Seeding Mesenchymal Stem Cells Onto PLGA/Collagen Compound Scaffold For Bladder Reconstruction In A Rabbit Model

Objective:Urinary bladder loss is one of the major problems due to congenital, trauma or malignancies.The traditional bladder reconstructive surgery with gastrointestinal segments has various complications.Therefore,we evaluated the alternative artificial bladder reconstructive surgery with seeding mesenchymal stem cells（MSCs）induced by Platelet-Derived growth factor-BB into PLGA/collagen compound scaffold in rabbit model,to investigate the feasibility and function parameters of tissue engineered bladders constructed by bone marrow stem cells seeded on PLGA/collagen compound scaffold.Methods:Total 21 male New Zealand White rabbits were randomly grouped:Group A（n=9）,Group B（n=9）,and Group C（n=3,control）with mesenchymal stem cells seeded into PLGA/collagen compound scaffold,small intestinal submucosa,and none,respectively.Part 1:MSCs isolation and culture.MSCs were extracted from the sternum or the iliac crest of anaesthetized New Zealand White rabbits,cultured and multiplied in vitro co-culturing with 0.1μg/L PDGE-BB for 7¹4 days to be used for cell seeding experiments.Part 2:Preparing the small intestinal submucosa（SIS）.Sections of rabbit jejunum were harvested within 4h of sacrifice.The fat was removed from rabbit’s jejunum by washing carefully with water and cut into lengths of approximately 10 cm.Then,the tunica serosa and tunica muscularis were removed mechanically.The decellularized SIS was washed with hypertonic saline or detergent to remove the residual cells then freeze-dried before use.Part 3:Seeding MSCs on scaffolds.Fourth passage PDGE-BB induced MSCs were seeded into the outside of PLGA/collagen or SIS scaffolds by placing a cell suspension（10⁵ cells/ml）into the matrix in a flask filled with DMED medium with low glucose,L-glutamine,110 mg/L Na-Pyruvate,pyridoxine HCl and 10%FBS and incubated at 37℃with5%humidified CO₂ for 7 days before implantation.Part 4:Bladder reconstruction.Under anesthesia,a defect was created in the dome of the bladder wall by surgical incision in 18 rabbits（A and B）,representing approximately 1×1 cm².The seeded PLGA/collagen compound Nanofiber scaffold,and SIS scaffold were sutured as patch into the dome of bladder with continuous 5-0 Vicryl suture in Group A and Group B,respectively.Nothing was done in Group C,sham operation.Part 5:Measurement of bladder volume,histology and immunohistochemistry.Three rabbits in each group were killed separately at 4,8,and 12 weeks after implantation.Bladder volume was tested preoperatively and at 4,8,and 12 weeks postoperatively along with cystography at the same time.Additionally,H&E staining and immunohistochemistry with CD31 and smooth muscle-actin（α-SMA）monoclonal antibody were performed to observe regeneration of urothelium and smooth muscle cells.Results:Our results exhibited a good biocompatibility with PLGA/collagen compound scaffold.The thickness of the grated segment were similar in Group A and B,close to native bladder tissue（Group C）at 12 weeks postoperatively.The bladder capacities in Group A were significantly better than that of the Group B（46.17+1.62ml vs.40.52+1.26ml,P<0.05）.Moreover,cystography revealed a better shape of reconstructed bladders in Group A.The histology and immunohistochemistry results of Group A demonstrated better well-regeneration of urothelium and smooth muscle cells,sustained positively for CD31 andα-SMA than that of Group B.Conclusion:In summary our study results suggested that seeding MSCs into PLGA/collagen compound scaffold promotes regeneration of the urothelium and smooth muscle cells in a rabbit model.Therefore,PLGA/collagen compound scaffold could be more suitable for bladder regeneration than SIS.