| Vaginal reconstruction is the main method to solve the problem of sexuality and gender roles for patients with no vagina caused by congenital and acquired diseases. Many techniques have been used for vaginal reconstruction but no single superior surgical approach can be accepted. The ideal method should not only form a vagina which is close to the physiological state of the native vagina in structure and function in order to meet the needs of the patient’s sex life, but also to minimize their own tissues damage. Currently the most common technique for vaginal reconstruction involves the creation of a canal between bladder and rectum, subsequently lining of the pelvic canal with a graft. The choice of graft largely determines the effect of surgery. Several materials have been used for graft, such as peritoneum, full-thickness or split-thickness skin grafts, oral mucosa, scalp, amniotic, and fetal skin, but many drawbacks were found. With the development of tissue engineering, a new idea for vaginal reconstruction is provided.In recent years, tissue engineering scaffolds have been used for vaginal reconstruction. Theses scaffolds itself can be used successfully for vaginal reconstruction but the epithelialization of neovagina was slow, increase the chance of infection and stenosis. Animal studies have confirmed that seeded scaffolds can promote the regeneration of tissue components in neovagina, which may construct a neovagina close to native vagina. Currently seed cells used for vaginal reconstruction include vaginal epithelial cells, smooth muscle cells and vaginal muscle-derived stem cells. Those cells can construct a better neovagina. however, they require higher culture conditions which limit its clinical applications. Bone marrow mesenchymal stem cells(MSCs) have adequate sources, easy to isolate and expand in vitro, have the potential for multi-lineage differentiation and paracrine functions, low immunogenicity, can be used for autologous and allogeneic tissue reconstruction. Many studies have confirmed the application of bone marrow mesenchymal stem cells can promote tissue repair. Small intestinal submucosal matrix(SIS)have wide variety of sources, the production method is relatively simple, and contains a variety of growth factors, which have been constructed for a variety of tissue engineering organization, is an ideal scaffold material. In this study, we attempts to use bone marrow mesenchymal stem cells seeded small intestinal submucosal matrix to reconstruct rat vagina and to evaluate the effectiveness of bone marrow mesenchymal stem cells in the vaginal reconstruction. Part one Bone marrow mesenchymal stem cells combined with SIS, and the labeling of bone marrow mesenchymal stem cells.Objective: To investigate the optimal condition for MSCs to combine with SIS. Select an appropriate MSCs labeling method for the following research.Methods: Preparation of SIS and observe it with an optical microscope and scanning electron microscopy. Rat bone marrow stem cells were isolated and cultured with the method of whole bone marrow adherent. Digested and collected the third generation of MSCs, adjust the cell concentration to 4×106/ml, 2×106/ml, 1×106/ml. MSCs in different concentration were seeded on the wet SIS in the 96-well cell culture plate respectively. After1, 3, 5, 7days the cells activity was evaluated by MTT protocol. The effects of different cell concentration and co-cultured time on cellular adhesion efficiencies were compared. Digested and collected the third generation of MSCs, adjust the cell concentration to 4×106/ml, 2×106/ml, 1×106/ml. MSCs in different concentration were seeded on the wet SIS in the 96-well cell culture plate respectively. After 2 days MSCs were seeded repeatedly in 2×106/ml, 1×106/ml. After1, 3, 5, 7days the cells activity was evaluated by MTT protocol. The effects of different cell seeding frequency on cellular adhesion efficiencies were compared. SIS was wetted with different method(new and old method). Digested and collected the third generation of MSCs, adjust the cell concentration to 4×106/ml、2×106/ml. MSCs in different concentration were seeded on the wet SIS which was treated with different method in the 96-well cell culture plate respectively. After 24 h the cells activity was evaluated by MTT protocol. The effects of different wet methods on cellular adhesion efficiencies were compared. Observe the MSCs seeded SIS with an optical microscope and scanning electron microscopy. MSCs were labeled with CM-Dil and the labeling effect was observed with a fluorescence microscopy.Results: SIS was observed with an optical microscope, no cells were remained on it. The results of scanning electron microscopy proved that the stratum compactum