Preparation Of Seed Cells For Vaginal Reconstruction By 3D Bio-printing In Rat

Part one Differentiation of BMSCs into endothelial cells,smooth muscle cells,epithelial cells and fibroblast cells in vitroObjective:Explore the methods to induce BMSCs differentiation into endothelial cells,smooth muscle cells,epithelial cells and fibroblast cells in vitro,to provide seed cells for 3D bio-printing to reconstruct vagina.Methods:Culture of rat BMSCs in vitro1 The effect of CoCl₂ on the BMSCs:To verify the ability of CoCl₂ to simulate hypoxic microenvironment,the effect of CoCl₂ on the growth of BMSCs was detected with MTT assay.Western blot was used to detect the expression of HIF-1?after treated with CoCl₂?2 To induce BMSCs differentiate into endothelial cells,smooth muscle cells,epithelial cells and fibroblast cells:To induced BMSCs differentiate into endothelial cells,BMSCs were pretreated with CoCl₂ for 24 h and then cultured with endothelial cells differentiation medium(containing with 3%FBS,50 ng/mL VEGF and 10 ng/mL bFGF)for 7/14 days.BMSCs were cultured with smooth muscle cells differentiation medium(containing with10%FBS,10 ng/mL PDGF-AB and 5 ng/mL TGF-?1)for 7/14 days to differentiate into smooth muscle cells.To induced BMSCs differentiate into epithelial cells,Air-liquid interface culture system and epithelial cells differentiation medium(containing with 3%FBS,50 ng/mL EGF and 10ng/mL KGF)were used to culture BMSCs for 7/14 days.For fibroblast cell differentiation,BMSCs were culture with fibroblast cells differentiation medium(containing with 10%FBS,5 ng/mL TGF-?1)for 7/14 days.3 The changes of cell morphology were observed with inverted micro-scope.4 Identification of the cells differentiated from BMSCs:Identification of the endothelial cells differentiated from BMSCs:Immunofluorescence assay was used to detect the expression of VEGFR1,VEGFR2 and v WF.The expression of VEGFR1 and VEGFR2 mRNA was detected by qRT-PCR.Tubular network formation assay in vitro was used to analysis the capillary formation of the cells.Identification of the smooth muscle cells differentiated from BMSCs:Immunofluorescence assay and Western blot were used to detect the expression of SMMHC11 and?-SMA.The expression of MYH11 and?-SMA mRNA was detected by qRT-PCR.Identification of the epithelial cells differentiated from BMSCs:Immunofluorescence assay and Western blot were used to detect the expression of AE1/AE3.The expression of AE1/AE3 mRNA was detected by qRT-PCR.Identification of the fibroblast cells differentiated from BMSCs:Immunofluorescence assay and Western blot were used to detect the expression of Collagen I.qRT-PCR was used to detect the expression of Collagen I?1 and Integrin?11 mRNA.Results:1.The effect of Co Cl₂ on the BMSCs:The results of MTT assay showed that CoCl₂ did not affect the viability of BMSCs with concentrations of?200?M and incubation times of 24 h.The results of Western blot showed that the protein level of HIF-1?increased after the cells treated with CoCl₂ in a concentration-dependent manner.There was no statistical difference between Co Cl₂ 100?M and 200?M groups.2.The changes of the morphology of the differentiated cells observed with inverted microscope:After the cells were induced for 14 days,the cytoplasm of endothelial cells was retracted,and the stereoscopic feeling of the cells increased.The smooth muscle cells assumed a spindle shape and a“hill-and-valley”growth pattern.In the epithelial cells,the cytoplasm was retracted and part of the cells was oval or polygonal in shape.Part of the cells on the transwell displayed cobblestone-like morphology.The fibroblast cells assumed long fusiform and arranged in a fibrous arrangement.3.Identification of the differentiated cells:Identification of the endothelial cells differentiated from BMSCs:Immunofluorescence staining data showed the cells expressed endothelial cell markers VEGFR1,VEGFR2,and vWF.Pretreated with CoCl₂ could increase the expression of VEGFR1,VEGFR2,and vWF.The result of qRT-PCR showed that the expression of VEGFR1 and VEGFR2 mRNA was increased in the group of cells pretreated with CoCl₂.The expression level significantly increased with the extension of incubation time(P<0.05).The result of tubular network formation assay showed that the differentiated cells arranged in circles on the ECM Gel.Identification of the smooth muscle cells differentiated from BMSCs:The results of immunofluorescence staining and Western blot showed that the cells treated with differentiated medium expressed SMMHC11 and?-SMA.The result of qRT-PCR showed that the expression of MYH11 and?