Dual-bioactive Factors Loaded Bilayer Composite Scaffold For Osteochondral Repair

Osteochondral damage is usually caused by congenital malformations,traffic accidents,and severe infections.Mechanical instability of the joint results in joint damage with a risk of degenerative joint & bone disease.Despite tremendous development in the field of regenerative medicine,repairing osteochondral interfaces and full-thickness articular cartilage defects remain a challenge due to the heterogeneous tissue structures of bone and cartilage.To overcome the limitations of complex joint lesions treatment,tissue engineering technology provides a promising way of constructing osteochondral tissues implants.In bone and cartilage tissue engineering,the constructs composed of seeding cells,growth factors and supporting substrate were served as graft materials for repairing osteochondral damage,showing great potentials.In recent years,hydrogels have attracted great attention from researchers because of their structural similarity to extracellular matrices(ECM)and their porous framework,which enables cell transplantation and proliferation.Compared with other synthetic biomaterials,hydrogels provide an appropriate microenvironment similar to the ECM,which reduces friction and mechanical effects on surrounding tissues and significantly improves the biological properties of materials.Meanwhile,it exhibits good biocompatibility,which does not affect the metabolic process of the living body,and the degradation products can be absorbed or excreted through metabolism in the body.Therefore,the development of bioactive hydrogels for bone and cartilage tissue engineering application is urgently needed.In this study,we constructed a bilayer hybrid scaffold with biomimicking articular cartilage-subchondral bone architecture for repair of osteochondral defect.In the cartilage layer,a natural polymer hydrogel loaded with the cartilage-inducing drug Kartogenin(KGN)was developed.In the subchondral layer,a multi-polymeric scaffold with nanofibrous structure was fabricated and treated with polydopamine coating for BMP-2 derived peptide(P24 peptide)immobilization.Then the physicochemical properties of the bilayer hybrid scaffold were studied,and the biocompatibility and osteochondral repair ability of thus developed bilayer scaffolds wereevaluated.The major contents of this study include the following three parts :(1)First,we study the effects of oxidized dextran content in gelatin/silk/oxidized-dextran composite on the physicochemical properties,including pore size,swelling property,mechanical property and degradation rate.The experimental results show that the higher content of oxidized dextran,the smaller the pore size of the hydrogel,the lower the swelling ratio,as well as the higher the mechanical strength and the slower the degradation rate.This is because the increase in oxidized dextran provides more aldehyde groups,which enhances the Schiff base reaction and increases the degree of crosslinking.(2)The Poly L-lactic acid(PLLA)/polylactic acid glycolic acid copolymer(PLGA)/polycaprolactone(PCL)nanofiber scaffolds were prepared by thermally induced phase separation(TIPS),and then the osteogenic factor P24 peptide was immobilized onto the nanofiber scaffold through the adsorption by polydopamine coating.The polydopamine coating can not only provide active groups to promote the immobilization of P24 peptide,but also greatly improve the hydrophilicity of material surface for increasing the affinity of the cells to the scaffold.Taking into account the performance of the hydrogel,we chose 6% oxidized dextran to prepare the hydrogel solution.The hydrogel solution was cast on the upper layer of the nanofiber scaffold,followed by transient vacuum treatment to allow the permeation of hydrogel solution into the interior of the nanofiber scaffold,and then integrated at room temperature to form the osteochondral scaffold.This semi-embedded bonding method allows the two layers of the hybrid scaffold to be more tightly bonded.(3)In vitro and in vivo evaluation of the bilayer hybrid scaffold.Cell proliferation results have shown that bone marrow mesenchymal stem cells(BMSCs)can adhere and grow on the cartilage layer and subchondral layer of scaffold.Cell differentiation results showed that the KGN-loaded hydrogel scaffold showed higher cartilage-related gene expression than the scaffold without growth factor loading.For the evaluation of osteogenic differentiation on the nanofiber scaffold,the results showed that P24 peptide-immobilized scaffold exhibited higher alkaline phosphatase(ALP)activity and higher osteogenic gene expression than the pure nanofiber scaffold.Subcutaneous implantation experiments in mice have shown that the hybrid scaffold possesses good biocompatibility,and the cells can grow into the scaffold and do not cause any inflammatory reaction.By implantation of the bilayer hybrid scaffold into the osteochondral defect on rabbit knee joint,the results indicated that the experimental group had better repair effect than blank control group and factor-free bilayer hybrid scaffold.In summary,the bilayer hybrid scaffold was constructed by integrating the hydrogel layer and nanofiber scaffold layer with the loading of KGN and P24 peptide in distinct layers,respectively.The in vitro and in vivo experimental results demonstrated that the bilayer integrated scaffoldcould be utilized for the regeneration of cartilage and subchondral bone.