Study On Repair Of Spinal Cord Injury By Using Tissue Engineering Method

Repair of spinal cord injury (SCI) is a difficult challenge in clinical medicine as well as a hot field of neuroscience research. According to the fundamental principles of tissue engineering, this thesis is focused on dealing with the basic problems in repair of SCI by studying the promising seed cells and scaffold biomaterials. Following are the main contents and results of the thesis:(1) Olfactory ensheathing cells (OECs) were isolated from adult rat's olfactory bulbs and highly purified OECs were obtained after purification. Immunocytochemical staining of purified OECs showed positive immunoreactivity to S-100β. Neural stem cells (NSCs) were isolated from the cortices of fetal rats on days 12 of embryonic development. Immunocytochemical staining results indicated that regardless of the form (single cells or neurospheres) NSCs showed positive immunoreactivity to nestin, an acknowledged protein marker for NSCs. The majority of NSCs differentiated into astrocytes when cultured alone in DMEM/F12 medium with 10% fetal bovine serum (FBS), however, co-culture NSCs with OECs demonstrated that OECs could promote NSCs to differentiate into neurons.(2) The SCI model was established by a right lateral 3/4 spinal cord incision at the T9 level in the adult rat. 7 days after SCI, four groups of rats were injected with DMEM/F12 medium, NSCs suspension, OECs suspension, and NSCs + OECs suspension respectively by a microinjector. Once every week after cell transplantation, the Basso-Beattie-Bresnahan (BBB) test was carried out in an open field to evaluate the recovery of locomotor function after SCI. The average BBB scale score of the co-transplantation group was significantly higher than that of the other three groups. Immunohistochemical staining and histological observation both showed new nerve fibers across the injured region. Co-transplantation of NSCs and OECs might have a synergistic effect on promoting neural regeneration and improving the recovery of locomotion function. Co-transplantation of NSCs and OECs was better than a single graft either by NSCs or OECs.(3) Chitosan films (Chi-F), chitosan porous scaffolds (Chi-PS), and chitosan multimicrotubule conduits (Chi-MC) were fabricated to investigate their effects on the differentiation and proliferation of NSCs. In the presence of 10% FBS, most NSCs cultured on Chi-F differentiated into astrocytes, NSCs cultured on Chi-MC showed a significant increase in neuronal differentiation, while Chi-PS somewhat promoted NSCs to differentiate into neurons. However, in serum free medium with 20 ng/mL basic fibroblast growth factor (bFGF), NSCs cultured on Chi-F showed the best proliferation behavior, and NSCs cultured on Chi-MC showed a moderate cell proliferation, but NSCs cultured on Chi-PS exhibited the least cell proliferation behavior. These observations indicated that chitosan topology structure could play an important role in regulating differentiation and proliferation of NSCs.(4) Chitosan-alginate hydrogel was fabricated as a carrier of OECs and NSCs. The Fourier-transformed infrared spectroscopy and X-ray diffraction results demonstrated that chitosan-alginate hydrogel was constructed due to the strong ionic interaction between the positively charged amino groups of chitosan and the negatively charged carboxyl groups of alginate. As two most promising cell types in nerve tissue engineering, both olfactory ensheathing cells and neural stem cells proliferated well on chitosan-alginate hydrogel compared with those on three other gels widely used in tissue engineering including nerve tissue engineering. All results indicated the good potential application of chitosan-alginate hydrogel for neural tissue engineering.