Objective: In this study, we investigate the regularity of connective tissue capsuleinduced by spiral PLA (Polylactic acid) stent and the effect of embedding intervals onbladder epithelial cells proliferation. Materials and Methods: Bladder biopsies wereprocured from Wistar rats, microdissected into pieces and treated with trypsin to harvestbladder epithelial cells. PLA (mean molecular weight1.5×10~5Da) was used to fabricatespiral stents. Flat films were also fabricated with PLA to study the effect of embeddingintervals on bladder epithelial cells proliferation. All of them were implantedsubcutaneously into Wistar rats. All animals were euthanized and the grafts wereharvested at designed time points (1,2or3weeks). Grafts which were retrieved fromrats were decellularized with1%Triton X-100solution in PBS with0.02%EDTA.Cells were seeded onto the prefabricated capsular stent in a static way at a density of4×10~4cells/cm~2. The proliferation of the seeded urothelial cells were determined byMTT assays. Results: At the end of1week, a thin wall of tissue film can be seen at thesurface of the spiral PLLA stent. The inflammatory response during this period wasmild and the reactive zone was limited to only a thin layer of polymorphonuclearleukocyts and occasional lymphocytes. Immunofluorescent staining of the freshretrieved connective tissue showed that there were no capillary networks in it. At theend of2weeks, a thin capsule of connective tissue formed around the PLLA stent, withno evidence of inflammation. A film sheet of connective tissue around the stent can beseen at the end of3weeks with fibroblastic proliferation, but the inflammatory responsewas entirely absent. Mssson`s trichrome staining results revealed that there were richcollagen components existing in the capsules in1,2and3weeks group. All essentialextracellular matrix molecules of basement membrane, such as associated collagenmolecules, laminin and fibronectin existed in such capsule of connective tissue.Scanning electron micrographs showed that the capsule presented with3dimension appearance and the seeded urothelial cells scattered on the surface of capsules formedrespectively after each imbedding intervals. The cells tended to spread from the3Dsurface in a flattened appearance with obvious pseudopods. MTT results showed thatthe entrapped cells in each group all remained consecutively proliferation duringincubation. Significantly greater amount of cells was detected in2week group,3weekgroup compared with1week group at day5and7. Conclusions: The results suggestthat the entrapped cells in all capsular stents grew well, lined up in continuous layersand remained continuous proliferation during incubation, especially with an embeddingtime from2to3weeks. We conclude that urothelial cells can be successfully harvested,cultured in vitro and seeded onto a prefabricated capsular stent with potential forureteral reconstruction. And the information obtained in this study should provideimportant information for fabricating capsular PLLA stent and construction of acell-based tissue engineered ureter in vitro. However, further studies are needed todetermine whether the cell-based capsular stents can effectively in situ replace of thedefect ureters. |