Tissue culture media were purchased from GIBCO BRL, fetal bovine serum (FBS) was from BioWhittaker. Rat tail collagen type 1, 99% pure, was purchased from Becton Dickinson Laboratories. All other reagents were purchased from Sigma Chemical Co. unless otherwise stated. Cells were expanded in permissive conditions (33°C in αMEM with 10% FBS, 100 units/ml penicillin, 50 µg/ml streptomycin, and 50 U/ml IFN-γ) on rat tail type I collagen-coated plates or gels or bovine type I collagen sponges. To induce osteogenesis, cells were plated at 80,000 cells/cm2 in osteogenic conditions (37°C with 50 µg/ml ascorbic acid and 4 mM β-glycerophosphate in the absence of IFN-γ). Collagen-coated surfaces were used because they were found to be effective at maintaining an osteocyte-like phenotype (10 (link)).
MLO-A5 cells, used as controls, are an established model of late osteoblasts with the ability to rapidly synthesize mineralized extracellular matrix (1 (link)). MLO-A5 cells are highly responsive to mechanical loading in 3D culture (15 (link)). MLO-Y4 cells, also used as controls, are an established model of osteocytes.
MLO-A5 cells, used as controls, are an established model of late osteoblasts with the ability to rapidly synthesize mineralized extracellular matrix (1 (link)). MLO-A5 cells are highly responsive to mechanical loading in 3D culture (15 (link)). MLO-Y4 cells, also used as controls, are an established model of osteocytes.
