Preparation And Biological Properties Of Silk Fibroin/Nano-hydroxyapatite/Hyaluronic Acid Composite Scaffold

Objective:The construction of a bioactive scaffold that can simulate the physiological microenvironment during tissue formation has always been a hotspot in biomedical research.In this study,silk fibroin（SF）,nano-hydroxyapatite（nHAp）and hyaluronic acid（HA）were blended,and 1-ethyl-（3-dimethylaminopropyl）carbodiimide hydrochloride（EDC）and N-hydroxysuccinimide（NHS）were added as cross-linking agents to prepare a porous SF/nHAp/HA composite scaffold.The physical and chemical properties and cell biocompatibility in vitro were studied to explore the effects of different contents of HA on the properties of scaffolds and osteogenic differentiation of bone marrow mesenchymal stem cells（BMSCs）,so as to provide new ideas for clinical materials related to repairing bone defects.Methods:1.Silk fibroin/nano-hydroxyapatite/hyaluronic acid（SF/nHAp/HA）porous composite scaffolds with different hyaluronic acid（HA）ratios were prepared by blending,cross-linking and freeze-drying.Five groups of sample materials were obtained:SF/nHAp,SF/nHAp/1.5wt%HA,SF/nHAp/2.5wt%HA,SF/nHAp/5.0wt%H and SF/nHAp/7.5wt%HA.Scanning Electron microscopy（SEM）,X-ray diffraction（XRD）,Fourier Transform infrared（FT-IR）,X-ray photoelectron spectroscopy（XPS）and water-soluble and universal materials testing machine were used to measure and analyze the morphology,secondary structure,pore size,porosity,water absorption capacity and mechanical properties of the scaffold,and to explore the influence of HA on the performance of the porous scaffold.2.After primary culture of rat bone marrow mesenchymal stem cells（BMSC）and flow identification,BMSCs were inoculated on scaffold materials of each group to evaluate the in vitro cytocompatibility of the materials.After 7 days of culture,the distribution and morphology of the cells were observed by SEM.After 1,3 and 7 days of culture,cell adhesion and proliferation were detected by CCK-8 method.After living/dead cell staining,the cell proliferation vitality was observed by Laser scanning confocal microscopy（LSCM）.3.After 4,7 and 14 days of osteogenic induction,BMSCs was inoculated on each group of materials,and the activity was determined by alkaline phosphatase（ALP）kit.After 7,14and 21 days of osteogenic induction,alizarin red staining and semi-quantitative analysis of calcium(Ca²⁺)were performed to evaluate the osteogenic ability of the materials in vitro.Results:1.The SEM observation shows that all the porous scaffolds have a typical three-dimensional porous structure with continuous and interconnected pores.This indicated that SF,nHAp and HA had good compatibility and no obvious agglomeration and precipitation in the composite scaffolds.After the introduction of HA into SF/nHAp porous scaffolds,the average pore size of SF/nHAp/HA porous scaffolds gradually decreased due to the high viscosity of HA,which inhibited the growth of ice cores.On the contrary,the porosity of porous scaffolds gradually increased.The results of XRD,FT-IR and XPS showed that all the porous scaffolds are mainly composed of Silk I and a small amount of Silk II structure,and the HA in the composite scaffolds was effectively cross-linked.The test results of water solubility and mechanical properties show that when the HA content was 7.5wt%,the porous SF/nHAp/HA scaffolds tended to be unstable in water because the Silk I content was insufficient to cover the high HA content.Howere when the amount of HA added is less than5.0wt%,especially the SF/nHAp/HA scaffold with a proportion of 5.0wt%has the highest water absorption and mechanical properties.The results of degradation experiments showed that the addition of HA reduced the degradation rate of composite scaffolds.2.According to the results of in vitro cell experiments such as SEM,LSCM and CCK-8,the cell number of the five groups of scaffold materials showed an increasing trend during the 7day cell culture,and all showed good cytocompatibility.However,BMSCs proliferated fastest on SF/nHAp/5.0wt%HA porous scaffolds,indicating that appropriate amount of HA could support cell adhesion and proliferation.3.The results of osteogenic induction differentiation in vitro showed that the activity of ALP gradually increased with the prolongation of time,and the ALP activity expression and calcium salt deposition of cells on the SF/nHAp/5.0wt%HA porous scaffold were higher than those of the SF/nHAp/1.5wt%HA,SF/nHAp/2.5wt%HA and SF/nHAp/7.5wt%HA porous scaffold groups at each time point,showing a good osteogenic induction ability.Conclusion:In this study,a kind of SF/nHAp/HA porous scaffold material was synthesized by freeze-drying and chemical grafting,and the synergistic effect of the three components in the scaffold and its application in tissue engineering were discussed.The overall water absorption capacity and mechanical properties of the scaffold can be effectively adjusted by introducing HA.The cell adhesion,viability and proliferation experiments showed that SF/nHAp/5.0wt%HA had better cytocompatibility in vitro than the other four groups of materials;in addition,the osteogenic induction and differentiation test in vitro also reflected that SF/nHAp/5.0wt%HA had better osteogenic ability in vitro.In conclusion,HA can improve the cytocompatibility and biological activity of the composites in vitro,which is expected to provide a new material for the repair and reconstruction of bone tissue.