surface of SIS was coarse and the abluminal surface was smooth. Rat bone marrow stem cells can be successfully isolated and cultured with the method of whole bone marrow adherent. The optimal condition for MSCs to combine with SIS is that the first day after MSCs seeded with new method treated SIS in the concentration of 2×106/ml. Observed with an optical microscope, there were many MSCs adhere to the stratum compactum surface of the SIS, while nearly no MSC adhere to the abluminal surface of it. Observed with a scanning electron microscopy, there were MSCs adhere to the surface of the SIS. The shape of MSCs on the surface of SIS appeared spindle or irregular shape. There were Granular material and microvilli on the MSCs surface. MSCs were labeled with 5μM CM-Di I. Observed with a fluorescence microscopy after 24 h, the cytomembrane of labeled MSCs were orange,nuclei was not labeled. Cell labeling rate was nearly 100%.Conclusions: The optimal condition for MSCs to combine with SIS is that the first day after MSCs seeded with new method treated SIS in the concentration of 2×106/ml(5×105 /cm2). MSCs were successfully labeled with CM-Di I. The MSCs seeded SIS can be used for the following research. Part two Influence of mesenchymal stem cells on rat reconstructed neovagina.Objective: Compare the difference between the neovagina reconstructed with SIS and MSCs seeded SIS. To investigate the influence of mesenchymal stem cells on rat reconstructed neovagina.Methods: 44 female Sprague–Dawley rats were randomly divided into two groups, SIS group and SIS+MSCs group. The vagina of rat was cut off and subsequently lining of the canal with SIS or MSCs seeded SIS. 6 female SD rats were randomly chose from each group and sacrificed at 2 weeks, 4 weeks and 12 weeks respectively. The neovagina tissues were prepared for MSCs tracing, histological evaluation with HE and immunohistochemistry stain. 4 remained SD rats from each group were sacrificed at 12 weeks, neovagina tissue was used for organ bath study, and another 6 female rats were used as control. High K+(124m M) solution was used first for the assessment of the activity of the vagina. Phenylephrine(10-6,10-5,10-4M) were add to the organ bath, contractile effects were observed. Blocked by phentolamine(10-4M), Phenylephrine(10-4M) was added again to observe the percent change in tone. EFS was used to elicit contractile effects where stimulation was made at varying frequencies(30, 60 and 90 Hz), for a period of 1 seconds with an amplitude of 30 V and a duration of 1 millisecond.Results: Two groups of vaginal reconstruction rat model were created successfully.(1) Gross observation:at 2 weeks, SIS can not be distinguished, the surface of neovagina was thin, pink, tough, inelastic. At 4 weeks, the surface of neovagina was thicker, pink and white, tough, no significant elasticity. At 12 weeks, the surface of neovagina was smooth, pink and white, softer than that at 4 weeks, a little flexibility. The surface of neovagina from SIS+MSCs group is smoother than that from SIS group. The neovagina harvested from SIS+MSCs group is thinner, softer and more flexible than that from SIS group, which was more obvious at 12 weeks.(2) Tracing of MSCs:Orange MSCs can be found in the neovagina at 2 weeks, 4weeks and 12 weeks. At low magnification, orange MSCs mainly distributed in the deep part of the tissue, only a little distributed in the surface layer. At high magnification, the cytomembrane of MSCs in tissue was orange, nuclei was black. Stained with DAPI, orange fluorescence can be found around the blue nuclei, cell structure is clear.(3) Histological observation:At 2 weeks, on the neovagina section from the two groups, regenerated epithelium can be found grown upward from the vaginal orifice, accompanied by inflammatory cell infiltration,regenerated epithelium grown faster in SIS+MSCs group. At 4 weeks, regenerated epithelium covered the whole neovagina, SIS+MSCs group accompanied by less inflammatory cell infiltration than SIS group. At 12 weeks, neovagina epithelium from both group became more mature, inflammatory cell infiltration was not obvious.(4) AE1/AE3 can be detected in the regenerated epithelium by immunohistochemistry. The result of α-SMA immunohistochemical staining showed that in neovagina tissue, some smooth muscle can be found at 4 weeks, smooth muscle bundle can be found at 12 weeks. There was more smooth muscle in the SIS+MSCs group than SIS group. The result of PGP9.5 immunohistochemical staining showed that in neovagina tissue, more nerve fibers can be found at 12 weeks in SIS+MSCs group, but still less than native vagina significantly.