Effects of transforming growth factor-beta and mechanical strain on osteoblast cell counts: An in vitro model for distraction osteogenesis

Factors known to regulate bone production during distraction osteogenesis i nclude mechanical strain on bone forming cells and up-regulation of transfo rming growth factor-beta (TGF-beta) during the distraction, or strain phase of distraction osteogenesis. In the present study, an in vitro model was u sed to evaluate the functional effect of exogenous TGF-beta 1 on mitogenesi s in murine-derived MC3T3 osteoblasts during the period of active mechanica l strain. The first hypothesis to be tested was that mitogenic suppression of MC3T3 osteoblasts by TGF-beta 1 is further enhanced when these cells are also subjected to mechanical strain. To test this hypothesis, MC3T3 osteob lasts were seeded on flexible and rigid membranes. These were subjected to cyclic, vacuum-induced strain, simulating physiologic stress loads. After 2 4 hours, all cells were transferred to media containing TGF-beta 1, and str ain was continued for an additional 48 hours. The study was repeated by usi ng two doses of TGF-beta 1. This study demonstrated that final cell counts were significantly decreased in the presence of TGF-beta 1 in both the nons trained and strained groups (p < 0.0001). The final cell count in the strai ned group was significantly less than that in the nonstrained group (p < 0. 0001) for both concentrations of TGF-beta 1 tested, confirming the initial hypothesis. The second hypothesis to be tested was that alteration in the m itogenic response of MC3T3 osteoblasts after strain is not directly due to autocrine factors produced by the strained osteoblasts. To test this hypoth esis, a proliferation assay was performed on nonconfluent MC3T3 osteoblasts by using conditioned media collected from strained and nonstrained osteobl asts. This study demonstrated no significant differences in cell counts aft er addition of conditioned media collected from strained versus nonstrained cells, confirming the latter hypothesis. The present study demonstrates th e functional significance of mechanical strain on osteoblast cell counts. F urthermore, this may help to explain the temporal relationship observed dur ing the early distraction (strain) phase of distraction osteogenesis in rod ent models in which peak up-regulation of TGF-beta 1 gene expression correl ates with peak suppression of osteoblast function as measured by gene expre ssion of extracellular matrix proteins.