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A Study of Bone Marrow Mesenchymal Stem Cells Combined with Small Intestinal Submucosa for Vaginal Reconstruction

Author: ZhangZhiQiang
Tutor: HuangXiangHua
School: Hebei Medical University
Course: Obstetrics and Gynaecology
Keywords: Bone Marrow Mesenchymal Stem Cells Cytocompatibility Vaginal Reconstruction Small Intestinal Submucosa Electrospun Silk FibroinScaffold
CLC: R318.0
Type: Master's thesis
Year: 2014
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Abstract

Part one: Study of cytocompatibility of small intestinal submucosa (SIS)and electrospun silk fibroin scaffold (ESFS) with bone marrowmesenchymal stem cells (MSCs).Objective: To compare the cytocompatibility of the two kinds ofbiomaterial, SIS and ESFS, and choose the better one for vaginalreconstruction.Methods: Rat bone marrow cells were isolated and cultured followingthe method of whole bone marrow adherent. The third generation of MSCswere tested for the markers CD29, CD90and CD45by flow cytometry.Osteoblasts induction and adipose induction were used to test whether thecells have the ability of multipotential differentiation. After induction, theosteoblasts induced cells were stained with Alizarin Red to evaluate themineralization capacity. The adipose induced cells were stained with Oil RedO to evaluate the lipid accumulation. MSCs at passage3were seeded onto twokinds of scaffolds and cultured. We used cell counting method to test celladhesion rate and used MTT to detect scaffolds’ toxicity to MSCs. MSCs atpassage3were labeled with BrdU and the labeling rate were measuredthrough cell counting method. MSCs at passage3were combined with SISand ESFS respectively. The MSCs’ morphology on the two scaffolds wasdetected with scanning electron microscopy. Scaffolds and MSCs-scaffoldcomplexes were implanted under cutis of rat back. At2and4weeks afterimplantation, the scaffolds with and without MSCs were assessed byhistological HE stain, Masson’s stain and immunohistochemical analysis.Result: MSCs shown fibroblast-like shape. When MSCs confluence achieved80%, they formed a spiral whorl-like outlook, Flow cytometryanalysis indicated the MSCs positively express CD29(99.91%) and CD90(99.95%), but did not express CD45. After induction, MSCs differentiatedinto osteoblasts and adipose cells respectively. The adhesion rate of MSCs onSIS was90.75%2.12%which had no significant difference with the rate onESFS(89.13%3.36%). SIS was thin, flexible and transparent. ESFS is somefragile, opaque and thicker than SIS. The results of scanning electronmicroscopy proved that the surface of SIS was smooth and compact and theESFS was comprised of fibres with different diameters. The shapes of MSCswere spindle on SIS but flat on ESFS. Both MSCs on the membranes of SISand ESFS had secretion granules and microvillus,. The proliferation rates ofMSCs on SIS and ESFS were88.35%and85.48%respectively. Both of themwere lower than the control group. But there was no significant differencebetween these two scaffolds. The toxicities of these two kinds of scaffoldswere all grade1. The BrdU labeling rate of MSCs was81.3%. At4weeksafter implanted under cutis of rat back, the MSCs-scaffolds’ surfaces werecovered with more new blood vessels than scaffolds without MSCs. Thedegradation speed of SIS was faster than ESFS. The numbers of new bloodvessels in MSCs-scaffolds and cells migrated into the inside ofMSCs-scaffolds were more than scaffolds without MSCs. The result ofMasson’s stain showed that SIS was light green and ESFS was red.Immunohistochemistry stain show that some MSCs migrated into thesurrounding tissue.Conclusion: Both SIS and ESFS have good cytocompatibility and can beused as vehicles for MSCs. SIS is softer and the degradation rate of SIS ishigher than ESFS, so SIS can easily attached to tissue closely and be insteadby new tissue. MSCs have the ability of promoting vascularization. We canuse SIS as carrier of MSCs to study vaginal reconstruction.Part two: Study of rat bone marrow mesenchymal stem cells combinedwith small intestinal submucosa in rat vaginal reconstructionObjective: To study the function of MSCs as seed cells in vaginal reconstruction and find a new way for vaginal reconstruction.Methods: Mature rats’ vaginae were cut off and SIS with or withoutMSCs were implanted into the surgical cavity holes. Upper ends of thescaffolds were sutured to the lateral sides of cervices, and the other ends weresutured together with the vaginal opening cut edges. After20and30days, thenew vaginae were obtained and assessed with HE stain, Masson’s stain andimmunohistochemical analysis.Results: Both rats implanted with two kinds of scaffolds grown newvaginae with certain elasticity. HE and Masson’s stains showed that the newvaginal epithelial cells grown from vaginal orifice upward to the upper endand spread to the full vaginal wall at last. Compared with single SIS group,new vaginal epithelial cells grown faster in the MSCs combined SIS group.And the new blood vessels were more in the new vaginal stroma of MSCscombined SIS group. Immunohistochemistry stain indicated that BrdU labeledcells existed in vaginal epithelium, muscles and new vessels and showncorresponding cell morphology. While MSCs existed in the surface of newvagina where there had not formed normal epithelium did not have vaginalepithelial shape.Conclusion: MSCs can accelerate vaginal epithelial regeneration andvasculogenesis. MSCs can different into epithelioid cells have epithelial cellshape, vessel endothelial-like cell and myoid cell. MSCs’ differentiation intovaginal epithelial cells might need the direct contact with vaginal epithelialcells.

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