Fabrication Of Silk Fibroin/Chitosan 3D Scaffolds And Preliminary Study On Cytology In Vitro

ObjectiveTo select the most suitable scaffold proportion for tissue engineering by investigating the characterization of 3D fibroin/chitosan composite scaffold and the behavior of MG-63 cells on scaffold.And to provide a new natural biomaterial for the treatment of bone tissue engineering.Methods:Part I: 1.Silk fibroin solution was extracted from cocoon by degumming,dissolving and dialysis,and its concentration was calculated.2.Three dimensional composite scaffolds with different proportions of fibroin protein and chitosan were prepared.3.The surface morphology of composite scaffolds with different proportions was detected by scanning electron microscopy(SEM).4.The conformation of scaffolds was detected by X-ray diffraction(XRD)and infrared spectrometer(FTIR).5.The expansion rate of the SF/CS scaffolds in PBS and the degradation rate of double-distilled water were studied.6.The porosity of the three-dimensional scaffold was detected by 75% alcohol solution.The transport capacity of nutrients in the scaffolds was measured by the permeation ability of alizarin red in the scaffolds.Part II: 1.In order to evaluate the cytotoxicity of the SF/CS scaffolds,the proliferation of MG-63 cells in the scaffold extracted media was detected by MTT assay and Hoechst method.2.MG-63 cells were seeded into 3D scaffolds that were sterilized by irradiation.The compatibility of the MG-63 cells with the SF/CS scaffolds was determined by measuring the adhesion ability of the cells in the scaffold and the proliferation of the cells on the scaffold by MTT assay.3.MG-63 cells were planted in different proportion scaffolds and embedded into slices.The morphology of the cells in the scaffold was detected by HE staining.4.The cell morphology in 40 SF ?50SFscaffolds was studied by scanning electron microscopy(SEM).5.Preliminary evaluation of osteogenic properties of MG-63 cells in SF/CS 3D scaffolds by detecting alkaline phosphatase activity(ALP).ResultsPart I: 1.Through the laboratory process,the fibroin solution of about 3% was prepared in 4 days.2.The chitosan solution with 3% was prepared.In 6 days,the following proportions were prepared: 100%SF?100%CS?50%SF-50%CS(50%SF)?20%SF-80%CS(20%SF)?30%SF-70%CS(30%SF)?40%SF-60%CS(40%SF).3.The SEM results showed that 50 SF and 40 SF have the most regular surface morphology.4.The results of XRD?FTIR showed that the crystallinity and conformation of SF were changed after adding CS.5.The results of expansion rate showed that the expansion rate was in direct proportion to CS content.6.The degradation rate experiment showed that the properties of SF/CS 3D scaffolds containing cross-linking agent were stable and the degradation solution was neutral 7.The porosity of 50 SF and 40 SF scaffolds were higher than other groups.8.Among the different ratio scaffolds,50 SF and 40 SF scaffolds had the best absorbability of protein.Part II : 1.MG-63 cells in 40 SF,50SF leaching liquid in the proliferation of the best,the remaining two were significantly lower.These groups without cytotoxicity.2.MG-63 cells in 40 SF,50SF stent adhesion rate and adhesion in normal culture without statistical difference.MTT test results showed that the rate of MG-63 cells on 50 SF and 40 SF scaffolds is better than other two groups,and 50 SF was best.3.HE showed that MG-63 cells on 50 SF and 40 SF scaffolds grew well,and the 50 SF stent survived more than the number of 40 SF.4.SEM results also showed that the number of MG-63 cells in 50 SF scaffolds was significantly higher than that of 40 SF cells.5.The ALP activity of MG-63 cells in 50 SF scaffold was higher than that in 40 SF group.The results showed that 50%SF-50%CS scaffold was more suitable for MG-63 cells culture.Conclusion:Silk fibroin/chitosan composite scaffold is easy to be prepared,and the surface morphology and physicochemical properties of the scaffolds different greatly.When the ratio of silk fibroin protein to chitosan is the closest,the scaffold has the best performance.The first part proves that the shape,pore structure,protein transport performance of 50%SF-50%CS and 40%SF-60%CS scaffolds are best.The second part of the related cell experiment proved that the 50%SF-50%CS scaffold was the most suitable for osteoblast adhesion and proliferation for a long time,and the osteogenic properties were preliminarily tested.In this study,the suitable biomaterials for tissue engineering were preliminarily discussed,which provided a new idea for the application of biomaterials in bone tissue.