-SMA mRNA of the cells treated with differentiation medium increased compared with the control group(P<0.05).But the expression level had none significant difference between of the cells induced for 14 days and for 7 days(P>0.05).Identification of the epithelial cells differentiated from BMSCs:The results of immunofluorescence staining and Western blot showed that the differentiated cells expressed AE1/AE3.Air-liquid interface culture promoted the expression of AE1/AE3.The data of qRT-PCR showed that the expression of AE1/AE3 mRNA increased in the differentiated cells compared with the control,and air-liquid interface culture promoted the expression of AE1/AE3mRNA(P<0.05).The expression level significantly increased with the extension of incubation time.Identification of the fibroblast cells differentiated from BMSCs:The results of immunofluorescence staining and Western blot showed that the cells treated with differentiation medium expressed Collagen I.The result of qRT-PCR showed that the expression of Collagen I?1 and Integrin?11mRNA of the cells treated with differentiation medium increased compared with the control group(P<0.05).The expression level significantly increased with the extension of incubation time.Conclusions:Hypoxic microenvironment established by pretreating the cells with Co Cl₂ combined with endothelial cells differentiation medium could induced BMSCs differentiation into endothelial cells.Smooth muscle cells differentiation medium containing with PDGF-AB and TGF-?1 could promote BMSCs differentiation into smooth muscle cells.BMSCs could differentiate into epithelial cells when cultured in air-liquid interface culture system and epithelial cells differentiation medium.Cultured with fibroblast cells differentiation medium could promote BMSCs differentiation into fibroblast cells.Part two The role of Wnt/?-catenin signaling pathway in the differenti- ation of BMSCs into endothelial cells,smooth muscle cells,epithelial cells and fibroblast cellsObjective:To illuminate the role of Wnt/?-catenin signaling pathway in the differentiation of BMSCs into endothelial cells,smooth muscle cells,epithelial cells and fibroblast cells.To explore the mechanism of differentiation and increase differentiation efficiency of BMSCs.Methods:1 The role of Wnt/?-catenin signaling pathway in the differentiation of BMSCs:Inducing the BMSCs differentiate into endothelial cells,smooth muscle cells,epithelial cells and fibroblast cells in vitro according the method described in part one.Western blot and qRT-PCR were used to detect the expression of GSK3?,TCF3 and?-catenin at the protein and mRNA level.2 Activation of Wnt/?-catenin signaling pathway by transfecting BMSCs with si DKK1:BMSCs were transfected with siDKK1.q RT-PCR was used to detect the expression of DKK1 mRNA after transfection and to investigate the optimum condition.3 The effect of activating Wnt/?-catenin signaling pathway on the differentiation of BMSCs into endothelial cells,smooth muscle cells,epithelial cells and fibroblast cells:To induced BMSCs differentiation into endothelial cells,BMSCs were pretreated with CoCl₂ for 24 h and then transfected with siDKK1 and cultured with endothelial cells differentiation medium for 14 days.For smooth muscle cells differentiation,BMSCs were transfected with siDKK1and then cultured with differentiation medium for 7 days.BMSCs transfected with siDKK1 were culture in air-liquid interface culture system and epithelial cells differentiation medium for 14 days to induce it differentiation into epithelial cells.To induce BMSCs differentiation into fibroblast cells,BMSCs after transfected with siDKK1 were culture with fibroblast cells differentiation medium for 14 days.Western blot and qRT-PCR were used to detect the expression of GSK3?,TCF3 and?-catenin at the protein and mRNA level of the differentiated cells.Detection of the expression of differentiated markers of the cells:Endothelial cells:Immunofluorescence assay was used to detect the expression of VEGFR1,VEGFR2 and vWF.The expression of VEGFR1 and VEGFR2 mRNA was detected by q RT-PCR.Smooth muscle cells:Western blot and Immunofluorescence assay were used to detect the expression of SMMHC11 and?-SMA.The expression of MYH11 and?-SMA mRNA was detected by q RT-PCR.Epithelial cells:Western blot and Immunofluorescence assay were used to detect the expression of AE1/AE3.qRT-PCR was used to detect the expression of AE1/AE3 mRNA.Fibroblast cells:Western blot and Immunofluorescence assay were used to detect the expression of Collagen I.The expression of Collagen I?1 and Integrin?11 mRNA was detected by qRT-PCR.Results:1.The role of Wnt/?-catenin signaling pathway in the differentiation of BMSCs:The results of Western blot and qRT-PCR showed that the expression level of GSK3?decreased in the treated group compared with that in the control group,and the expression levels of?-catenin and TCF3 increased.In the endothelial cells inducing groups,the change was more evident in the cells pretreated with Co Cl₂.In the epithelial cells inducing groups,the change was more obvious in the cells cultured in air-liquid interface culture.2.The data of qRT-PCR showed that the expression of DKK1 mRNA decreased after BMSCs were transfected with si DKK1.Transfected with 50nM siDKK1 for 24 h was the optimum condition.3.The effect of activation of Wnt/?