Article

Smad3‐Deficient Chondrocytes Have Enhanced BMP Signaling and Accelerated Differentiation

Center for Musculoskeletal Research, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
Journal of Bone and Mineral Research (Impact Factor: 6.59). 02/2006; 21(1):4-16. DOI: 10.1359/JBMR.050911
Source: PubMed

ABSTRACT All studies were performed under the auspices of the University Committee on Animal Resources. Smad3−/− mice derived from a C57/B6 lineage, in which exon 8 of the Smad3 gene is deleted (a kind gift from Dr CX Deng, NIH, Bethesda, MD, USA), were bred using heterozygote pairs. (22, 37) Three-day-old neonatal mice were killed, and genotyping was performed on tail snips obtained at the time of death. The anterior rib cage and sternum were harvested en bloc, washed with sterile PBS, and digested with pronase (Roche Laboratory, Nutley, NJ, USA) dissolved in PBS (2 mg/ml) in a 37°C water bath with continuous shaking for 60 minutes. This was followed by incubation in collagenase D (Roche) solution (3 mg/ml dissolved in serum-free DMEM) for 90 minutes at 37°C. The soft tissue debris was carefully removed. The remaining sterna and costosternal junctions were further digested in fresh collagenase D solution in petri dishes in a 37°C cell incubator for 4–6 h with intermittent shaking. This step allows remnant fibroblasts to attach to the petri dish while the chondrocytes remain afloat in the medium. The digestion solution was filtered through swinex to remove residual tissue fragments. The solution was centrifuged, and the cells were resuspended in complete medium (DMEM with 10% FBS, 1% penicillin/streptomycin, 100 mM L-glutamine, and 50 μg/ml ascorbic acid, pH 7.1). Chondrocytes were counted and plated at appropriate concentrations. To remove any remaining fibroblasts, cells were treated with 0.05% trypsin for 1 minute after plating for 24 h, which lifts the chondrocytes from the culture dish while allowing the fibroblasts to remain attached. Where indicated, recombinant murine noggin (R&D Systems, Minneapolis, MN, USA) was added to the cultures at a concentration of 500 ng/ml.

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