Gradient Constructs Made Of Layered Electrospun Fibrous Meshes And Cell Sheets For Osteochondral Regeneration

Cartilage is an avascular tissue,and the chondrocytes are differentiated and mature cells,which makes cartilage damage difficult to self-repair.At the same time,large-area cartilage defects are often accompanied by damage to subchondral bone,and the treatment of osteochondral defects remains a significant challenge in clinical therapy.Tissue engineering technology is a feasible strategy for the regeneration of osteochondral cartilage,but the heterogeneous structure of osteochondral imposes requirements on the construction of osteochondral scaffold with biomimetic gradient.The preparation of three-dimensional constructs by stacking different cell layers is a well-known technique,and it is a convenient method that can be used to construct gradient structures.Electrospun fibrous meshes have been proved to be excellent supporting materials for incubating cell layers.Therefore,in this paper,composite fibrous meshes with different compositions were prepared by electrospinning technology,imitating the natural osteochondral gradient composition.Bone marrow mesenchymal stem cell(BMSCs)layers with multi-differentiation potential were incubated on different composite fibrous meshes,and the BMSCs layers were induced to differentiate into cartilage,calcified cartilage and osteoblast.Finally,the three-dimensional construct is obtained through gradient stacking,which can be used for osteochondral regeneration.In this study,polylactide/glycosaminoglycan,polylactide/gelatin,polylactide/gelatin/glycosaminoglycan,polylactide/gelatin/hydroxyapatite composite fibers were prepared by electrospinning their blending solutions.The glycosaminoglycan components as hyaluronic acid and chondroitin sulfate with the ability to promote cartilage differentiation were selected,and the addition amounts of hyaluronic acid(HA),chondroitin sulfate(CS)and hydroxyapatite(HAp)in the composite fibers were changed.Next,researches on cell layer incubation on different fibrous meshes and induced differentiation were carried out.Among them,particularly,the electrospinning process for the preparation of composite fibers containing glycosaminoglycan was optimized,and the effects of fiber compositions on the chondrogenic differentiation of BMSCs and chondrocytes were comparatively investigated.The results show that all the composite fibrous meshes are non-cytotoxic,have high hydrophilicity and excellent cell affinity,and support the adhesion,proliferation and differentiation of cells.Among them:1)BMSC layer was incubated onto the polylactide/gelatin/HA/CS composite fibrous meshes and chondrogenically induced in vitro,which was applied as the cartilage layer in stacking the gradient osteochondral construct;2)The BMSCs layer incubated on the polylactide/gelatin composite fibrous mesh was applied as the intermediate part of the gradient osteochondral construct,after it was in vitro induced with 1:1 mixed chondragenically inducitve medium and osteogenically inductive medium;3)The BMSC layer incubated on the polylactide/gelatin/HAp composite fibrous mesh served as the bone layer in stacking the gradient osteochondral construct,after it was treated in vitro with osteogenically inductive medium.Consequently,in order to explore the mechanism of calcified intermediate layer and in vitro pre-differentiation culture on the regeneration of osteochondral tissue,the three kinds of cell/mesh complexes were stacked to get gradient constructs to carry out in vivo studies,which well mimicked the heterogeneous gradient structure of natural joint osteochondral tissue.The control group is using a cell/mesh complex without pre-differentiation as the intermediate layer.In vivo evaluations were conducted using both the subcutaneous implant model and the knee articular osteochondral defect model in New Zealand rabbits,to carry out assessments on the biocompatibility and the regenerative ability of the implants.In summary,in this paper,by controlling the compositions of the fibrous meshes and combining with the method of pre-differentiation culture in vitro,the obtained gradient osteochondral construct is highly expected to achieve good outcomes in regenerating osteochondral defects,which will provide guidance in understanding the mechanism of the osteochondral regeneration.