| Objective:With the rapid development of bone tissue engineering, scaffold and guided tissue regeneration (GTR) membrane was constructed to assist the regeneration of new bone. As the raw material has a wealth resource and the complication is rare, the use of scaffold and GTR membrane will replace the traditional bone repair method. In general, the commercial GTR membrane can be divided into nondegradable membrane and degradable membrane. The nonderadable membrane requires a second operation to take it out, which bring great inconvenience to people. The degraded membrane has the problem that its degradation rate couldn’t be well regulated and it may be collapsed due to the lack of mechanical strength. As carboxymethyl chitosan existed in the aqueous solution in a form of ampholyte, the formation of different space conformation endowed it with some special biological activities. Compared with chitosan, carboxymethyl chitosan processes much good solubility in water and biocampability. A good antibacterial property has been reported recently, which all suggest that carboxymethyl chitosan may be a promising material for GTR membrane. Due to the good water solubility and the quick degradation rate, we aimed to find a sufficient method to crosslink the carboxymethyl chitosan, and evaluate the GTR effect of the membrane.Method:The whole marrow method was used to culture the bone marrow mesenchymal stem cells (BMSCs); MTT assay was used to detect the stimulation effect of carboxymethyl chitosan, sulfated chitosan, chondroitin sulfate and N-acetyl glucosamine on BMSCs; MTT assay was used to detect the sufficient propotion of Nano-HA added into the membrane, and then, phase invasion method was used to construct an asymmetry membrane. The water absorption quality, swelling property, porosity, tensile strength, enlongation at break and the biocompability was detected. By implanting the membrane into the skin and muscle of rats, we evaluated the degradation property and the histocompatibility of the membrane. The skull defect model of the rat was established to evaluate the function of the GTR membrane. The rat was sacrificed at 4,8, and 12 weeks after the operation, and the tissue in the defect area was taken, HE staining and masson staining was performed after decalcification and fixation.Result:BMSCs can be purified by whole marrow method, and the MTT assay proved that the carboxymethyl chitosan and the chondroitin sulfate can promote the prolification of BMSCs greatly; after assured that the sufficient ratio of Nano-HA is 10%, we constructed an asymmetry membrane with phase inversion method and then crosslinked the membrane with CaCl2 and Butyl glycol diglycidyl ether. The water absorption rate was 360.69%, swelling rate was 174.41%, tensile strength was 0.640MPa, enlongation at break was 54.424% and the cytotoxicity was 1 grade, and the cell can adhesion, extend and prolification on the membrane well. The in vivo degradation experiment showed that the membrane can keep its integrity in vivo for at least 8 weeks and there was slight inflammatory response occurred at the first and second week, and it vanished from the 4th week. The bone defect on the skull of rat showed that there was certain new bone regenerated at the 4th week in the experiment group, the new bone occupied the whole defect area at the 8th week, and the defect was almost fixed in the 12th week; in the control group, there was only a small amount of new bone generated in the 12th week.Conclusion:Carboxymethyl chitosan and the chondroitin sulfate can promote the prolification of BMSCs greatly; the membrane we constructed in the study has good biocompability and biodegradability, it can inhibit the invasion of the fibrous connective tissue into the defect area and promote the repair of the bone defect. |
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