The Study On The Application Of Chitosan In Peripheral Nerve Regeneration

Using a biodegradable conduit to bridge nerve gap is a promising method for peripheral nerve regeneration. Chitosan is an ideal biomaterial for preparing nerve conduits. This thesis is focused on the application of chitosan in peripheral nerve repair. The main contents of the thesis are as follows: (1) Expansion of adult Schwann cells from rat peripheral nerves. An effective technique was presented for isolation and expansion of Schwann cells. Schwann cells were isolated from the predegenerated sciatic nerves and seeded on the flasks coated with laminin. During the cell culture, the nerve fragments were reserved and the endogenous mechanisms of Schwann cell expansion were exploited. By the method a large number of Schwann cells can be obtained from small amounts of adult nerve fragments within a short period of time. (2) Effects of the degree of deacetylation (DD) on the properties of chitosan. Six kinds of chitosan samples with similar molecular weight but different DD were prepared from one stock chitosan material and fabricated into films. Their swelling and mechanical properties were measured and for the first time Schwann cells were used to evaluate the nerve cells affinity of chitosan. The results suggested that chitosan with DD higher than 90% could be considered as a promising material for application in peripheral nerve regeneration. (3) Regulation on the swelling, degradation and mechanical properties of chitosan. Three kinds of crosslinked chitosan films were prepared with hexamethylene diisocyanate (HDI), epichlorohydrin (ECH) and glutaraldehyde (GA) as crosslinking agents, respectively. The physical and mechanical properties, biodegradability and Schwann cell affinity of the crosslinked films were investigated. A significant decrease in the degradation rate in lysozyme solution and a large change in the mechanical properties were observed compared with uncrosslinked chitosan films. However, ECH and GA crosslinked films showed poor Schwann cells affinity while the HDI crosslinked chitosan films enhanced the spread and proliferation of Schwann cells. Therefore, crosslinking chitosan with HDI is a promising method to decrease the degradation rate of chitosan. In addition, poly (3-hydroxybutyrate) (PHB) was used to modify chitosan by an emulsion blending technique for the first time. With the incorporation of PHB, the swelling property of chitosan decreased while the tensile strength, the breaking elongation and the cytocompatibility increased. Furthermore, these properties could also be regulated by the selecting of the composite ratio of chitosan and PHB. (4) Manufacture and sterilization of chitosan conduits. For the first time the key technologies in the manufacture of chitosan conduits by freeze-drying were further studied from the viewpoint of engineering science. And an effective technique was obtained, by which chitosan conduits with the controllable inner diameter, the controllable wall thickness and the length of more than 15 cm could be prepared. Subsequently chitosan conduits were sterilized with 60Co γray radiation at a dose of 25 kGy and the influence of sterilization radiation on their properties was explored. After γray irradiation, the swelling index of chitosan conduits and the degradation rate in lysozyme solution was considerably reduced while the tensile strength and elastic modulus were markedly increased. These properties changes benefit the application of chitosan conduit in peripheral nerve repair and, consequently, γray radiation sterilization is suitable for chitosan conduits.