Using Tissue Expander Capsule To Prefabricate Vascularized Tissue Flap

Background and Objectives During the last two decades,tissue engineering has become a rapidly growing field of research in biotechnology and medicine.The key challenge in tissue engineering is the establishment of an efficient vascularization for tissue constructs guaranteeing long-term survival and function.The lack of vascularization may lead to the shrinkage of the tissue after implantation.Previous studies have clarified that the axial-type capsular flap induced by a tissue expander or silicone block has adequate vascularity and that the induced vascularized capsule can be used as a robust vascular bed for composite flap prefabrication.Encouraged by successful results with autologous cells transplanted onto the prefabricated capsule pouch and skin grafting on an induced capsular tissue,we began to evaluate the efficacy of using a tissue expander capsule as an induced vascular bed to prefabricate axial vascularized tissue flaps for bladder and urethral reconstruction in a rabbit model.Materials and Methods1.Silicone tissue expanders were inserted superficial to the superficial circumflex iliac(SCI)vessels(branches of the femoral artery and vein)underneath the bilateral inguinal skin.After wound closure,the expanders were filled with 3 ml of saline intraoperatively and on days 10,13,16 and 19 postoperatively to achieve the final volume of 15 ml.One week after full inflation,the tissue expanders were removed and the femoral artery was cannulated and subsequently flushed with 20 ml heparinized Indian ink solution.The capsular tissue containing axial vessels was transected and fixed for histologic examination.2.After removal of the tissue expander,buccal mucosa grafts(BMGs)were harvested and placed in front of the SCI artery and vein and stretched and sutured on the capsule vascularized by the SCI artery and vein.To help mucosa attach to the capsule and facilitate mucosal spreading,the water-filled tissue expander was placed back into the capsule pouch.One month after prelamination,the mucosal grafts were scrutinized macroscopically,and India ink was used to examine blood perfusion in the prefabricated capsule buccal mucosa composite flap.3.To investigate whether BM can be expanded by tissue expansion,the original silicone tissue expander was replaced by a bigger one(Volume: 30 ml)at one month after BM prelamination.The flap expansion procedure was started 7 days after surgery and was continued twice weekly by injecting 15 ml of normal sterile saline through the port of the tissue expander until sufficient expansion(30 ml in volume)was obtained.After measurement of the mucosal area,each BM-lined flap was perfused with Indian ink and was transected for histologic examination.4.Urethral defects were created and the prefabricated capsule buccal mucosa composite flaps were trimmed and tubularized over an 8F urethral sound to construct a neourethra.The neourethra flap were tunneled without tension toward the urethral defects and were then aligned and anastomosed with the two urethral ends.5.Bladder smooth muscle cells(SMCs)and peripheral blood endothelial progenitor cells(EPCs)were isolated and cultured,respectively.SMCs and EPCs were co-cultured on polystyrene culture plates at a ratio of 6:1 to harvest the prevascularized smooth muscle cell sheet.Then 3 layers of the cell sheet were stacked together and were transplanted to the vascularized capsular tissue with PET sheets.Two days later,another 3 layers of the stacked cell sheet were transplanted to the vascularized smooth muscle with the same method.One month after transplantation,the smooth muscle tissue were analyzed for vascularization and histological study.6.Bladder muscle tissue defects were created,the prefabricated axial vascularized smooth muscle tissue flap was used for bladder wall defects reconstruction.The effects of the reconstructed bladder wall was evaluated at1 and 3 months after transplantion.Results1.Visual inspection revealed that a highly vascularized capsule in the shape of a hollow viscus with a smooth surface was induced by the tissue expansion process.The SCI vessels were located in the center of the capsule and remained pulsatile.India ink injection studies showed many new blood vessels originating from the axial vessels and extending to the periphery of the capsule.2.The BMG attached to the vascularized capsule well.After 1 month of incubation,capillary vessel extensions into the overlying mucosa were evident,and fibrous tissue in the capsule without buccal mucosa lining became denser,which easily distinguished the mucosa from the adjacent native capsular tissue with remarkable boundaries.The axial vascular bundle remained pulsatile,and the prefabricated capsule buccal mucosa composite flap could be raised from the abdominal wall.H&E and Masson’s trichrome staining revealed that the mucosa still showed the characteristic features of oral mucosa with papillae and stratified squamous epithelial structure.Indian ink filled vessels,and the axial vascular bundle was evident in the submucosa layers of the flap.3.In the expansion group,a total of 8 flaps were evaluated.The mucosal surface area in all 8 flaps exceeded the original size after the expansion schedule.The new mucosal area was significantly larger than the original mucosal graft. 4.Circumferential urethral defects were created and repaired by the prefabricated capsule buccal mucosa composite flap.Buccal mucosa formed the lining in the neourethra and kept a wide urethral caliber for 3 months.5.EPCs showed a typical cobblestone-like morphology and SMCs showed a spindle-like morphology.SMCs and EPCs were mixed at a ratio of 6:1 and were co-cultured for 8 days to harvest the prevascularized smooth muscle cell sheets.The cell sheets can be stacked one by one.Three layers of the stacked cell sheets were transplanted onto the axial vascularized capsule.Two days after transplantation,large amount of capillary vessels were extended into the overlying cell sheets and the cell sheets showed a complete survival.A second triple-layer tissue was overlaid on the first graft after 3 days incubation and the whole construct was perfused for a further 2 days.A six-layer smooth muscle cell sheet tissue was overlaid on the vascularized capsule and cultured for 1 month.Thicker engineered tissues can be produced in vitro by overlaying additional triplelayer cell sheets.A thick 6-layer construct is produced by overlaying triple-layer cell sheets two times on a vascularized capsule.6.The prefabricated capsule smooth muscle tissue flap were long enough for bladder wall reconstruction.The smooth muscle survived well and the tissue were infiltrated with capillary vessels.Conclusions1.The capsule may serve as an induced vascular bed for buccal mucosa-lined flap prefabrication.The prefabricated buccal mucosa-lined flap may serve as a neourethra flap for circumferential urethral replacement.2.The capsule may serve as an induced vascular bed for vascularized smooth muscle tissue fabrication and the fabricated thick pedicled smooth muscle tissues can be used for bladder wall reconstruction.