Bone Matrix Gelatin as Scaffold for Cartilagetissue Engineering

Introduction . Articular cartilage has a limited intrinsic capacity for repair, therefore, methods to repair or reconstruct osteochondral defects have been sought by many labs (). The search for the ideal cellular matrix has resulted in the development and testing of both artificial and natural scaffold materials(). Current cellular scaffolds, however, are limited by the presence of inflammatory or immunologic reactions to the scaffold material. The history of demineralized bone matrices dates back to 1965, when Urist () observed cartilage and heterotopic bone formation after implanting allogenic demineralized bone matrix in intramuscular sites in rodents. Bone matrix gelatin (BMG) is obtained by demineralizat ion of bone in an acid medium. Studies into the mechanism of bone and cartilage induction BMG have shown that native insoluble BMP contains noncollagenous proteins capable of induingmesenchymal cells into differentiate towards mature chondrocytes in vitro . Despite this potentially useful property of BMG, this material has not been used as a scaffold for osteochondral regeneration to our knowledge. In the present study, we report our preliminary results of in vitro culture articular chondrocytes on autolyzed antigen-extracted allogenic BMG. . Materials and Methods. Demineralized bone matrix gelatin was prepared from distal femur segments harvested from freshly euthanized adult New Zealand white rabbits. Passage 1 (P1) chondrocytes were dynamically seeded onto BMG. Briefly, BMG scaffolds were pre-wetted in culture medium and cut into 5mm×5mm×1mm pieces. BMG was placed at the bottom of a 50ml Falcon centrifuge tube and, 2×10 cells were plated on BMG and cultured in 10ml of Dulbecco’s modified Eagle’s medium containing 4.5gm/liter glucose, 20% fetal bovine serum, 50mg/liter ascorbate, 10mM HEPES, 100 units/ml penicillin, and 100 mg/ml streptomycin for 6 weeks. Histological properties and organization of the engineered cartilage were evaluated by light microscopy after staining with hematoxylin and eosin (H&E) and safranin -O. Type I collagen and type II collagen were immunolocalized in engineered cartilage. Engineered cartilage was also subjected to scanning and transmission electron microscopy to study collagen fibril formation. Expression of cartilage extracellular genes was determined on engineered cartilage at different time points. Amino sugars, as indicators of glycosaminoglycan (GAG), were measured colorimetrically after 3weeks and normalized to total DNA in the culture system.