An Experimental Study On Repairing Nerve Defects With Tissue Engineering Bioartificial Nerve

[Objective] With the development of microsurgery, though the repair following injury of peripheral nerve has achieved a great progress in the recent years, the results of the repair are still not satisfied. Even after severe nerve injuries, resection of tumor or congenital deformity,the loss of nerve substance resulting in a significant gap between the nerve stamps could not be repaired by direct epineural suture. The regeneration and function recovery of peripheral nerve injury are focus issues in the field of neuroscience. The preferred approach to repair peripheral nerve defects is to bridge the two ends of the injuried nerve with a segment of autologous nerve graft in clinic; as a result, this approach incurs some donor site sense dysfunction and usually involves a prolonged operation time and so on. The main drawback of this approach is insufficient functional outcome after nerve repair. Explored by numerous researchers, up to now, many materials including bio-material, nature polymer and synthetic polymer were used to substitute for the auto nerve grafts. Theses materails were made into conduits to repair the peripheral nerve defects and it has been proved that these materials could improve the regeneration of peripheral nerve. While the repairing result is still not very satisfied. With the development of tissue engineering, in order to diminish the patients' suffering of losing autologus tissue and multi-operation and to improve the rate of function recover, it is important to find a more effective substitute of autologous nerve. Highpolymer such as PDLLA and PLGA have been used to make into tissue engineering bio-artificail nerve.but these bio-artificial nerves lacked of active cells and extra cellular matrix and nerve nutrient factors, and they cold not mimic the contracture of nerve which would influence the nerve regeneration. The objective in this study include:(l)To investigate the cultural methods of a large amount of purified Schwann cell of nonfatal SD rats.(2) To investigate the growth and migration behavior of Schwann cell during co-culture of Schwann cells with the PLGA microfilaments and with the inner sides of PDLLA nerve conduits. To study the promote proliferation effect and validity of the delayed release microspheres incorporating bFGF on Schwann cells.(3) Schwann cells seeded into 30%ECM/DMEM gel at 1X 107/ml and delayed release microspheres incorporating bFGF injected into the PDLLA permeable nerve conduits with 480 PLGA microfilaments inside to construct tissue engineering bio-artificial nerve. To bridge 15mm sciatic nerve defects of rats with the tissue engineering bio-artificial nerve ,then to study the effect of the tissue engineering bio-artificial nerve on peripheral nerve repair, and to analyze the interaction of Schwann cells, PLGA microfilaments and delayed release microspheres incorporating bFGF on peripheral nerve regeneration. [Methods] (1) Schwann cell culture were established from the sciatic nerves and brchial plexus nerves of 3-5d neonatal SD rats with tissue explant-reexplantation technique and repeated enxymolysis technique. The growth of Schwann cell was observed under the phase contrast microscope. (2) To purify the Schwann cells, different ways were used, such as the different adhesion time technique, the repeated different adhesion time technique combined with lOOmg/L G418 culture medium working for 3 daysor 9days.Then to observe the purity of the cultured cells which were stained with S-100 protein with immunochemical method. (3) To observe passage purification of Schwann cells controlled digestion under the phase contrast microscope, 0.125% trypsinase or 0.25% trypsinase were used. When Schwann cells were passaged, the cells was digested by the 0.125% trypsinase under the phase contrast microscope or directly digested by 0.25 trypsinase. Then to observe the purity of the cultured cells which stained with S-100 protein with immunochemical method. (4) Schwann cells seeded into 30%ECM/DMEM gel at lxlO7/lml or Schwann cell suspension at lx 107/lml were implanted into 48 hole plates with PLGA microfilaments, to observe the Schwann cell growth or migration on the microfilaments under the phase contrast microscope for 10 days. After 10-day co-culture, the microfilaments were observed under the scan electro microscope (SEM). (5) Schwann cells seeded into 30%ECM/E)MEM gel at 1X 107/lml or Schwann cell suspension atl x 107/lml were implanted into 48 hole plates with PDLLA nerve conduits for co-culture for 10 days, then to observe the Schwann cell growed on the inside of conduits under the SEM. (6)Secondary purified Schwann cells at 1X 104/ml were seeded in the 96-well culture plates and divided into three groups according to the different ingredients being added to the DMEM culture mediun: blank control group, free bFGF group and delayed release microsphere incorporating bFGF group. After 1,3,5,7,9 culture days, the proliferative cells were collected from there groups individually. Then the number of Schwann cells was measured with cell counting method,the viability of Schwann cells were measured with MTT method .