Construction Of BMSC Cell Sheet On Fibrous Mesh For Osteochondral Defect Regeneration

Osteoarthritis,cartilage wear and osteochondral defect are common remaining problems in clinical,and osteochondral tissue engineering is one of the most promising treatment for osteochondral defects.The key of osteochondral tissue engineering is to maintain the viability of seeded cells and to prepare the biomimetic-structured scaffolds.The traditional cell harvest methods have the risk to destroy fibronectins on cell membrane and cell junction.Besides,cell loss is frequently associated with the conventional procedures for cell seeding.Cell sheet engineering can avoid these shortcomings without the need to digest cells,and the well-preserved extracelluar matrix will facilitate tissue regeneration,which makes it possible application in treating osteochondral defects.In order to endow the cell sheet with good operation and regulation,this study is supported by the fibrous meshes prepared by the electrospun spinning method,incubates the cell sheet of bone marrow mesenchymal stem cells(BMSCs),and BMSCs is a adult stem cell with multipotential differentiation potential,and the structure of the biomimetic natural extracellular matrix of the fibrous meshes is created a good microenvironment for the proliferation and differentiation of BMSCs.In this paper,the chondrogenic,calcified cartilage and osteogenic differentiation of BMSCs cell sheets in vitro were studied in this paper.A three-dimensional gradient osteochondral prosthesis was implanted into the knee osteochondral defect of New Zealand white rabbit,and the gradient induced BMSCs cell sheets/fibrous mesh composite was applied to osteochondral.And evaluating the feasibility of osteochondral regeneration by cell sheet/fibrous mesh composite.First,the non-woven poly(lactic acid/gelatin)composite fiber mesh was prepared and inoculated on BMSCs and obtained a tightly packed cell sheet/fibrous mesh composite.The osteogenic and chondrogenic differentiation properties of the cell sheet/fibrous mesh composite were studied by using the different proportions of osteogenic induction and chondrogenic differentiation,and the best in vitro induction conditions of calcified cartilage were explored.The results showed that compared with the tissue culture plate,the fibrous meshes had obvious promoting effect on cell adhesion and proliferation,enhanced the osteogenesis and chondrogenic differentiation ability of the cell sheets,and determined that the cell sheets showed significant calcified cartilage under the joint action of 50%osteogenic differentiation and 50%cartilage induced differentiation medium.Secondly,the cell sheet/fibrous mesh complex,which was induced by different induction conditions,was stacked in the order of cartilage-calcified cartilage-osteogenesis,and the three-dimensional gradient prosthesis of the physiological structure of the osteoid cartilage was obtained and placed in the region of the osteochondral defect of the knee joint of New Zealand white rabbits.At 6,12 and 24 weeks after the operation,Micro-CT,histological staining and immunohistochemical staining were used to analyze the effect of osteochondral defect regeneration.The results showed that the implantation of the three-dimensional gradient cell sheet/fibrous mesh composite accelerated the calcification of the bone matrix and enhanced the regeneration and repair ability of the osteochondral defect of the knee,compared with the cell sheet/fibrous mesh implants without gradient induction,and the calcified cartilaginous layer introduced between the osteogenic area and the cartilaginous region.The isolation and contact of two phases can better maintain the microenvironment of cartilage and bone and facilitate the formation of intact cartilage layer.In summary,BMSCs cell sheets can be readily obtained on electrospun fibrous meshes,which can be induced osteogenic,chondrogenic,and calcified cartilage-like differentiation in vitro.Then the cell sheet/mesh complexes can be used to build three-dimensional construct with gradient design mimicking natural osteochnodral structure via layer-by-layer stacking strategy,which promote the regeneration of osteochondral defects in vivo.This kind of three-dimensional construct is expected as a new type of repairing material for tissue regeneration.