| Chitosan, composed ofβ-D- (1→4)-linked 2-amido-2-deoxy-glucose, is a natural polycation biological polysaccharide. Owing to their excellent biocompatibility and biodegradability, chitosan and its derivatives are widely used in the biomedical field. Artificial skin, artificial kidney film made of chitosan have been successfully developed. As repair materials of nerves, tendons, bones, teeth and other organs as well as scaffold materials in the development of a liver, kidney and other organs, chitosans are showing a wide range of applications. With the improvement of industrial mechanization and the development of transport, the incidence of peripheral nerve injury is increasing significantly, about two million cases per year in China. Currently, microsurgical technique is usually used to end-to-end suture the outer membrane injury of short-range peripheral nerve defect, but the break greater than 5mm can not be directly sutured. Furthermore, getting nerves from other parts to transplant will result in loss of new features, thus it has been a tough problem in the field of neurosurgery. The use of tissue engineering to repair will be an effective means. However, systematic research in the nerve conduit material selection, design and production of seed cells is needed. This is the focus of our project listed as follows.1 Study on the characteristics of chitosan as repair materials for peripheral nerve injury The degradability, hydrophilicity of chitosans with different molecular weight and degree of deacetylation, as well as their cellular affinity with Schwann cells and bone marrow mesenchymal stem cells have been investigated in vitro. The results indicaded that chitosans with molecular wieght of 50kD, 200kD, and deacetylation degree of 85% had faster degradation (50% and 40% degraded during a period of 6 weeks, respectively), worse hydrophilicity as well as lower cellular affinity. On the contrary, chitosans with molecular wieght of 500kD, 100kD, and deacetylation degree of 95% had slower degradation (30% and 20% degraded during a period of 6 weeks, respectively), better hydrophilicity as well as higher cell affinity, which is comparable to expensive poly-L-lysine. In order to meet the both requirements of degradation and cellular affinity, chitosan with molecular wieght of 1000kD, deacetylation degree of 95% was ultimately selected to act as the outer catheter material, while the molecular weight of 500kD, deacetylation degree of 95% was selected to as the inner catheter.2 Design and the characteristics of chitosan nerve conduitMolds of bilayer multi-channel and single-channel chitosan conduit have been designed, furthermore, the two conduits were made using selected chitosans. 45 parallel channels with diameter of 0.1mm are evenly arranged in the longitudinal nerve conduit. Among the conduit channels there were cross-cutting, anisotropic micropores with diameter of 5 - 50μm. The in vivo degradation time of such nerve conduits were up to 6-8 weeks, so they can meet the requirements of peripheral nerve repair, moreover, their mechanical properties ,swelling property also fully meet the requirements of transplantation and surgery.3 Study on chitosan nerve conduit and composite bone marrow-derived mesenchymal stem cells in repairing peripheral nerve injury in ratsRepairing effect of nerve injury of five groups are studied, including blank control group, single-channel chitosan conduit group, bilayer multi-channel chitosan conduit group, bilayer multi-channel chitosan conduit combined with bone marrow-derived mesenchymal stem cells (marked by nuclear dye Hoechst 33258) group and autograft group. According to sciatic functional index and the general observation, the regenerative effects were ranked as follows, autograft group, multi-channel composite bone marrow-derived mesenchymal stem cell group, multi-channel chitosan conduit group, single-channel chitosan conduit group, blank control group. From the results of HE staining and toluidine blue staining it can be seen that the groups of chitosan conduits had satisfactory histocompatibility. The density of the regenerated nerve fibers from high to low were: multi-channel composite bone marrow-derived mesenchymal stem cell group, multi-channel chitosan conduit group, autograft group, single-channel chitosan conduit group, blank control group. In the decending order of regenerated nerve fiber diameter , the groups are arranged as follows,: autograft group, multi-channel chitosan conduit composite bone marrow-derived mesenchymal stem cell group, multi-channel chitosan conduit group, single-channel chitosan conduit group, blank control group. All groups expressed nestin from the immunohistochemical analysis, the decending order of expressing levels was: blank control group, single-channel chitosan conduit group, multi-channel chitosan conduit group, multi-channel chitosan conduit composite bone marrow mesenchymal stem cells, autologous transplantation group. Fluorescent tracer and immunofluorescence assay revealed that some of bone marrow mesenchymal stem cells co-grafted with conduits expressed neistin indicating that they had differentiated to neural stem cell.The main achievements obtained in the study are as follows:1 The rules between the molecular weight, degree of deacetylation of chitosan to the degradation and cell affinity was dicovered, which provided the basis for the selection of a suitable chitosan for preparation of tissue engineering scaffolds or catheter. Two chitosan were chosed for preparation of conduits.2 Single-channel and multi-channel chitosan conduits were made. The latter showed d better ultrastructure as well as mechanical properties and could be further used for repairing nerve damage.3 The effect of chitosan conduit composite bone marrow mesenchymal stem cells used to repair peripheral nerve injury in rats was evaluated. The result showed the existence of chitosan conduit helped to prevent non-nerve tissue invasion. Multi-channel conduit, due to greater specific surface area could support the regeneration of nerve fibers better. Bone marrow-derived mesenchymal stem cell,when co-grafted with the nerve conduit they differentiated to neural stem cells in the injury microenvironment All of above contributes to the excellent regenerative effect of multi-channel composite bone marrow-derived mesenchymal stem cell group. |
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