(7) Schwann cells seeded into 30%ECM/DMEM gel at 1X 107/mland delayed release microspheres incorporating bFGF(contain bFGF 2.5/ig/ml) were injected into the PDLLA permeable nerve conduits with 480 PLGA microfilaments inside to construct tissue engineering bio-artificial nerve, which to bridge 15mm sciatic nerve defects of rats with these tissue engineering bio-artificial nerve ,then to study the effect of tissue engineering bio-artificial nerve on peripheral nerve repair (E group). The blank PDLLA conduits group (B group), PDLLA conduits inside with PLGA microfilaments (C group), PDLLA conduits inside with Schwann cell/ECM gel and PLGA microfilaments (D group). 4,8,12,16,20,24 weeks after operation, to analysis the gaits of the experimental animals and determine the sciatic nerve function index (SFI). 12 and 24 weeks after operation evaluated the status of regeneration with general observation, gastrocnemius muscle measurement, electro-physiological examination, histological observation and transmission electron microscope (TEM) observation. The regenerative nerve area, total myelinated fibers, density of reinnervation and thickness of myelin sheath were analysed by Mias-1000 image analysis system. [Results] (1) By using the double-enzyme repeated enrymolysis technique, the nerve tissues could be used more effectively with little influence on adhesion and growth of Schwann cells. By using the different adhesion time technique, most of the fibroblast cells could be removed. A large amount of purified Schwann cells could be harvested by using tissue explant-reexplantation technique without using antimitogenic drugs (G418), but this culture technique need more time.(2) To purify the Schwann cells, the repeated adhesion time technique combined with lOOmg/L Geneticin culture medium work for 3 days. The purity of the cultured cells stained with S-100protein with immunochemical method achieved 90%. (3) When Schwann cells were passaged, the cells was digested by the 0.125% trypsinase under the phase contrast microscope. The purity of the cultured cells could be improved further.(4)When Schwann cells were seeded into 30% ECM/DMEM gel and co-cultured with PLGA microfilaments, with the formation of gel ,most of the Schwarm cells adhered on the filaments to form Btingner band-like structures of Schwarm cells columns.When simple Schwann cells suspension was co-cultured with PLGA microfilaments and a little of cells were adhered and grown on PLGA microfilamets, most of the cells were grown on the bottom of the culture plates. (5) When Schwann cells were seeded into 30% ECM/DMEM suspension and co-cultured with inner side of PDLLA conduits, most of the Schwarm cells adhered on inside surface of PDLLA conduits and to form Biingner band-like structures of Schwarm cells columns. When simple Schwann cells suspension was co-cultured with inside surface of PDLLA conduits and a little of cells were adhered and grown on the surface. (6) The in vitro cellular study showed: at 3 days after plate culture, the cell number and viability of free bFGF group were much better than delayed release microsphere group and blank control group, and the microsphere group is better than the blank group; at 5 days after plate culture, the cell number and viability of free bFGF group and microsphere group were much better than blank control group, and there is no difference between the microsphere group and free bFGF group; at 7^ 9 days after plate culture, the cell number and viability of microsphere group was much better than free bFGF group and blank control group, and free bFGF group was much better than blank control group. (7) At 12 and 24 weeks afteroperation, the results of SFI, electrophysiological examination, gastrocnemius muscle measurement, histological observation, transmission electron microscope (TEM) observation showed: the repair effect of E group (Schwann cells seeded into 30%ECM/DMEM gel at 1X 107/ml and delayed release microspheres incorporating bFGF (contain bFGF 2.5/Ltg/ml) were injected into the PDLLA permeable nerve conduits with 480 PLGA microfilaments inside) was approaching to that of the nerve autograft, and better than the other groups. The repair effect of D group was better than than that of C group. The repair effect of B group is worst. [Conclusions] (1) Combined using of the methods of mechanical disssection, repeated enzymolysis technique, different adhesion time technique, antimitogenic drugs G418 could harvest a large amount of purified Schwann cells in a short time to fulfill the requirement of constructing tissue engineering binartificial nerve. 2) ECM gel could hold most of the Schwann cells to adhere on PLGA microfilaments. The Schwann cells on microfilaments could grow in three dimensions to form Biingner band-like structures of Schwann cell columns when co-cultured with PLGA microfilaments. The ECM gel was a good integrating substance for constructing tissue engineering bioartificial nerve. (3) Free bFGF can promote the proliferation of Schwann cell, but the effective duration is very short. Delayed release microspheres incorporating bFGF can sustain release active bFGF, and can promote the proliferation of Schwann cells in a long period. (4) We developed a tissue engineering bioartificial nerve which was constructed with Sehwann cells /ECM gel and delayed release microspheres incorporating bFGF (contain bFGF 2.5/ig/ml) injected into the PDLLApermeable nerve conduits with 480 PLGA microfilaments inside. This tissue engineering bioartifscial nerve could promote nerve regeneration and the effect was approaching to that of the nerve autograft when used to repair 15mm length defect of rat sciatic nerve. And the Schwann cells/ECM gel, PLGA microfilaments and delayed release microspheres incorporating bFGF have synergistic effects in peripheral nerve regeneration.