Preparation Of Bone And Cartilage Acellular Matrix And Repairing Effect In Animal Experiments

Background and objectivesArticular cartilage constitute with hyaline cartilage, subchondral bone and chondro-osseous junctional (COJ) structure between the former two structures. The frequent use of joint, trauma, tumor and inflammation can lead to cartilage or osteochondral injury. Cartilage injury will develop to synovium, joint capsule and subchondral bone, resulting in osteochondral damage, osteochondral damage is usually accompanied by changes in mechanical stress, which can lead to degenerative arthritis and lower quality of life. Articular cartilage, containing only a single kind of cells, lacking local progenitor cells and having high extracellular matrix cell rate, is avascular, aneural and alymphatic and has poor self-repair capacity. Subchondral bone mainly play a supportive role on the articular cartilage and insufficient vascularation of subchondral bone lead large bone defects repair difficult. The chondro-osseous junctional region between the bone and hyaline cartilage contains deep zone of hyaline cartilage, tide line, calcified cartilage, cement line and the upper zone of subchondral bone, is thin and complex and has important physiological functions. Articular cartilage and subchondral bone damage have poor self repair capacity, treatment of these injury become one of the problems of joint surgery.There are a lot of osteochondral defects repair methods, autologous and allogeneic osteochondral transplantation may be the most effective method; currently matrix-induced chondrocyte implantation (MACI) may be the promising method to repair cartilage lesion in clinic, which have the chance to regenerate hyaline cartilage. MACI is unavailable to osteochondral defects and sources of osteochondral grafts are limited, but MACI and osteochondral transplantation imply that a graft containing cartilage scaffold, subchondral bone and COJ structure, combined with seed cells, may be a potential osteochondral repair approach. Ideal tissue engineering scaffold should simulate the structure of the natural joint and then simulate its normal function, but current method including construction by layers and integration construction in tissue engineering is difficult to simulate the structure of cartilage, COJ and subchondral bone. Cells in minced cartilage matrix can be removed by advanced acellular technology, while retain the cartilage extracellular matrix.On these basis, we suppose that osteochondral tissue(containing COJ)and cartilage tissue can be decellularized respectively, then osteochondral scaffold can be constructed by the acellular matrix, the scaffold includes deep hyaline cartilage zone, tide line, calcified cartilage layer, cement line, the subchondral bone plate. Cartilage scaffold was cross-linked by porous acellular cartilage matrix, seeded with bone marrow-derived mesenchymal stem cells, then was implanted in animal osteochondral defect model and the effect of cartilage formation, cell biology and subchondral bone changes was observed; and bone marrow-derived endothelial progenitor cells (EPCs) were seeded into acellular bone matrix to repair large bone defects in animals, vascularization of the bone scaffold may provide a new method of repair subchondral bone. This study will biomimetic osteochondral composite scaffolds, and create the conditions to build a more complex organization. This study mainly include: (1) cells components are removed from minced cartilage and osteochondral block respectively, the acellular degree of bone and cartilage tissues are verified; (2) Construct osteochondral scaffold which containing COJ structure; (3) the osteochondral scaffold are seeded with bone marrow MSCs, implanted to osteochondral defect of weight-bearing area of the sheep knee, then grossly and histologically evaluate the repair tissue. (4) large bone defect of rabbit ulna is repaired by acellular bone matrix through vascularization promoted by bone marrow derived endothelial progenitor cells.Methods1. The fresh human cartilage are crushed to particles when immerced in protease inhibitor, then separated by centrifugal and treated with acellular process, then verified by histological evalution, quantitative analysis and biochemical properties.2. Prepare a 8mm diameter osteochondral block which contain only about 100μm hyaline cartialge, then treated with acellular process and have relative detect. Osteochondral scaffold was structured by extrcellular matrix with freeze-drying and crosslinking, then seeded with sheep bone marrow derived mesenchymal stem cells, had histology, histocompatibility and cell toxicity detect after cultured 10 days in vitro.3. Osteochondral defects was prepared in weight loading area of sheep, defects were separated into scaffold-cells group, scaffold group and blank group, implanted scaffold-cells composite, scaffold and nothing respectively, then the animals were sacrificed 3 months after the operation and the specimens have grossly and histologically evaluation.4. Decalcified bone matrix (DBM) was prevascularized by seeding fibrin gels with EPCs that are derived from rabbit bone marrow mononuclear cells (BMMNCs). The compound was then transplanted autologously into bone defects of rabbits to observe the vascularization in vivo. 2,4 and 8 weeks animals were sacrificed and have histological biochemical analysis.Results:1. No cell debris are left in cartilage particles after acellular treatment in HE, cartilage tissue are positive in toluidine blue, Safranin O and type II collagen immunohistochemical staining, cartilage matrix microstructure reserved; biochemical quantitative analysis show that the DNA decrease significantly.2. No cell debris are left in osteochondral tissue after acellular treatment, cartilage part are positive in toluidine blue, Safranin O and type II collagen immunohistochemical staining, the basic structure and function of COJ reserved, biochemical quantitative results show that the DNA decrease significantly. Acellular osteochondral scaffold connect well in histology, sheep bone marrow mesenchymal stem cells grew well and secret extracellular matrix in the scaffold under electron microscopy and HE, cartilage part of the scaffold are positive in Safranin O and Type II collagen immunohistochemical staining.3. Cell scaffold composite group had better repair in weight-bearing area osteochondral defect in sheep model, subchondral bone have repaired in scaffold group, fibrous tissue repair in the blank group with the cartilage degeneration in defects’edge.4. Bone marrow derived endothelial progenitor cells can culture and expand in vitro, At 2, 4 and 8 weeks after grafting, the microvessel density of new bone tissues was significantly higher in the seeded EPCs group than in the control group (p<0.05), DBM seeded with bone marrow derived EPCs is able to partly repair large bone defect of rabbit ulna. Conclusion:Minced cartilage tissue can be decellularized to maintain their extracellular matrix; the cellular components can be removed in osteochondral tissue containing only 100μm thick hyaline cartilage and retain the structure and function of COJ; Extracellular matrix osteochondral scaffold can be made by freeze-drying and chemical cross-linking, which include COJ structure, porous cartilage part and acellular subchondral bone, the scaffold have good biocompatibility with no significant cytotoxicity; Extracellular matrix osteochondral scaffold seeded with MSCs can repair better than control group in loading area osteochondral defects of sheep; BMMNC-derived cells seeded into DBM may be suitable for repairing large bone defect of rabbit ulna and improving vascularization.