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Objective:To prepare the whole bladder acellular matrix (BAM) using the self-designed perfusion decellularization system, and evaluate the feasibility of constructing the tissue engineering bladder with the adipose-derived stem cells (ADSCs).Methods:This study was conducted from October 2020 to April 2021. The self-designed perfusion decellularization system was used, and four different decellularization protocols (group A, group B, group C and group D) were formulated, according to the flow direction of the perfusate and the action time of different decellularization solutions. Among them, the urethral orifice of the bladder tissue was used as the outflow tract of the perfusion fluid in groups A and B. The top of the bladder was cut off and used as the outflow tract of the perfusion fluid in groups C and D. In groups A and C, 1% Triton X-100 was treated for 6 h, and 1% sodium dodecyl sulfate (SDS) was treated for 2 h. In groups B and D, 1% Triton X-100 was treated for 7 h, and 1% sodium dodecyl sulfate (SDS) was treated for 1 h. In addition, the tissue in the normal bladder group was directly obtained from the natural bladder tissue, which did not require perfusion, cryopreservation and thawing. The fast and efficient decellularization protocol was screened out through HE, DAPI, Masson trichrome and Alcian Blue staining and quantitative analyses to prepare the whole bladder scaffold. The prepared BAM was used as the scaffold material, and the ADSCs were used as the seeding cells to construct the tissue engineering bladder. HE and DAPI staining were used to observe the distribution of ADSCs on the BAM.Results:HE and DAPI staining showed that there was no obvious nuclear residue in the group C. Masson trichrome and Alcian Blue staining showed that the collagen structure and glycosaminoglycan were well preserved in the group C. There was no significant difference in bladder wall thickness between the group C and the normal bladder group [(975.44±158.62)μm vs.(1 064.49±168.52)μm, P > 0.05]. The DNA content in the group C [(43.59 ±4.59) ng/mg] was lower than that in the normal bladder group, group A, group B and group D [(532.50±26.69), (135.17±6.99), (182.49±13.69) and(84.00±4.38)ng/mg], and the difference was statistically significant ( P<0.05). The collagen content [(10.98 ± 0.29)μg/mg] and glycosaminoglycan content [(2.30±0.18)μg/mg] in group C were not significantly different with those in the normal bladder group [(11.69±0.49) and (2.36±0.09)μg/mg, P>0.05]. Scanning electron microscopy showed that a large number of pore structures could be observed on the surface of the prepared BAM in groups A-D and were facilitated to cell adhesion. The isolated and cultured ADSCs were identified by flow cytometry to confirm the positive expression of CD90 and CD29, and the negative expression of CD45 and CD106. Live/dead staining and CCK-8 detection confirmed that the prepared BAM in the group C had no cytotoxicity. HE and DAPI staining showed that a large number of ADSCs were distributed on the surface and inside of the tissue engineering bladder. Conclusions:The whole bladder shape BAM prepared by the self-designed perfusion decellularization system could be used as the scaffold material for bladder tissue engineering, and the constructed tissue engineering bladder could be used for bladder repair and reconstruction.
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Objective: To study the vascularization of tubular graft of bone marrow mesenchymal stem cells (BMSCs) and bladder acellular matrix (BAM) wrapped up in omentum. Methods: We planted BMSCs on the BAM and made it into tubular grafts. Thirty healthy New Zealand white rabbits were randomly divided into three groups with 10 rabbits in each. Rabbits in experimental group received BMSCs-BAM tubular graft wrapped in omentum. Rabbits in control group received BAM tubular graft wrapped in omentum. Rabbits in blank group were planted with silicone catheter wrapped in omentum. Vascular endothelial growth factor (VEGF) protein, CD34 expression and microvessel density of the graft were measured at weeks 4 and 8, respectively. Results: ① The expression of VEGF protein was higher in the experimental group than in the control group and the blank group, and the expression of VEGF protein was higher in the control group than in the blank group; the differences among the three groups were statistically significant [F(2,15)=5.314, P=0.017]. ② The microvessels with positive CD34 expression were denser in the experimental group than in the other two groups, and the blood vessels were closely connected with each other. The microvessels with positive CD34 expression were also much denser in the control group than in the blank group. ③ The microvessel density was higher in the experimental group than in the control group and the blank group, and it was also higher in the control group than in the blank group. The differences among the three groups were statistically significant [F(2,12)=6.172, P=0.021]. Results: Omental wrapping promotes vascularization of BAM, and planting BMSCs on the BAM is more conducive to vascularization of BAM.
