A Double-layer Scaffold With Improved Cell Adhesion And Distribution For Integrated Osteochondral Repair

Damage cartilage is difficult to heal itself and its surgical repair is a hard problem in clinical practice.In recent years,3D printing of hydrogel scaffold has become a hot topic in the research of tissue engineering for cartilage repair.However,due to the poor cell adhesion ability of the large and interconnected pores,the cells seeded on the top of scaffold always leak to the bottom.Furthermore,cells are difficult to migrate and grow into the hydrogel block.As a result,the inhomogeneous cell distribution leads to poor repair effect.In view of the above problems,the studies carried out in this work are listed as follows.(1)Polydopamine modification of 3D printed Gel MA/SA hydrogel scaffold for bone repair application.First,polydopamine(PDA)solution is synthesized by prepolymerized for 12 hours.Second,the printed Gel MA/SA composite hydrogel scaffold is coated with PDA nanoparticles by immersing in the PDA solution.Third,the PDA modified Gel MA/SA hydrogel scaffold is mineralized in simulated body fluid.Last,the composition,structure,and peropertis of the asprepared scaffolds are characterized and tested.The results show the following.(i)The prepolymerization process avoids the cytotoxicity of inadequately polymerized DA monomers.(ii)PDA coating enhances the adhesion ability of bone marrow mesenchymal stem cells(BMSCs)by about 4 times.Also,PDA coating improves the mechanical properties and mineralization ability of Gel MA/SA scaffolds.(iii)Compounding nano-hydroxyapatite(n HA)particles in the scaffold further increases the compression modulus of scaffolds.(iv)PDA coating and n HA particles together improve the expression of osteogenetic differentiation related genes(ALP,COL Ⅰ,OCN,OPN,RUNX2)and promote osteogenesis differentiation of BMSCs.(2)SDF-1α and Kartogenin(KGN)loaded Gel MA/HAMA block hydrogel scaffold for cartilage repair application.First,KGN molecules are embedded in the modified β-cyclodextrin cavity to form hydrogel with methylacrylated hyaluronic acid(HAMA).Then,the hydrogel is cut into microblocks.Second,the modified β-cyclodextrin composite with KGN and HAMA microblocks are added into Gel MA respectively.After a secondary cross-linking,the composite is soaked in stromal-derived factor-1alpha(SDF-1α)solution to form a hydrogel scaffold.Last,the composition,structure,and properties of the as-prepared scaffolds are characterized and tested.The results indicate the following.(i)SDF-1α molecules release from Gel MA hydrogel quickly and recruit BMSCs from the surrounding environment.(ii)HAMA microblocks degrade and generate pores during cell culture,which allows the cells to grow into the composite hydrogel about 400μm depth within 5 days.By contrast,cells cultured on Gel MA hydrogel surface without HAMA microblocks only grow into about 90μm depth.(iii)The modified β-cyclodextrin molecules extend the releasing time of hosted KGN molecules and improve the compression modulus of Gel MA/HAMA hydrogel as the crosslinking agent.(iv)KGN molecules release after HAMA and β-cyclodextrin degrade,and upregulate the expression of cartilage differentiation related genes(ACAN,COL Ⅱ,SOX9)and promote the differentiation of BMSCs recruited by SDF-1α into chondrocytes.(v)HAMA and KGN synergistically significantly reduce the chondrocytes hypertrophy.(3)Primary studies on double layer hydrogel scaffold of 3D printed Gel MA/SA and Gel MA/HAMA.The Gel MA solution mixed with HAMA microblocks is poured onto the precooled Gel MA/SA scaffold,and then immediately forms a double layer hydrogel at a low temperature.There is a small amount of solution perfuses into the upper layer of the 3D printed scaffold about 600μm depth,forming an integrated scaffold with the upper layer for cartilage repair and the lower layer for bone repair.After implanting the double layer scaffold in the cartilage defect,the following is predicted to happen successively.(i)SDF-1α releases to recruit autologous BMSCs;(2)PDA improves the adhesion of BMSCs on the scaffold surface;(3)HAMA microblocks degrade and generate pores;(4)BMSCs grow into the scaffold;(5)Lower layer mineralizes and induces the osteogenic differentiation;(6)Upper layer releases KGN fromβ-cyclodextrin and induces the chondrogenic differentiation.This work fabricates an osteochondral scaffold by combining the chemical modification,3D printing,surface coating,encapsulation,and perfusion bonding technologies.The BMSCs behaviors,including recruitment and adhesion on the scaffold surface,as well as migration,proliferation and differentiation in the scaffold,are regulated by various methods,such as sequential releasing factors,surface modification,and degradation pores.In vitro studies show that the osteochondral scaffold has good bioactivity,which is a potential implant for tissue engineered cartilage repair.