Effect Of EDC And Genipin Cross-linking On Chitosan/Calcium Silicate/Silk Fibroin Scaffolds

PurposeArticular cartilage injury caused by infection or trauma is common in clinical diseases,which seriously reduces the quality of patients’ life.However,once the articular cartilage is damaged,it is difficult to repair the damages on its own due to the lack of nerves,blood vessels and other structures.Therefore,it is an effective solution to design an implant material that meets the requirements of articular cartilage tissue engineering according to the principle and technology of articular cartilage tissue engineering.Previous studies have shown that chitosan / calcium silicate / silk fibroin composite scaffolds can meet the requirements of articular cartilage tissue engineering,but due to the low elastic modulus,the clinical application of these scaffolds is limited.If the elastic modulus can be improved,the composite scaffolds will be better used in articular cartilage tissue engineering.N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride(EDC)and genipin are both cross-linking agents with good biocompatibility,therefore,in this experiment,EDC and 0.01%,0.05%,0.1%,0.5%,1% genipin were respectively used to cross-link the chitosan / calcium silicate / silk fibroin composite scaffolds.The effects of the two kinds of cross-linking agents and different cross-linking concentrations on the performance of scaffolds were compared in order to select a more appropriate cross-linking agent and a more compatible cross-linking concentration to improve the overall performance of the scaffolds.MethodsFirst,prepare the silk fibroin solution,mix the chitosan,calcium silicate and silk fibroin solution uniformly,and then add EDC and different concentrations of genipin respectively into the mixed liquid.After the solutions were cross-linked at 4°C and37°C respectively for 24 hours,they were pre-frozen,and then freeze-dried to prepare composite scaffolds.Then the scaffolds were soaked in ethanol and sodium hydroxide solution successively and freeze-dried again.Finally,the prepared scaffolds were observed under direct vision,analyzed by scanning electron microscope and energy dispersive X-ray spectroscopy,and the pore size,porosity,elastic modulus,compressive strength,water contact angle,swelling rate and cytotoxicity were determined.Results1.The cross-linked and uncross-linked chitosan / calcium silicate / silk fibroin scaffolds were successfully prepared.All the scaffolds were regular cylinders.The pores of the scaffolds changed after adding cross-linking agents into the scaffolds.The pores of the EDC cross-linked scaffolds changed from round to flat.The pores of the genipin cross-linked scaffolds were irregular in shape,and the pore connectivity of the 0.5% genipin cross-linked scaffolds was poor,with most regions simply layered.2.Cross-linking increased the pore size of the scaffolds and reduced the porosity.Among them,the pore size of 0.1% genipin cross-linked group was significantly larger than that of non cross-linked group,but the porosity of 0.1% genipin cross-linked group was significantly lower than that of other genipin cross-linked groups.In genipin cross-linked groups,0.01%、0.05% and 1% genipin cross-linked groups were more suitable for tissue engineering.There was no significant difference in pore size between EDC cross-linked group and genipin cross-linked groups,however,the porosity of EDC cross-linked group was higher than that of 0.05% and1% genipin cross-linked groups(P < 0.05),and significantly higher than that of 0.1%genipin cross-linked group(P < 0.01).3.Cross-linking increased the elastic modulus of the scaffolds.Among them,the elastic modulus of 0.01%、0.05%、0.1% and 1% genipin cross-linked groups increased in turn,while the elastic modulus of EDC cross-linked group was significantly lower than that of genipin cross-linked groups(P < 0.01).4.Compared with non cross-linked group,the compressive strength of 0.01%genipin cross-linked group decreased(P < 0.05),the compressive strength of 1%genipin cross-linked group increased significantly and was higher than that of other cross-linked groups(P < 0.01),while the compressive strength of EDC、0.05%、0.1%genipin cross-linked groups had no significant difference compared with non cross-linked group(P > 0.05).5.EDC cross-linking enhanced the hydrophilicity of scaffolds,while genipin cross-linking weakened the hydrophilicity of scaffolds.Among them,there was no significant difference in water contact angle between genipin cross-linked groups(P >0.05).6.EDC cross-linking didn’t change the swelling rate of the scaffolds,while genipin cross-linking reduced the swelling rate.Among them,the decrease range of1% genipin cross-linked group’s swelling rate is the smallest.7.All of the scaffolds were biosafety and had no obvious cytotoxicity to L929 cells within 72 hours.Moreover,at the same time,the viability of L929 cells cultured in extracts of EDC、0.01%、0.05% and 1% genipin cross-linked scaffolds respectively had no significant difference.ConclusionThe chitosan/calcium silicate/silk fibroin scaffolds were cross-linked using EDC and genipin respectively,and the regular cylindrical scaffolds were made successfully.It is found that after cross-linking,the pore size of scaffolds increases,the porosity of scaffolds decreases and the elastic modulus of scaffolds increases.EDC cross-linking has no effect on the compressive strength,hydrophilicity and swelling rate of scaffolds,while genipin cross-linking weakens the hydrophilicity and swelling rate of scaffolds,and changes the compressive strength of scaffolds.Among them,the compressive strength of 1% genipin cross-linked scaffolds is significantly higher than that of other genipin cross-linked scaffolds and EDC cross-linked scaffolds.The 0.5%genipin cross-linked scaffolds are not compared with other cross-linked scaffolds due to the lack of connected pores.It is found that among the genipin cross-linked scaffolds,1% genipin cross-linked scaffolds are the best.EDC cross-linked scaffolds are better than 1% genipin cross-linked scaffolds in structure、porosity、hydrophilicity and swelling.1% genipin cross-linked scaffolds are better than EDC cross-linked scaffolds in elastic modulus and compressive strength.There is no significant difference in pore size between these two scaffolds,and both of them have good biological safety.