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Objective: To explore the effects of the microenvironment of rabbit bladder acellular matrix graft (BAMG) on proliferation, cell surface markers, and molecular protein level of human bone marrow mesenchymal stem cells (hBMSCs). Methods: We prepared BAMG immersion fluid medium and detected its effect on the proliferation of hBMSCs by MTT method. The expressions of CD44, CD45, CD73 and PDGFRβ were detected by flow cytometry. The expressions of PPAR, OCN and α-SMA were detected by RT-PCR, and the expression of OCT was detected by Western blot. Results: hBMSCs had good compatibility with BAMG. The MTT method showed that BAMG and BAMG immersion medium did not affect the proliferation capacity of hBMSCs. The surface of hBMSCs cells cultured with immersion fluid still expressed CD44, CD73 and PDGFRβ, but not CD45. RT-PCR showed that OCN, PPAR, and α-SMA were all expressed. Western blot test also showed the positive expression of OCT-4. Conclusion: hBMSCs can still keep their original biological characteristics in the microenvironment of rabbit BAMG. It can be the seed cells and combined substrate materials for urinary system tissue engineering.
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ObjectiveTo assess the feasibility of seeding adipose-derived stem cells (ADSCs) onto bladder acellular matrix grafts (BAMGs) for bladder reconstruction in a rabbit model.MethodsAutologous ADSCs were isolated,expanded and identified by flow cytometry.In the experimental group,ADSCs were seeded onto BAMGS for reconstructing bladder defects in 12 male rabbits.Unseeded BAMGs were used for bladder reconstruction in the control group of 12 rabbits.Cystography was performed at 24 weeks after grafts implantation.Following cystography,the animals were scarified and grafts were harvested； H&E and immunohistochemical staining were performed with cytokeratin AE1/AE3,smooth muscle α-actin and S-100 markers.ResultsFlow cytometry demonstrated that the ADSCs expressed CD90,CD44,CD105,CD166 and CD34,but not CD45 or CD106.The cells demonstrated good biocompatibility with BAMGs.At 24 weeks,in the experimental group,the reconstructed bladders reached a mean volume of (94.68 ± 3.31 )％ of the precystectomy bladder capacity.Complete regeneration of smooth muscle and nerve tissue was evident.Regenerated SMCs,urothelium and nerve cells stained positively for α-smooth muscle actin,AE1/AE3 and S100.In the control group,the mean bladder volume was (69.33 ± 5.05 )％ of the pre-cystectomy volume.Histologically,the control group was characterized by multi-layered urothelium without evidence for organized muscle or nerve tissue.Conclusion The tissue engineering bladder constructed by ADSCs and BAMG can be used as an ideal biomaterial to replace and repair the bladder.
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Objective To investigate the feasibility of replacing urinary epithelial cells with oral keratinocytes by being seeded on bladder acellular matrix graft(BAMG). Methods Twenty-four male rabbits were randomly divided into 2 groups:experimental group and control group.A length of 2.0 cm and width of 0.8 cm penile urethral mucosal defect was induced in the anterior urethra 2.0 cm awav from the urinary meatus in all the rabbits.Oral keratinocytes were isolated from a small buecal mucosa(1.0 cm×0.4 cm)of the 12 rabbits in experimental group and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin(i3T3).Passage 2 oraI keratinocytes cuItured with i3T3 were expanded and seeded onto sterilized BAMG(2.2 cm×1.0 cm)to repair the de-fects of the urethra.Urethroplasty was performed with BAMG with no cell seeding in the controlgroup.Catheter examination and retrograde urethI ography was done in 1,2 and 6 months after graft-ing.The urethral grafts were harvested and analyzed by histological staining. Results Oral kerati-nocytes seeded onto a feeder layer of i3T3 had better morphous and amplification capability.The corn-patibility of the compound graft was assessed by HE staining and scanning electron microscope.Twelve rabbits in the experimental group could void without difficulty.Catheter examination and ret-rograde urethrogram revealed that a wide urethral caliber was maintained with no sign of strictures and fistula formation.Histological examination showed the grafted urethra was covered with smooth mu- cosa with no strictures at 1,2 and 6 months.The oral keratinocytes of the graft still existed even 6 months after grafting.On contrast,gross and urethrogram demonstrated urethral stricture in the con-trol group.And inflammatory reactions could be found in the area of the stricture through light micro-scope. Conclusions Oral keratinocytes have good compatibility with BAMG and the compound graft could be used for urethral reconstruction in the animal model.