-catenin signaling pathway on the differentiation of BMSCs:The results of Western blot and qRT-PCR showed that the expression level of GSK3?decreased and the expression levels of?-catenin and TCF3increased in the cells transfected with siDKK1 compared with that in the negative control group and the group without transfection.Endothelial cells:Immunofluorescence staining datas showed expression of VEGFR1,VEGFR2,and vWF increased in the group of cells transfected with siDKK1.The result of qRT-PCR showed that the expression level of VEGFR1 and VEGFR2 mRNA was higher in the group of cells transfected with siDKK1 compared with the other groups(P<0.05).Smooth muscle cells:Western blot and immunofluorescence staining data showed the expression of SMMHC11 and?-SMA increased in the group of cells transfected with siDKK1 compared with the other groups.The result of qRT-PCR showed that the expression level of MYH11 and?-SMA mRNA of the cells transfected with siDKK1 were higher than the other groups(P<0.05).Epithelial cells:The results of Western blot and immunofluorescence staining showed the expression of AE1/AE3 increased in the group of cells transfected with siDKK1 compared with the other groups.The result of qRT-PCR showed that the expression of AE1/AE3 mRNA of the cells transfected with siDKK1 were higher than the other groups(P<0.05).Fibroblast cells:Western blot and immunofluorescence staining data showed the expression of Collagen I increased in the group of cells transfected with siDKK1 compared with the other groups.The result of qRT-PCR showed that the expression level of Collagen I?1 and Integrin?11 mRNA of the cells transfected with siDKK1 were higher than the other groups(P<0.05).Conclusions:The Wnt/?-catenin signaling played a positive role in the differentiation of BMSCs into endothelial cells,smooth muscle cells,epithelial cells and fibroblast cells.Activation of Wnt/?-catenin signaling by si DKK1 could promote the differentiation of BMSCs into endothelial cells,smooth muscle cells,epithelial cells and fibroblast cells.Part three The safety detection of the endothelial cells,smooth muscle cells,epithelial cells and fibroblast cells differentiated from BMSCsObjective:To detect the biological safety,tumorigenicity and acute toxicity for nude mice of the endothelial cells,smooth muscle cells,epithelial cells and fibroblast cells differentiated from BMSCs,in order to evaluate its safety and further applied value in vivo.Methods:Inducing the BMSCs differentiate into endothelial cells,smooth muscle cells,epithelial cells and fibroblast cells in vitro according the methods described in part two.1 The cells were inoculated directly in thioglycollate medium and improved martin culture medium and were detected whether or not contaminated with bacterial and fungi.Mycoplasma and endotoxin in the differentiated cells were detected by mycoplasma staining test kit and Gel method endotoxin rapid detection kit.2 The cells were inoculated in the nude mice subcutaneously to detect the tumorigenicity of the cells.HE stain was used to detect the histological change of the inoculated position.3 The cells were injected into the tail vein of nude mice to detect the acute toxicity of the cells.The effect of the cells on the growth of nude mice was observed.Results:1.The biological safety of the cells:No bacterial and fungi contamination was found in which the cells differentiated from BMSCs were inoculated in thioglycollate medium and improved martin culture medium for 3 days.The result of detection of mycoplasma by mycoplasma staining test kit was negative.The detection of endotoxin by Gel method endotoxin rapid detection kit was also negative.2.The differentiated cells had no effect on the growth of nude mice after inoculated in the nude mice subcutaneously.There was no tumor could be found in the inoculated position.No visible lesions could be found in the organs of the nude mice after inoculate for 8 weeks.The results of HE staining showed that there was some inflammatory cell infiltration at the inoculation site after cells inoculated for 3 days.There was no significant difference between the groups inoculated with cells and the control group in the pathological feature of injection site after transplantation for 2 weeks and 8weeks.3.There was no such abnormality as death of the mice could be found after the differentiated cells were injected into the tail vein of nude mice for 2weeks.The weight of nude mice injected with differentiated cells increased gradually,and have no statistic difference compare with the control group(P>0.05).No visible lesions could be seen in the organs of the nude mice after injected with differentiated cells for 2 weeks.Conclusions:No contamination of bacterial,fungi mycoplasma and endotoxin of the cells differentiated from BMSC was found in vitro.Tumorigenicity and acute toxicity of the differentiated cells neither been found on nude mice.