Preparation Of Crosslinked Chitosan And Its Applications In Osteoarthritis

Part Ⅰ Preparation and performance test of crosslinked chitosanObjective A new type of long-acting crosslinked chitosan was prepared by modifying carboxymethyl chitosan and its properties were studied.Methods The chitosan dry powder was dissolved and stirred evenly,and then lysine and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride(DMTMM)were added in turn.After mixing,the pH value of the reaction is adjusted with HCl solution,the obtained mixture is homogenized,and ethanol is added under stirring state to obtain white powder,which is dried in vacuum and dissolved in phosphate buffer,After full stirring,the pH value was adjusted with HCl solution to obtain transparent and uniform crosslinked chitosan gel.The properties of crosslinked chitosan were evaluated by testing the appearance,chitosan content,pH value,osmotic pressure,dynamic viscosity,modification ratio and Fourier transform infrared spectroscopy(FTIR)of crosslinked chitosan.Results The novel crosslinked chitosan gel prepared by the above steps is colorless and transparent colloid,The average content of chitosan was 12.32 mg/mL,pH value 6.8~7.6,osmotic pressure 270 mOsmol~350 mOsmol,dynamic viscosity(3000~5000)mPa·s,modification ratio(9±2)%.The variation curve of the dynamic viscosityηvalue of the sample with frequency was measured by a rotating rheometer.The results showed that carboxymethyl chitosan without lysine modification could not be sterilized at the end,but the critical gel could realize the terminal sterilization,and there was no change in dynamic viscosity before and after sterilization.Fourier transform infrared spectroscopy(FTIR)was used to detect the cross-linked chitosan and the carboxymethyl chitosan,and it was found that the critical gel of carboxymethyl chitosan modified by lysine occurred on the carboxyl group.The original structure of carboxymethyl chitosan was retained,and the inherent biocompatibility and biological activity of carboxymethyl chitosan were retained.Conclusion The new crosslinked chitosan hydrogels successfully prepared in this experiment not only retain the original biocompatibility and biological activity,but also have better viscoelasticity,solve the problem of terminal sterilization.Part Ⅱ Experimental study of crosslinked chitosan in the treatment of knee osteoarthritis in rabbitsObjective To investigate the effect of intraarticular injection of crosslinked chitosan in the treatment of knee osteoarthritis in rabbits to explore a new method for the treatment of osteoarthritis.Methods Thirty-two female New Zealand white rabbits weighing(2.5 ±0.5)g were randomly divided into 4 groups(groups A,B,C,and D;8 rabbits in each group).The knee osteoarthritis models were prepared by anterior cruciate ligament transection in the left hind in groups A,B,and C.At 4 weeks after operation,the rabbits were received intraarticular injection of 0.6 m L crosslinked-chitosan in group A,0.3 m L chitosan(once per 2 weeks)in group B,and 0.3 m L saline(once per 2 weeks)in group C.The rabbits in group D were treated with sham operation in the left hind.At 8 weeks,the macroscopic observation,histological examination(HE staining,Safranin-fast green double staining,and Mankin score),scanning electron microscopy(SEM)observation,and immunohistochemical staining of collagen type Ⅱ were performed.Results Macroscopic and SEM observations showed that the cartilage in group D was basically the same as normal and better than those in groups A and B,and the abrasion of cartilage in group C was the most serious.The histological observation results in groups A and B were slightly similar and better than that in group C,but not up to the structure of group D.The macroscopic score and Mankin score of groups B and C were significantly higher than that of group D(P<0.05),and there was no significant difference between groups A and B(P>0.05).Immunohistochemical staining results showed that the collagen type Ⅱ positive percentage of chondrocytes was significantly higher in group D than that in groups B and C,and no significant difference was shown between groups A and B(P>0.05).Conclusion The crosslinked chitosan can significantly improve the osteoarthritis of the rabbit knee,delay the pathological changes of osteoarthritis,and decrease the number of injection.