| Objective: In this study,octacalcium phosphate/silk fibroin/sodium alginate(OCP/SF/SA)composite porous scaffold was prepared as the bone layer and CS/SF/SA scaffold containing chondroitin sulfate(CS)with oriented pore structure was prepared as the cartilage layer by lyophilization and directional freezing technique.The physicochemical properties and biological behaviors of scaffolds were evaluated in vitro and the proper scaffolds for bone and cartilage layer were selected to construct the osteochondral biomimetic scaffold with a good interface.These experiments would lay a foundation for osteochondral repairing in vivo.Methods:(1)Preparation of scaffold and analysis of its physicochemical properties: 5 groups of porous bone scaffolds with the mass percent of OCP/(SF + SA)as 0:100,10:90,25:75,50:50and 75:25 were prepared by lyophilization;4 groups of CS/SF/SA cartilage scaffolds with oriented pore structure respectively containing 0,1%,2% and 10% CS were prepared by directional freezing technique and lyophilization;the osteochondral biomimetic scaffold was prepared by combining proper bone and cartilage layer scaffolds;the morphology of scaffolds was observed by SEM;the crystal structure of scaffolds was detected by XRD;the molecular structure of the scaffolds was determined by FTIR;the scaffold was immersed in simulated body fluid(SBF)to observe the mineralization;the water absorption rate,degradation rate,calcium ion release from the bone layer scaffold and chondroitin sulfate release from the cartilage layer scaffold were detected by soaking the scaffold in phosphate buffer solution(PBS);the mechanical properties of the scaffold were tested by dynamic mechanical testing machine.(2)Cytological analysis of scaffolds in vitro: after m BMSCs were cultured on the bone layer scaffolds,CCK-8 was used to detect cell proliferation;SEM and CLSM were used to observe cell adhesion on scaffolds;ALP staining,ALP activity test and osteoblast differentiation related gene expression test were used to evaluate the osteoblast differentiation of m BMSCs;after ATDC5 cells were co-cultured with the extracts of the cartilage layer scaffolds,CCK-8 was used to detect cell proliferation;Live-dead staining was used to detect cell viability;alcian blue staining and chondrogenic differentiation related gene expression detection were applied to evaluate the chondrogenic differentiation of ATDC5.Result:(1)Analysis of physicochemical properties of the scaffolds: SEM photographs showed that the bone layer scaffolds had cellular porous structure with the diameter ranging from 81±42 μm to 219±56 μm and the aperture decreased with the increase of OCP content;In the mineralization experiment on day 14,a large number of flake crystals like hydroxyapatite(HA)were found in the scaffolds of 10:90 and 25:75group;the cartilaginous scaffold had a directional pore structure,with the pore size ranging from 130±39 μm to 444±53 μm,and the pore size decreased with the increase of CS content;the bone layer of the biomimetic bone-cartilage scaffold was a cellular porous structure and the cartilage layer was a directional pore structure;the two layers gradually changed to form the bone-cartilage interface;FTIR results showed that absorption peaks of β-sheet structure in silk fibroin appeared in each group of bone and cartilage layer scaffolds;XRD results showed that the characteristic peaks of OCP appeared in the OCP/SF/SA composite scaffolds,and the peak strength increased with the increase of OCP content;the water absorption experiment results showed that: 0:100,10:90,25:75 group of scaffolds had good water absorption rate,which was significantly better than other groups,each cartilage layer scaffold had good water absorption rate,which increased with the increase of CS conten;The bone layer scaffold can release Ca2+ and Cartilage scaffold can release CS in PBS,In addition,the incorporation of OCP improved the mechanical properties of the scaffolds and reduced scaffold degradation rate.(2)Osteogenic activity of subchondral scaffold in vitro: CCK-8 detection showed that m BMSCs could proliferate in each group of scaffolds,especially in 25:75 group;SEM and CLSM showed that BMSCs adhered well in each group of scaffolds;ALP activity test and staining results showed that ALP activity in 10:90 and 25:75 groups was higher than that in other groups;The q PCR showed that moderate OCP could promote the expression of osteogenic differentiation related genes(ALP,Col-I)of m BMSCs and high content of OCP promoted the genes expression of OC and OPN.(3)Chondrogenic activity of chondrogenic scaffold in vitro: CCK-8 results showed that ATDC5 could proliferate well in each group,especially in the 2% CS group;the results of Livedead staining showed that each groudp of scaffolds had good cytocompatibility;the results of Alixin blue staining showed that ATDC5 secreted more polysaccharide in each group than in the pure SF/SA group;the results of q PCR showed that proper amount of CS could promote the expression of chondrogenic differentiation related genes(Col-II,Sox9,aggrecan).Conclusion: in this study,we successfully constructed an osteochondral biomimetic scaffold with good physicochemical properties and biocompatibility.OCP in the bone layer promoted the osteogenic differentiation of m BMSCs and CS in the cartilage layer promoted the chondrogenic differentiation of ATDC5.The development of this study can lay a foundation for osteochondral repair experiment in vivo. |
