Construction Of Organic-inorganic Composite Gradient Scaffold For Osteochondral Regeneration And Repair

Osteochondral injury is a common and intractable joint disease.The existing methods(such as autograft/allograft transplantation,autologous chondrocyte implantation,etc.)have limited therapeutic effects with high expense.In recent years,osteochondral tissue engineering has been considered a promising technology for osteochondral regeneration.Considering that the osteochondral defect often involves both cartilage and subchondral bone damage,tissue scaffolds must have a discrete gradient or a continuous gradient in terms of cell composition,growth factor,material composition,structure,mechanical properties,and cell culture conditions.Thus,the difficulty in repairing osteochondral tissue is how to simulate the complex gradient structure of osteochondral in order to match the regeneration requirements of cartilage to bone interfacial tissue.In order to cope with this problem,this research focused on the design and construction of gradient scaffolds according to the anatomical structure of the native osteochondral tissue.We constructed two types of biomimetic scaffolds:(1)double-network hydrogel compositing with continuous magnetic hydroxyapatite(HAp)gradient and(2)the trilayered gradient aerogels simulating cartilage-calcified cartilage-bone interfacial tissue with depthdependent physical and chemical properties resembling osteochondral interface.Additionally,the treatment of osteochondral injury requires plenty of stem cells migrating into defects,which promotes the repair process.Clinically,subchondral bone drilling are usually applied to release BMSCs into the trauma site.In regards,the implantation should support the migration and gathering of BMSCs,otherwise the regeneration would be impeded.Constructing a continuous gradient scaffold using the GelMA hydrogel system is a promising strategy to avoid these problems by simplifying the preparation process and mimicking the osteochondral gradient structure more precisely.Compared with synthetic materials,hydrogel is more similar to the extracellular matrix and can better integrate with surrounding tissues.Therefore,based on the magnetic field induced gradient formation technology,a composite hydrogel with a continuous magnetic HAp gradient was fabricated.Under the action of an appropriate magnetic field,the prepared magnetic HAp nanoparticles(FeHA)with suitable saturation magnetization moved a certain distance within the gel by controlling the magnetic field strength,magnetic field shape and other parameters,and a continuous gradient was obtained after the FeHA nanoparticles were fixed in the gel.The gradient FeHA distribution was expected to induce BMSCs to differentiate down specific lineages between chondrocytes and osteocytes according to their positions and secrete osteochondral-like extracellular matrix(ECM).The domain with high incorporated FeHA concentration had high mechanical properties and could promote calcium deposition and bone formation.Under an external static magnetic field environment,the ferroferric oxides inside FeHA were magnetized,further promoting osteogenesis.GelMA has abundant adhesion sites,but its hydrogel has poor mechanical properties,insufficient strength,high rigidity,low relaxation ability,and brittleness.In order to amplify the induction effects from the gradient stimulation,the hydrogel matrix was modified to endow with suitable relaxation ability and cell adhesion.Therefore,acrylate βcyclodextrin was introduced to form host-guest crosslinks with large-sized groups such as benzene rings on the gelatin backbone.Such a viscoelastic network allowed stem cells to migrate into the hydrogel and differentiate under guidance from the microenvironment.Moreover,the cyclodextrin could quickly integrate with surrounding tissues post-implantation and accelerate the regeneration of osteochondral interface tissue.The concept of continuous gradient is based on the previous research of the three-layered gradient scaffold.Briefly,we build a gradient mineralized and cell-affinity three-dimensional aerogel via electrospinning,homogenization,freeze-drying and other technologies.Aerogel for cartilage regeneration(A-C)is made of PLLA/Gel/HA/CS fibers,while A-M and A-B layers treated by the gradient biomineralization process are made of A-U aerogels.With the increase of minerals,the ability of the materials to promote osteogenic differentiation was in the order of A-B>A-M>A-U>blank plates(TCPs).With the addition of CS and HA,the aerogel promoted chondrogenic differentiation.Then,the A-C,A-M,and A-B were stacked and adhered together using methacrylate gelatin(GelMA)to form a gradient aerogel scaffold(A-G)in terms of biochemistry and biophysics,which could effectively promote the tidemark formation and the maturation of new osteochondral tissue after its implantation into the defect.In order to recruit stem cells from the bone marrow cavity,this study further modified A-G with BMSCs affinity peptide(E7)to obtain a cell-affinity gradient scaffold A-E7G,which had significantly accelerated the process of osteochondral repair and regeneration.In summary,composite scaffolds with composition,structure and mechanical gradients can be considered as a promising treatment for fullthickness osteochondral defects.Compared with single-phase and biphasic scaffolds,three-phase gradient scaffolds more benefit the reconstruction of osteochondral integration.However,the layered structure cannot perfectly match the continuous gradient heterogeneous structure of osteochondral,and faces the problem of stress concentration at the layered interface.The continuous gradient scaffolds can more adequately mimic the original osteochondral structure,regulating BMSCs differentiation and facilitating the regeneration and maturation of cartilage-bone interface tissue.