Experimental Study Of Prevascularized Tissue Engineering Artificial Nerve

An ideal tissue engineering artificial nerve is an organic unity including Seed cells, biomaterial, extracellular matrix and the nerve growth factors In our earlier arlierstage work, we had already investigated Schwann cell culture and successfully achieved a large number of highly purified Schwann cells. After that we obtained an ideal nerve scaffold by improving the method of chemically extracted acellular allograft nerve bridging. The followhing research in vitro about the histocompatibility of Schwann cell and the nerve scaffold showed SCs came from fetus rabbit can grow well in the extracted never graft in which they can form cables and migrate along the extracellular matrix in vitro. On the basis of previous studies, we are going to do further research to prevascularize tissue engineering artificial nerve.So, we are going to do the research as follows:1. Plant fetus rabbit Scs into the extracted acellula nerve of homogeneity and made into’a nerve bridging-complex and to repair defective sciatic nerve with the complex.2. Investigate the optimal ratio of Schwann cells and vascular endothelial cells in co-culture system in vitro.3. Explore the feasibility of chemically extracted acellular allograft nerve complex after prevascularization for the repair of long-distance sciatic nerve defect in rabbits and effects of vascularized graft in early stage on the recovery of neurological function.Part 1 The Experimental Study of Repairing Nerve Defect by the Complex of Schwann Cells from Fetus RabbitObjective:The aim of this part is to plant fetus rabbit Scs into the extracted acellula nerve of homogeneity and made into a nerve bridging-complex and to repair defective sciatic nerve with the complex.Methods:This experiment includes following steps:1 Preparation of the Scs 2 Preparation of Extracted Nerve Grafts (eNG) 3 Animal experiment. Enzyme digestion (trypsin/collagenase) was applied to culture Scs. The Scs will be micro-injected into the eNG(treated by 3% Triton X-100 totally for 96hs). Experimental group was transplanted nerve to repair the rabbit defective sciatic nerve. The control group was transplanted extracted nerve graft of homogeneity (without the Schwann cells cultured from fetus rabbit). we observed the changes of the ulcers on the rabbits’feet, detected the regeneration of axons of rabbit sciatic nerves with light microscope, and measured NCV by EMG on 4,8 and 12 weeks after operation.Results:At the fourth, eighth and twelfth week after operation, the rejection was not found in the operating field of two groups’animals. The animals in experimental group were better than the control group in healing of ulcers with more nerve fibers. Fourth week after operation, the nerve conduction velocity (NCV) were not different between the two groups. Eighth week after operation, the NCV were 24.80±1.27 in experimental group, 18.72±0.81 in the control group(T=-10.91, P<0.05); and twelfth week after operation NCV was 37.95±2.43 in the experimental group and 32.58±1.89 in the control group (T=-5.01, P<0.05). There was significant difference between two groups. The number of nerve fibers:Fourth week after operation, the number of nerve fibers were 750.65±74.24 in experimental group, 543.67±61.42 in the control group(T=-5.30, P<0.05). Eighth week after operation,2069.83±231.65 in experimental group,1804.50±156.65 in the control group(T=-2.2, P<0.05). Twelfth week after operation,3434.33±124.48 in experimental group,2495.17±154.95 in the control group(T=-11.25, P<0.05). There was significant difference between two groups by statistic analysis.Conclusions:The experiment confirmed that the nerve bridging-complex not only could offer more favorable supporting effects for regeneration of axons but also could induce and promote the regeneration of neuraxises more effectively.Part 2 Study on the Co-culture of Schwann Cells and Vascular Endothelial Cells in VitroObjective:The aim of this study was to investigate the optimal ratio of Schwann cells and vascular endothelial cells in co-culture system in vitro.Methods:Culture Schwann cells and vascular endothelial cells respectively using enzymatic digestion method and then put them together with the ratio of 1:1(group A),2:1 (group B) and 4:1 (group C) in a co-culture system in vitro. Schwann cells were cultured solely in the control group (group D).Count the Schwann cells and draw its growth curve at the first, third, 5th,7th day respectively.Results:The number of Schwann cells in each group increased with culture time. At the first day, there was no significant difference in cell number between experimental groups and control group (P>0.05).Significant difference were showed from the third day between group A and D as well as group C and D (P<0.05). But no significant difference appeared between group B and D (P>0.05).When comparing in experimental groups, the number of