The Construct And Application Of Tissue Engineering Nerve Conduit For Peripheral Nerve Injury

Chitosan is a kind of natural gather cationic biological polysaccharide. It is biodegradable and non-toxic. However, its application is restricted for its higher brittleness and poor mechanical property. PLA has better mechanical property. Meanwhile it has been approved by the U.S. food and drug administration (FDA) to be used as tissue engineering, drug release carrier and repairing material. Nevertheless, it has drawbacks such as slow degradation rate, local inflammatory response and hydrophobicity which limited its use. In our research, PCLA materials were successfully fabricated by grafting lactic acid onto the amino groups in CS by vacuum freezer drier and vacuum reaction. The poly(chitosan-g-lactic acid)(PLCA) material has their advantages and overcome their shortages.Electrostatic self-assembly (ESA) technology has drawn the attentions of the researchers for easily operating and controlling. It is based on the adsorption of opposite electric charge with polyelectrolyte. It has the characteristics of easily operate, good stability and controlled size and shape. Now it has been widely used in the surface modification of biomedical material, drug release, preparation of biological sensors, etc. In our research, electrostatic self-assembly technology was adapted to fixed chitosan and heparin into the surface of5/0surgical suture line in order to prepare nerve regeneration microfilament. What’s more, the1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was used to crosslink nerve growth factor to the surface of microfilament and conduit scaffold. Finally, Bone marrow mesenchymal stem cells (BMSCs) were used in the regeneration of peripheral nerve injury. As the development of transport and industrial mechanization, the incidence of peripheral nerve injury is rising every year. At present microsurgical techniques are used for surgical treatment in the clinical. However, autologous can’t meet the regeneration in more than5mm damage when nerve can’t direct suture. So it has been a difficult in the field of neurological surgery for many years. Using tissue engineering method to repair injury nerves is an effective way.The main issues in the field include:①construction and surface modification of nerve conduit material.②nerve conduit material combined with neurotrophic factor.③lect and add the suitable seeds cells. The subject work commenced in these areas, including:1The construction and study of poly(chitosan-g-lactic acid)The PCLA scaffold was obtained by grafting lactic acid (LA) onto amino groups on chitosan (CS) without a catalyst. The PCLA scaffolds were characterized by fourier transform infrared spectroscopy (FT-IR) and scanning electron microscope (SEM). The porosity, mass loss in vitro, pH value fluctuated during the in vitro incubation, degradation in vivo of PCLA scaffolds were investigated as a function of feed ratio of LA/CS. Bone marrow mesenchymal stem cells (BMSCs) culture was performed to evaluate cell proliferation on the copolymer scaffolds. Histological examination was done in vivo which indicated the PCLA copolymers have fine biocompatible property. The results showed that PCLA was a kind of biodegradable and biocompatible material which was promising for tissue engineering scaffold.2The construction and study of nerve engineering conduits2.1The construction of poly(chitosan-g-lactic acid) nerve conduitThe poly(chitosan-g-lactic acid) nerve conduit was made by the self-made mold, using of freeze-drying and high-temperature vacuum drying method. The mechanical properties and ultrastructure were determined in order to meet the need. The results showed that the PCLA nerve conduit can effectively improve the mechanical properties compared with the chitosan never conduit, what’s more the structure of the nerve conduit can effectively prevent the ingrowth of surrounding tissue and provide a good microenvironment.2.2The electrostatic self-assembly modification of microfilamentThe biodegradable5/0polyglycolic acid surgical suture line was coated with polylactic acid as base material of electrostatic self-assembly modification. The chitosan/heparin multilayer was prepared on the surface of microfilament, and detected by infrared spectroscopy, X-ray photoelectron spectroscopy and scanning electron microscopy. The results show that the chitosan/heparin multilayer was already located on the surface of the microfilament. It can effectively increase the internal surface area of the nerve conduit combined with the nerve conduit scaffold.2.3Fixation of NGFThe NGF was fixed on the nerve conduit and microfilament by the EDC in order to achieve sustained release of NGF during nerve repairment. The results show that, by the release of the pre-show there was a sudden release of NGF at the beginning of culture, as the time wnet by it showed a smooth release. The fixed amount of NGF on the nerve conduit was significantly more than the microfilament as a result of surface area.2.4Isolation and fluorescent labeling of BMSCs as stem cellThe BMSCs was isolated by commercially available kit as a method of density gradient centrifugation separation. The BMSCs was combined with nerve conduit to built tissue engineering nerve conduit, which was applied to the sciatic nerve injury repairment. The BMSCs was marked with Hoechst33258and detected by immunohistochemical methods. The results showed that the BMSCs still alive after6weeks of the surgery. What’s more, the expression of nestin indicated that the BMSCs have differentiated into neural cells.3The study of tissue engineering nerve conduit for peripheral nerve injuryIn this part, repairing effects of nerve injury are studied, including blank control group, autologous group, tissue engineering group, nerve growth factor group, BMSCs group, NGF fixed on micro filament group, ESA modify microfi lament group, Pyrroloquinoline quinone group, PCLA group, CS group. PLA group, injury group. The repairing effects were evaluated by the nerve electric physiology index, sciatic nerve function index (SFI) and histological sections. The regenerative effects were ranked as follows:autologous group, tissue engineering group, nerve growth factor group, BMSCs group, NGF fixed on microfilament group, ESA modify microfilament group, Pyrroloquinoline quinone group, PCLA-Ⅱ group, PCLA-Ⅰ group, PCLA-Ⅲ group CS group, PLA group. Fluorescence tracer and immunofluorescence assay revealed that BMSCs had differentiated to neural stem cell under the indicated of NGF, which lead to the expression of nestin.The main achievements obtained in our research are as follows:1The PCLA scaffold was obtained by grafting lactic acid (LA) onto the amino groups on chitosan (CS) without a catalyst. The physical and chemical properties, degradation both in vivo and in vitro, cell affinity and histological examination were performed to evaluate the PCLA, which can be used as guidance to choose appropriate biomaterials for tissue engineering.2. Electrostatic self-assembly technique was used to fix heparin/chitosan multilayer on the surface of surgical suture line and their physical and chemical properties were characterized. EDC was use as crosslink to fix nerve growth factor, in vitro test showed that its can slowly release nerve growth factor; the result of mechanical test showed that PCLA-Ⅱ group has the best mechanical properties, which can be applied to further repair of nerve injuries.3. The influence factors for rats in repairing of peripheral nerve injury were evaluated, and the regenerative effects were ranked as follows:autologous group, tissue engineering group, nerve growth factor group, BMSCs group, NGF fixed on microfilament group, ESA modified microfilament group, Pyrroloquinoline quinone group, PCLA-Ⅱ group, PCLA-Ⅰ group, PCLA-Ⅲ group CS group, PLA group. The results show that the role of each factor as follows:NGF group, BMSCs group, microfilament group, Pyrroloquinoline quinone group.