Tissue Engineering Epidural Fat Graft To Prevent Scar Formation

Objective: Preadipoeytes can differentiate directly into mature adi- pocytes,and Fibrin glue is compatible with many sorts of cell.Therefore this experiment will be pre-fat cells and fibrin glue composites implanted in vivo, and explore in the body preadipocyte proliferation and differentiation of the feasibility. For tissue engineering of fat transplants to prevent epidural scar formation and provide a theoretical basis.The objective in this study include:to culture preadipocytes in vitro and seeded fibrin glue,to study the adhesion,proliferation and differentiation of preadipocytes on fibrin glue.To exploring the complex in vivo implantation in rats to prevent the effects of epidural scar formation.Method:(1)To 80 rats were randomly divided into 4 groups. A group of fat cells- protein gel fiber composite stent implantation group. Group B particles of autologous fat and fibrin glue mixture implant group. C group alone groupautologous fat. D control group.(2)Rat preadipocytes cultured cell lines: A group of rats to 10% respectively by intraperitoneal injection of chloral hydrate anesthesia (0.35 mg/100g) after.Remove sterile epidi- dymal adipose tissue of the Department of groin. Cut dipose tissue in the larger blood vessels and connective tissue,cut the epididymis with the Department of Ophthalmology and the Department of mixed inguinal adipose tissue fragments cut of around 1mm3. Digestive juice by adding collagenase, 37℃to digest 1 hour, 5 minutes each time oscillation. Through the aperture of 100um and 25um nylon sieve, centrifugal filtrate collected for 10 minutes (600g). Abandoned to the supernatant by adding red blood cell lysis buffer, Army uniform, standing at room temperature for 10 min- utes.Centrifuge again 5 minutes away to the supernatant by adding serum- free culture medium, Army uniform, that is, to repeat the former fat cells. Fat cells to digest the first passage to an area of 25cm2 culture at the end of the bottle, by adding complete culture medium (DMEM +10% FBS +100 U / ml of green, streptomycin), cells at 37℃incubator (5% CO2) in the training, culture medium replaced every two days time, the cultured cells to the number of experiments required.(3)Preadipocyte with a mixture of fibrin glue preparation. Culture bottle in good condition first growth period in the growth of the number of pre-fat cells of 0.25% trypsin digestion, centrifuga- tion, and then counting cells under a microscope plate count, and then with 10% fetal bovine serum and induced differentiation agent The DMEM culture medium into a density of 1×105/ml back-up of the single-cell suspension. Will be adding fibrinogen serum DMEM culture medium containing fully dissolved 75mg/ml fibrinogen preparation as a solution of thrombin containing 40mmol / L of dissolved calcium chloride solution dissolved to a final concentration of thrombin 100u/ml. 24-hole plate to each hole before the instillation of fat cell suspension 100ul, adding pre-heating to 37℃fibrinogen of 200ul, and then joined the thrombin solution 100u1, culture shock after mixing board Add 37℃incubator within a few minutes to see the formation of gel. Each hole to join the culture medium and then placed inside the incubator, to be implanted in rats A laminectomy defect.(4)Tissue engineering in vivo fat .1) Preparation of animal model of laminectomy defect and the transplanted fat tissue engi- neering of the SD rats in each group are 10% chloral hydrate intra- per- itoneal anesthesia, sterile surgical conditions. Check the back of the middle longitudinal incision, resection of spinous process and the L1-L3 lamine- ctomy, laminectomy 0.3cmx1.0cm caused by defects in the ligamentum flavum resection, removal of epidural fat, dura mater intact. Laminectomy defect model made. After complete hemostasis, can remove all to see under the microscope fiber soft tissue and bone-like film.Defects when implanted in the region covered with appropriate closure of incision, the laminectomy defect area periodically to observe the organization of the scar. 2) specimens to observe the project, testing methods and indicators.1) general observation: 4,8,12 weeks after the respective merits in three rats, origin- ally under the operating microscope in the surgical approach for anatomical observation. To the naked eye to see the improvements in accordance with the epidural scar Redell-Balazs [18] standards for the degree of adhesion score. (2) light microscope. Ibid based point in time, each of four animals each. Complete spinal surgery removed, placed in 10% of nitric acid - formalin fixed mixture solution 3d, then 50% of the formic acid decalcified at room temperature, and then the middle of specimens derived, conven- tional dehydration and paraffin-embedded, 5μm serial sections of each biopsy specimen 2, in which a line HE staining, light microscope observa- tion, according to improved standards Nussbaum [18] histological score line. VG staining a trip to observe the nature of scar collagen and their distribution. (3) electron microscopy at eight weeks under the microscope generally cut specimens in each group of epidural scar, about 1mm×1.5mm×2mm size fixed by glutaraldehyde and post-production ultra-thin sections under the transmission electron microscope to observe the ultrastructure.Result:Was successful from the rat isolated and cultivated before the fat cells, a large number of in vitro amplification and induced into mature fat cells. Observed under an optical microscope before the fat cells can be seen in the fibrin glue has a good adhesion, growth capacity. Purification of rat preadipocyte and fibrin glue composite implanted in rat laminectomy defect area 4 weeks after the epidural adipose tissue survival can be seen more, no epidural adhesions, 8,12 weeks after subdural survival is still visible adipose tissue, epidural adhesions light. A group of epidural scar adhesion less than the other group, B group of the second, D the largest group.Conclusion: Fibrin gel with the fat cells have a good biocompatibility and can be used as the carrier before the fat cells, fat is a viable tissue engineering scaffold material. Preadipocyte autologous fibrin glue made with a mixture of implants to prevent epidural scar formation and good adhesion of the effect of a preventive epidural space after laminectomy scar formation and adhesion in a good way .