(5) In organ bath study, a dose-dependent contraction in neovagina was found after the administration of phenylephrine(10-6,10-5,10-4M). Contractile effects of phenylephrine and electrical field stimulation have been expressed as percentage of the tension elicited by high K+(124m M). Contractile effects of phenylephrine(10-6,10-5,10-4M) in native vaginas, SIS group neovagina and SIS+MSCs group neovagina was 18.471%, 81.753%, 141.196%; 63.354%, 148.058%,170.243%'52.572%,165.558%,181.171%。There was significant difference among the three groups(P<0.001). Further study showed that the contractile effects in SIS group and SIS+MSCs group neovagina were stronger than the native vagina, but no significant difference was found between the two groups. The contractile effects decreased when added phentolamine(10-4M), contractile effects in native vaginas, SIS group neovagina and SIS+MSCs group neovagina were 39.777%,48.540%,40.466% of the contractile effects of phenylephrine(10-4M) without phentolamine(10-4M). There was no significant difference among the three groups(P=0.672>0.05). Contractile effects of native vaginas, SIS group neovagina and SIS+MSCs group neovagina to electrical field stimulation(30v, 1s, 30, 60, 90Hz) was 113.732%,222.179%,254.331%;11.256%,18.214%,20.251%'28.348%,51.406%,57.535%. There was significant difference among the three groups(P<0.001). Further study showed that the contractile effects in SIS group and SIS+MSCs group neovagina were significantly less than the native vagina, but the contractile effects in SIS+MSCs group neovagina were significantly stronger than that in SIS group neovaginaConclusions: MSCs can accelerate vaginal epithelial, smooth muscle and nerve regeneration. The contractile effects in SIS group and SIS+MSCs group neovagina were stronger than the native vagina, but no significant difference was found in the percent changes in tone when added phentolamine, which implied that there may be more adrenergic receptors in the regenerated smooth muscle. Under electrical field stimulation,the contractile effects in SIS+MSCs group neovagina were significantly stronger than that in SIS group neovagina, which implied the fact that MSCs can accelerate the regeneration of smooth muscle. Part three The mechanism of accelerated repair of the neovagina using mesenchymal stem cells seeded SIS.Objective: To explore the mechanism of accelerated repair of the neovagina using mesenchymal stem cells seeded SIS.Methods: Microvessel density(MVD) in neovagina was detected by immunohistochemistry stain with CD34. Microvessel density(MVD) in neovagina of SIS+MSCs group was detected by immunofluorescence, the relation between engrafted MSCs and microvessel was observed. The expression of VEGF m RNA and protein expression in neovagina tissue was detected with RT-q PCR and western blot.Results:(1) At 2 weeks, MVD in neovagina of SIS group and SIS+MSCs group was 28.278±6.259 and 36.347±4.054, significant difference was found between the two group(P=0.024<0.05). At 4 weeks, MVD in neovagina of SIS group and SIS+MSCs group was 22.250±4.385 and 31.325±6.064, significant difference was found between the two group(P=0.014<0.05). At 12 weeks, MVD in neovagina of SIS group and SIS+MSCs group was 20.833±2.246 and 25.347±4.077, significant difference was found between the two group(P=0.039<0.05).(2) Immunofluorescence showed that vascular in neovagina expressed CD34 and was stained green, some CM-Dil labeled MSCs can be found around the vascular, but MSCs didn’t express CD34.(3) Fold change of VEGF m RNA expression in neovaginas of SIS+MSCs group normalized to that in neovaginas of SIS group was 1.490±0.436 at 2 weeks, 1.562±0.375 at 4 weeks and 1.432±0.319 at 12 weeks. The P value was 0.040 at 2 weeks, 0.009 at 4 weeks and 0.022 at 12 weeks, significant difference was found between the groups.(4) At 2 weeks, the VEGF protein expression in SIS+MSCs group neovagina(0.156) was higher than that in SIS group neovagina(0.146), but no significant difference was found between them(P=0.817>0.05). At 4 weeks, the VEGF protein expression in SIS+MSCs group neovagina(0.156) was significantly higher than that in SIS group neovagina(0.104)(P=0.039<0.05). At 12 weeks, the VEGF protein expression in SIS+MSCs group neovagina(0.145) was significantly higher than that in SIS group neovagina(0.091)(P=0.023<0.05).Conclusions: MVD in SIS+MSCs group neovaginas was significantly higher than that in SIS group neovaginas. Engraft MSCs didn’t differentiate to endothelial cells, but some MSCs can be found around the vascular. VEGF m RNA and protein expression in SIS+MSCs group neovagina tissue were significantly higher than that in SIS group neovagina. MSCs may accelerate the tissue regeneration through angiogenesis and the local micro-environment improvement. |
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