Substrata Prepared from Bone Matrix for Chondrogenesis in Tissue Culture

The literature on chondrogenesis in vitro is rich in knowledge of the products of differentiation but presents little information about agents which may initiate cartilage cell differentiation or conditions which control cartilage tissue morphogenesis . Previous investigations by our research group (18, 19) demonstrate that the character of the substratum may control differentiation . For example, an outgrowth of mesenchymal cells from muscle on to nonbiologic substrata (plastic or millipore membrane) produces fibrous connective tissue cell differentiation, while outgrowths on to a special preparation of undenatured, demineralized rat bone matrix differentiates into cartilage (30) . If the substratum consists of bone matrix denatured by demineralization in a solution of 0 .6 N HCI in 70% alcohol, the mesenchymal cells differentiate only into fibrous connective tissue (30) . This communication demonstrates experimental modifications of a bone matrix substratum which either permit, retard, or prevent differentiation of cartilage . INTRODUCTION to obtain pieces approximately 0 .5 mm in size . In some cultures, minced muscle was substituted with trypsinized muscle. The medium was CMRL-1066 with 15% heatinactivated newborn calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) (all from Grand Island Biological Co .) . Approximately 0 .75% of the medium was changed every 48 h and maintained for 3-14 days (and extended to 30 days for special experiments) on the reversed grid in a Falcon organ culture dish (Falcon Plastics, Div . of B-D Laboratories, Los Angeles, Calif .) at 37°C in a CO a incubator (5% CO 2 in air, model 3221, National Applicance Co ., Portland, Ore .) as described previously (19) . The initial pH of the medium was 7 .3 . Cultures prepared as described above were grown on matrix denatured by demineralization at 2°C in 0 .6 N HCI in 70% alcohol for 24 h . Cultures of muscle mesenchyma were also made on millipore membranes (8-μm pore size) or sandwiched between millipore membranes in order to simulate the condition of a crevice in the matrix . Outgrowths of bone marrow cells, buffy coat cells, lymph node, spleen, thymus, or skin were also cultured on bone matrix substrata . Bone marrow cells were obtained from the femur of 2-wk old rat and buffy coat cells were obtained by means of heart puncture of 3-mo old rat . MATERIALS AND METHODS