Rapid cell culture and pre-clinical screening of a transforming growth factor-beta (TGF-beta) 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-beta1, 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 microg 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-beta1 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 microg or 192 microg 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

Local Application of Ibandronate/Gelatin Sponge Improves Osteotomy Healing in Rabbits

Delayed healing or non-union of skeletal fractures are common clinical complications. Ibandronate is a highly potent anti-catabolic reagent used for treatment of osteopenia and fracture prevention. We hypothesized that local application of ibandronate after fracture fixation may improve and sustain callus formation and therefore prevent delayed healing or non-union. This study tested the effect of local application of an ibandronate/gelatin sponge composite on osteotomy healing. A right-side distal-femoral osteotomy was created surgically, with fixation using a k-wire, in forty adult male rabbits. The animals were divided into four groups of ten animals and treated by: (i) intravenous injection of normal saline (Control); (ii) local implantation of absorbable gelatin sponge (GS); (iii) local implantation of absorbable GS containing ibandronate (IB+GS), and (iv) intravenous injection of ibandronate (IB i.v.). At two and four weeks the affected femora were harvested for X-ray photography, computed tomography (CT), biomechanical testing and histopathology. At both time-points the results showed that the calluses in both the ibandronate-treated groups, but especially in the IB+GS group, were significantly larger than in the control and GS groups. At four weeks the cross sectional area (CSA) and mechanical test results of ultimate load and energy in the IB+GS group were significantly higher than in other groups. Histological procedures showed a significant reduction in osteoclast numbers in the IB+GS and IB i.v. groups at day 14. The results indicate that local application of an ibandronate/gelatin sponge biomaterial improved early osteotomy healing after surgical fixation and suggest that such treatment may be a valuable local therapy to enhance fracture repair and potentially prevent delayed or non-union