Preparation Of Collagen Type ? Composite Cartilage Scallfold

The study established a new method for extracting collagen type II.Bovine cartilage was used as raw material,dealt with a mixture of chloroform and methanol to degrease,pepsin to remove periosteum,guanidine hydrochloride to remove proteoglycans,pepsin to digest CII,NaCl to sault out and deionized water to dialysis,and finally CII sample was achieved by Vacuum Freeze Drying Plant.The CII sample and CII standards were electrophoresed by SDS-PAGE,and results showed that CII sample from bovine cartilage was of high purity.Then,four different ratios of CII,HA,and CS were applied to construct scallfolds,and four scallfolds were crosslinked by EDC/NHS.The morphology,crosslink degree,pore diameter,porosity,absorption rate and degradation rate were determined to evaluate properties of four scallfolds and the ratio 9:1:1 was optimal.TGF-?1 loaded chitosan microspheres were prepared by emulsification and cross-linking methods.The morphology,absorption rate,degradation rate were determined and TGF-?1cumulative release curve in vitro was drawn.Based on TGF-?1 cumulative release curve,drug loading and entrapment efficiency were calculated.Results showed that the morphology of microsphere was round and uniform,mean diameter was about 100 nm,absorption rate was up to 983.73±4.38%.When the microsphere was incubated under the condition of 10~7 U/L lysozyme,the degradation rate was only 51.0±1.8%on day 28.From the cumulative release curve,TGF-?1 was released quickly at initial 24 h,slowly progressively later and reached plateau phase on hour 120.The drug loading and entrapment efficiency were 25.6±0.23?g/g and 80.21±0.02%respectively.In order to evaluate the biological property of the scallfold,control chitosan microspheres and TGF-?1 loaded chitosan microspheres were incorporated into the scallfolds respectively,and then ATDC-5 were inoculated into the two composite scallfolds.MTT assay and Fluorescence staining test showed that the scallfolds composed of CII,HA and CS were quite suitable for ATDC-5 growth and proliferation,and showed that TGF-?1 loaded chitosan microspheres could significantly promote the growth and proliferation of ATDC-5.