Biofunctionalized Gelatin-silk Fibroin Composite Cryogel Scaffold For Osteochondral Repair Applications

Clinical treatment for osteochondral injury cannot achieve satisfactory long-term repair effect,while cartilage tissue engineering technology is limited by the difficulty in regulating stem cells proliferation and differentiation.In contrast,in situ promoting osteochondral regeneration by cell-free bioactive scaffolds has excellent clinical application prospect.In recent years,Natural polymer cryogels have attracted widespread attention because of their unique advantages such as similarity to natural extracellular matrix and macroporous structure.However,the drawback of mechanical strength and relatively limited biofunction have greatly constrained its application for osteochondral regeneration.To address this challenge,lots of effort were focus on enhancing the mechanical strength and promoting materials-tissue interaction of gelatin methacryloyl(GelMA)cryogel with excellent bioactivity.We hope to design and fabricate customized cell-free osteochondral regenerative cryogel scaffold with stable abilities of BMSCs recruitment and promoting the osteogenesis /chondrogenic differentiation of stem cells.GelMA-co-SF-GMA composite cryogel(Gel/SF)was prepared at-20℃ and reinforced by treatment with methanol to induce β-sheet transformation of SF-GMA.The concentration and feed ratio of biopolymer can regulate the microstructure and properties of cryogels by affecting the freezing concentration process.The Gel/SF cryogels have macroporous structure(over than130μm),improved compressive Modulus(0.523 MPa,increased by 9.1 times compared with GelMA cryogel)and lower degradation rate(20% remained after 28 days).In addition,when co-culture with mBMSCs,the result showed that Gel/SF cryogels can promote cell adhesion,spreading,migration and proliferation.Subsequently,Multilayer or 3D customized Gel/SF cryogel scaffolds were prepared by conbining appropriate low temperature and customized PLA mold.Results showed that there is no interface in Multilayer cryogel scaffolds and solution can rapidly diffuse into the 3D customized scaffolds.E7@cryogel with the stable ability of BMSCs recruitment was obtained by modifying the BMSCs-specific-affinity peptide(E7)to the Gel/SF cryogel.E7@cryogel has excellent biocompatibility to mBMSCs and can significantly promote BMSCs to up-regulate the expression of SDF-1α.When increasing the concentration of E7 peptide,the ability of E7@cryogel to promote osteogenesis differentiation was increased firstly and then decreased.It’s suitable for E7@cryogel(100μg/mL)to be candidate for subchondral bone repairFinally,with the target for enrichment of endogenous TGF-β1 and further promoting chondrogenic differentiation of BMSCs,TGF-β1 affinity peptide(LPL)was grafted on Gel/SF cryogel.The ability of LPL@cryogel for promoting proliferation was weaker than pure Gel/SF cryogel.However,The ability of promoting proliferation was upregulated when conbining E7 and LPL peptides to the cryogel(named as EL@cryogel).LPL peptide is the key factor for enrichment of TGF-β1 in vitro.Both LPL@cryogel and EL@cryogel can achieve the controlled release of TGF-β1 for more than 7 days.E7@cryogel can promote the chondrogenic differentiation and inhibit hypertrophy of BMSCs,while LPL@cryogel can promote chondrogenic differentiation by enriching TGF-β1.EL@cryogel has the potential to be used for cartilage regeneration.