate(10-5~2*10-4M),Kaba choline(10-6~10-4M),atropine(10-5~10-4M)were added to the organ bath respectively and then access the contractile effects of the new vaginas.Result: Rat BMSCs can be successfully isolated and cultured by the method of the whole bone marrow adherent,which showed fibroblast-like shape.The BMSCs formed a spiral whorl-like outlook when the cells fused to 90%.Gross observation found that there was no significant difference between the composite cell group and the simple AVM group.After 8 weeks of operation,the new vaginas of two groups were mucosal and similar to the normal vagina.Masson staining showed: 1 week after operation,a few of the mucosal epithelium formed in composite cell group,while just incompleted degradation scaffold materials were observed in simple AVM group.There were many inflammatory cell infiltration in both group.At 2 weeks after operation,the epithelial tissue of composite cell group covered the whole scaffold.Simple AVM group 3 weeks after operation on full-thickness epithelial tissue.At 4 weeks after operation,the epithelial layer of the two groups increased,composite cell fibers arranged more neatly.At 8 weeks after surgery,smooth muscle tissue was formed in composite cell group,only tangled muscle fibers was observed in simple AVM group.Immunofluorescence staining found that BMSCs can differentiate into muscle cells,vascular endothelial cell and nerve cell.The artific ial vagina in composite cell group was better than the simple AVM group in the drug and electrical stimulation.Conclusion: Both cell group and simple scaffold group can construct tissue engineering vagina.The morphology and function of the cell group were superior to that of the simple AVM group.BMSCs can promote the reconstruction of tissue engineering vagina by direct differentiation.