Preparation Of Carboxymethyl Chitosan Thermosensitive Hydrogel And Study On Its Biocompatibility

BackgroundPeriodontal disease is one of the most common diseases in human oral cavity, and has comparatively high prevalence in the world. The final purpose of curing periodontal disease is to fulfill the periodontal regeneration and form periodontal new attachment. For many years, people are seeking the method to increase periodontal new attachment effectively and accelerate the periodontal regeneration. In order to get this goales people has made periodontal bone transplantation, growth factor application and guide periodontal tissue regeneration curing method. Although the researches have achieved certain development, these can still hardly fulfill the complete periodontal tissue regeneration. In recent years research on tissue engineer using hydrogel biomaterials has developed rapidly. It is proved by many researches that chitosan hydrogel is a kind of biomaterial with good biocompatibility and degradability in vivo and bears universal application prospect as the support of periodontal tissue engineer cell. Carboxymethyl Chitosan (CMCS) is water-soluble polysaccharide turned from chitosan by carboxy methylation. Compared with chitosan, carboxymethyl chitosan is of higher water-solubility, nontoxic, antigenic free, degradable and good biocompatibility, and it is used widely in medicine and health food. There are many reports about the research on chitosan temperature sensitive gel in recent years, but research focusing on carboxymethyl temperature sensitive gel is rather few.Objective Choosing various influencing factor to optimize formula composition while self make carboxymethyl chitosan temperature sensitive gel, and studying its physicochemical property and its toxicity on mice's lung fibroblasts L929, moreover, probe into its tissue compatibility through animal experiments which can provide the fundamental experiment proof for further clinical application.MethodsThe research can be divided into two parts1 Preparation of carboxymethyl chitosan temperature sensitive gel The CMCS disinfected withγRay and weigh it certain amount, then put it into 45ml hydrochloride solution, and dissolute for 2h in temperature(23±1)℃. Weigh certain amount of GP to dissolute it into ultra pure water, and add them slowly into Carboxymethyl Chitosan filtrate with continuous mixing, then the clear and transparent complex solution comes into being. Continue mix it even for 20mix for back up. Take the test tube with CMCS/GP complex solution into water bath with temperature 30℃～70℃until it reaches the initial gelation temperature(IGT), then keep the same temperature for a while to get hydrogel. Through adjust hydrochloric acid concentration, substitution degree of carboxymethyl, GP concentration and carboxymethyl concentration research on their effect toward carboxymethyl chitosan temperature sensitive gelcan be achieved, so as to optimize the formula composition and make the medical material which can be gelatificated under the body temperature.2 Evaluate the biocompatibility of carboxymethyl chitosan temperature sensitive gel2.1 Effect of cell toxicity of carboxymethyl chitosan temperature sensitive gel on mice lung fibroblasts L929Adopt MTT method to measure the cell toxicity that carboxymethyl chitosan temperature sensitive gels of different concentration produced on L929 cell cultured in vitro. Measure the OD value after 24h, 48h, 72h, and 96h and calculate RGR, then calculate the cell toxicity grade.2.2 The subcutaneous implantation of the mouseCarrying out regular disinfection, paving the table, and cuting a wound with 1cm long on the outside skin of mice's leg, and transplant carboxymethyl chitosan temperature sensitive gel 0.5ml in its subcutaneous tissue, while no transplantation is carried out on the blank comparison mice. Then suture fascia and skin. Observe the appearance difference of each experiment mice group on its muscle tissue surrounding where gel were injected, and kill them separately on the third, 7th and 14th day. Remove the transplantation part together with the surrounding tissue, and put them into formalin to be fixed, and make regular tissue slice, then HE dye and observe the inflammatory response on the surrounding tissue of gel, finally evaluate the tissue biocompatibility and degradability of the gel of each group.Results1 The hydrogel which we prepared has many characteristics, it is a soft, moist and with the ambient temperature changes can occur reversibly. After formula composition is optimized by the orthogonal experiment. When the degree of substitution(DS) of CMCS is 0.3, the concentration of CMCS is 5.0%, the concentration of HCL is 0.1 mol/m3, the concentration of GP is 6.3%, IGT is 37℃which is the closest person oral temperature.2 The biocompatibility of the Carboxymethyl chitosan thermosensitive hydrogel.2.1 To evaluate the the cytotoxicity effects of the Carboxymethyl chitosan thermosensitive hydrogel on mouse lung fibroblasts(L929).2.1.1 After dosing 24h, it is statistical difference between 0.4 times group and other groups, 0.08 times group and other groups, P<0.05; it is no statistical difference between the other experimental groups, P>0.05. 2.1.2 After dosing 48h, it is no statistical difference between 50 times group and negative group, 50 times group and positive group, P>0.05; it is statistical difference between the other experimental groups and negative group, P<0.05.2.1.3 After dosing 72h, it is no statistical difference between 50 times group and negative group, P>0.05; it is statistical difference between 50 times group and positive group, negative group and positive group. P<0.05; it is statistical difference between the other experimental groups and negative group, P<0.05.2.1.4 After dosing 96h, it is statistical difference between 50 times group and negative group, P<0.05; it is statistical difference between 50 times group and positive group, negative group and positive group, P<0.05; it is statistical difference between the other experimental groups and negative group, P<0.05.2.2 The subcutaneous implantation of the mouseThe research histology shows that, on the 3dth after histology injecting, the experimental group is mainly in the acute inflammation, the Large accumulation of neutrophils, lymphocytes is more, and see macrophages and capillary congestion; on the 7dth after histology injecting, the experimental group had a small amount of wavy collagen fibers, capillaries decreased, vascular congestion is decreased, the number of inflammatory cells are significantly reduced, newborn capillaries are not found; on the 14dth after histology injecting, The Inflammatory cells disappeared in the experimental group, it descriptions that carboxymethyl thermosensitive hydrogels have good biocompatibility.Conclusion1 Studying on preparation technique of carboxymethyl chitosan, it was prepared by a substance that is liquid at room temperature, it will become gelatination under the body temperature environment and it have thermosensitive characteristic medical material.2 We have carried on the preliminary appraisal for biocompatibility of Carboxymethyl chitosan thermosensitive hydrogel, and proved that it has no toxicity to L929 cells, in which 0.4 times leaching liquor have the cell-multiplication function obviously. After the experiment of Hypodermic buries plants to mouse for 14 days, we can not see the obvious difference between inflammatory cells with compare group, which can proved Carboxymethyl chitosan thermosensitive hydrogel have well biocompatibility.