Rapid cell culture and pre-clinical screening of a transforming growth factor-β (TGF-β) inhibitor for orthopaedics

Background: Transforming growth factor-beta (TGF-beta) and bone morphogenetic proteins (BMPs) utilize parallel and related signaling pathways, however the interaction between these pathways in bone remains unclear. TGF-beta inhibition has been previously reported to promote osteogenic differentiation in vitro, suggesting it may have a capacity to augment orthopaedic repair. We have explored this concept using an approach that represents a template for the testing of agents with prospective orthopaedic applications. Methods: The effects of BMP-2, TGF-beta 1, and the TGF-beta receptor (ALK-4/5/7) inhibitor SB431542 on osteogenic differentiation were tested in the MC3T3-E1 murine pre-osteoblast cell line. Outcome measures included alkaline phosphatase staining, matrix mineralization, osteogenic gene expression (Runx2, Alp, Ocn) and phosphorylation of SMAD transcription factors. Next we examined the effects of SB431542 in two orthopaedic animal models. The first was a marrow ablation model where reaming of the femur leads to new intramedullary bone formation. In a second model, 20 mu g rhBMP-2 in a polymer carrier was surgically introduced to the hind limb musculature to produce ectopic bone nodules. Results: BMP-2 and SB431542 increased the expression of osteogenic markers in vitro, while TGF-beta 1 decreased their expression. Both BMP-2 and SB431542 were found to stimulate pSMAD1 and we also observed a non-canonical repression of pSMAD2. In contrast, neither in vivo system was able to provide evidence of improved bone formation or repair with SB431542 treatment. In the marrow ablation model, systemic dosing with up to 10 mg/kg/day SB431542 did not significantly increase reaming-induced bone formation compared to vehicle only controls. In the ectopic bone model, local co-administration of 38 mu g or 192 mu g SB431542 did not increase bone formation. Conclusions: ALK-4/5/7 inhibitors can promote osteogenic differentiation in vitro, but this may not readily translate to in vivo orthopaedic applications.