Experimental Study Of Neotype Abdominal Wall Repair Patch Constructed By Tissue Engineering

Objective:(1)To study the feasibility of three-dimensional,contractile skeletal muscle with parallel muscle fiber constructed by myoblasts seeded on collagen I/PLGA under simulated mechanical circumstance and tissue blood supply.(2)To study the feasibility of abdominal wall defect repaired by tissue engineered(TE) patch consist of small intestinal submucosa(SIS) with fibroblasts seeded on it and TE skeletal muscle.(3)To compare the effect and regenerating organ of TE patch and SIS on repairing abdominal wall defect in animal models.To look for the potential approach to upgrade constructing.Method:(1)Preparation of seed cells and scaffold.①Utilize different methods,such as enzymatic digestion,adherence,flow cytometer and so on to isolate,purify,culture skeletal myoblasts from the skeletal muscles of neonate rat.②Utilize enzymatic digestion to isolate,purify, culture dermatic fibroblasts from neonate rats.③Manufacture porcine small intestinal submucosa following the method invented by Abraham.④Identify the myoblasts,fibroblasts, SIS through the observation of the shape of the cell with inverted microscope and immunocytochemistry.(2)Construction of TE patch and repair abdominal wall defect in animal models.①skeletal myoblasts were suspended into a collagen/PLGA gel,under simulated mechanical circumstance inside mechanical cell stimulator for 3 weeks in vitro.②Suspend fibroblasts on SIS and co-cultivate for 3 weeks in vitro.③The above-mentioned two,combined with omental vasoganglion to form TE patch.④All animal underwent creation of a 3×2.5cm full-thickness abdominal wall defect and the defect was repaired by TE patch or biomaterial(3 layers of SIS).⑤Animal's recover information and electrophysiological characteristic of repaired abdominal wall postoperatively in two sets were recorded.⑥The mechanic strength of repaired abdominal wall postoperatively in two sets was measured.⑦The observation of tissue structure,cell density,cell component of the centre of repaired body wall was performed to confirm whether there was muscle layer similar to normal skeletal muscle in appearance,whether anagenetic muscle fiber could confluence with host muscle fiber,whether H-chain of myoglobulin could be detected.⑧Statistical analysisResults:(1) Three-dimensional skeletal muscles with parallel and ordered muscle fiber,potential to contract were constructed successfully.(2)TE patch,consist of TE skeletal muscle and fibroblast seeded on SIS,was superior to biomaterial in the effect of repairing abdominal wall defect.in two sets were both execelent and similar.Anagenetic body wall in TE set was thicker,with fibrous tissue layer and muscle layer,displayed higher in cell density with better cellular infiltration.H-chain of myoglobulin was detected and electromyography evidenced single motor-unit potentials,sometimes grouped into arithmic discharges in t TE set.While Sixty days after surgery,biomaterials were completely replaced by fibrous tissue,no electric activity detected.(3)The thickness of myoblast-seeded patches and intensity of their myoelectric activity were inferior at day 90 to 60 after implantation,suggesting a loss of contractile muscle fibers.Conclusion:(1)Construction of three-dimensional TE skeletal muscle,with similarity to normal skeletal muscle in appearance and potential to contract,is feasible.(2)The therapeutic efficacy of TE patch to abdominal wall defect is superior to biomaterial.TE patch represent a very promising tool for body-wall defect repair and can retrieve biomaterial in scar replacing repaired body wall,low recovery rate,high long-term rate of heria,since they are repopulated by skeletal muscle cells and fibroblasts,show positive responses in the electrophysiological studies.(3)Studies shouid be under way to identify strategies to improve and maintain the structural and functional integrity of implants for a longer time,especially in innervation.Innovation:(1)To our knowledge,this experiment is the first exploration of tissue engineering used in repairing of abdominal wall defect(certification affiliated).(2)This experiment is also the first construction of tissue engineered skeletal muscle in China.