Preliminary Research Of Adipose-derived Stem Cells Combined With Small Intestinal Submucosa For Vaginal Reconstruction

Objective: To study the effect of Adipose-derived stem cells(ASCs)in artificial vaginal reconstruction,and then find a new way for vaginal reconstruction with tissue engineering.Methods: The ASCs were isolated from adipose tissue of SD rats,which was digested with type?collagenase.The third generation of ASCs were analyzed for the surface antigens: CD44,CD90,CD29,CD105,CD34 and CD45 by flow cytometry.The Adipogenic and Osteogenic induction were tested to ensure that the cells have the multipotential differentiation ability.The third generation of ASCs were combined with SIS with the cell density of 2×105/cm2,4×105/cm2.The cell viability was tested 72 h,3d,5d and 7d after combination.The second generation of ASCs were labeled by CM.dil,and the labeling rate was tested with the fluorescence microscope.1.5×106 labeled ASCs were taken to combine with SIS,and then cultured 5 days.Remove the natural vagina of the female SD rats whose weight was about 200-250 g,and then the pure SIS or the SIS combined with ASCs were transplanted into the residual cavity of the removed vagina.The transplants' top were fixed to the cervix by anastomosis,and the bottom were sutured with the surrounding skin of the incision.Remove the artificial vagina after transplantion 7,14 and 28 days,and test the vestigial of the ASCs which were labeled by CM.dil using the fluorescence microscope.Both the two groups have to take HE and Masson's stain test,in order to compare the histological differences between the groups,then we can infer which one was better for vaginal reconstruction.Result: The ASCs showed fusiform usually.When the cell confluence reached 80%,the cells present the spiral arrangement.The expression rates of the ASCs' surface antigens: CD44,CD90,CD29,CD105,CD34 and CD45 were 97.48%,99.91%,99.99%,100%,58.05% and 0.79% respectively.The ASCs differentiated into osteoblasts and adipose cells successfully.Five days after ASCs combined with SIS,the absorbance of MTT reached the highest one.The cell membrane of ASCs which were labeled by CM.dil presented orange under the fluorescence microscope,and a round hypofluorescein showed in the cytoplasm and cell nucleus.And the labeling rate could reach 100%.Both the two groups could form new artificial vagina.We could see the labeled ASCs exist in all layers of the combined groups' newborn vagina,especially basal layer.And after HE and Masson's staining,we could see the nonkeratinized stratified squamous epithelium cells grow from vagina orifice upward to the external cervical orifice and then spread to the full wall of newborn vagina after transplanted 28 days.The epithelium cells of the ASCs combined with SIS groups grew and spread faster than the single SIS groups.In the combined groups we can see more blood vessels and less inflammatory cells compared with the single SIS groups.So that we can included that the SIS combined with ASCs have better effect in vagina reconstruction.Conclusion: ASCs have effect not only in promoting vaginal epithelium cells regeneration and angiogenesis but also in inhibiting the inflammatory reaction.The SIS combined with ASCs can form new artificial vagina more successfully.