Schwann cells in group A, group C were significantly less than group B (P<0.05).Conclusions:Schwann cells can culture with vascular endothelial cells in co-culture system in vitro and vascular endothelial cells can promote the proliferation of Schwann cells when their ratio is 1:2 in co-culture system.Part 2 Study on the Co-culture of Schwann Cells and Vascular Endothelial Cells in VitroObjective:The aim of this study was to investigate the optimal ratio of Schwann cells and vascular endothelial cells in co-culture system in vitro.Methods:Culture Schwann cells and vascular endothelial cells respectively using enzymatic digestion method and then put them together with the ratio of 1:1(group A),2:1 (group B) and 4:1 (group C) in a co-culture system in vitro. Schwann cells were cultured solely in the control group (group D).Count the Schwann cells and draw its growth curve at the first, third, 5th,7th day respectively.Results:The number of Schwann cells in each group increased with culture time. At the first day, there was no significant difference in cell number between experimental groups and control group (P>0.05).Significant difference were showed from the third day between group A and D as well as group C and D (P<0.05). But no significant difference appeared between group B and D (P>0.05).When comparing in experimental groups, the number of Schwann cells in group A, group C were significantly less than group B (P<0.05).Conclusions:Schwann cells can culture with vascular endothelial cells in co-culture system in vitro and vascular endothelial cells can promote the proliferation of Schwann cells when their ratio is 1:2 in co-culture system.Part 3 Experimental Research on Repairing of Sciatic Nerve Gap in Rabbits with Revascularized Allogenenous Neural ComplexObjective:The aim of this study was to explore the feasibility of chemically extracted acellular allograft nerve complex after prevascularization for the repair of long-distance sciatic nerve defect in rabbits and effects of vascularized graft in early stage on the recovery of neurological function.Methods:Vascular endothelial cells and Schwann cells were cultured together and transplanted into the chemically extracted acellular allogeneous nerve graft. The pre-revascularized nerve grafts were used to repair the rabbits’defective sciatic nerve in experiment group. Acellular allogeneous nerve grafts were transplanted Schwann cells only in control group.The electrophysiological studies were carried out at 4th,8th,16th week respectively. After that, the allogenous neural were cut down. The light microscope and transmission electron microscope were performed to observe the myelin formation and the sciatic nerve regeneration in every group. Image analysis system was used to do quantitative analysis on the number of regenerated nerve fibers, axon diameter and the myelin sheath thickness.Results:1 After operation, neither the whold body nor the operating field showed reject reaction. The experimental group was better than the control group in healing of ulcers.2 Recovery of nerve conduction velocity at the 8th week after operation were respectively 23.35±1.05 m/s in experimental group and 23.11±0.80 m/s in control group. There was significant different in the two groups(T 2.13, P<0.05). Recovery of nerve conduction velocity at the 12th week after operation were respectively 39.52±1.0m/s in experimental group and 38.34±0.15 m/s in control group. It also had significant difference in the two groups (T 2.14, P<0.05).3 The wet weight of tibialis anterior at the 4th,8th and 12th week after operation were respectively 2.22±0.13g,3.38±0.11 g and 6.10±0.22 g in experimental group and 2.20±0.16g,3.28±0.07g and 5.97±0.15g in control group. There was no significant different in two groups (T 0.04, P>0.05) at the 4th week after operation. But significant different was showed at the 8th,12th week after operation(P<0.05).4 The number of nerve fibers at the 4th,8th and 12th week after operation were respectively 922.97±25.06,1736.44±36.28 and 2840.91±20.93 in experimental group and 901.48±12.39,1713.51±25.00 and 2823.74±14.42 in control group. There was significant different in two groups (P<0.05) at each time.5 The axon diameter at the 4th,8th and 12th week after operation were respectively 5.83±0.16μm,6.01±0.09μm and 6.11±0.08μm in experimental group and 5.58±0.42μm,5.87±0.16μm and 6.00±0.07μm in control group. There was significant different in two groups (P<0.05) at each time.6 The thickness of myelin sheath of medullated nerve fibers at the 4th,8th and 12th week after operation were respectively 1.95±0.14μm,1.99±0.11μm and 2.03±0.05μm in experimental group and 1.70±0.22μm,1.85±0.16μm and 1.90±0.06μm in control group. There was significant different in two groups (P<0.05) at each time.Conclusion:Pre-revascularized allogenous neural complex could significantly promote the functional recovery of injured nerve in repairing rabbit sciatic nerve defect.