A novel low-molecular-weight compound enhances ectopic bone formation and fracture repair

Abstract

Background: Use of recombinant human bone morphogenetic protein-2 (rhBMP-2) is expensive and may cause local side effects. A small synthetic molecule, SVAK-12, has recently been shown in vitro to potentiate rhBMP-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype. The aims of this study were to test the ability of SVAK-12 to enhance bone formation in a rodent ectopic model and to test whether a single percutaneous injection of SVAK-12 can accelerate callus formation in a rodent femoral fracture model.

Methods: Collagen disks with rhBMP-2 alone or with rhBMP-2 and SVAK-12 were implanted in a standard athymic rat chest ectopic model, and radiographic analysis was performed at four weeks. In a second set of rats (Sprague-Dawley), SVAK-12 was percutaneously injected into the site of a closed femoral fracture. The fractures were analyzed radiographically and biomechanically (with torsional testing) five weeks after surgery.

Results: In the ectopic model, there was dose-dependent enhancement of rhBMP-2 activity with use of SVAK-12 at doses of 100 to 500 μg. In the fracture model, the SVAK-12-treated group had significantly higher radiographic healing scores than the untreated group (p = 0.028). Biomechanical testing revealed that the fractured femora in the 200 to 250-μg SVAK-12 group were 43% stronger (p = 0.008) and 93% stiffer (p = 0.014) than those in the control group. In summary, at five weeks the femoral fracture group injected with SVAK-12 showed significantly improved radiographic and biomechanical evidence of healing compared with the controls.

Conclusions: A single local dose of a low-molecular-weight compound, SVAK-12, enhanced bone-healing in the presence of low-dose exogenous rhBMP-2 (in the ectopic model) and endogenous rhBMPs (in the femoral fracture model).

Clinical relevance: This study demonstrates that rhBMP-2 responsiveness can be enhanced by a novel small molecule, SVAK-12. Local application of anabolic small molecules has the potential for potentiating and accelerating fracture-healing. Use of this small molecule to lower required doses of rhBMPs might both decrease their cost and improve their safety profile.

Fig. 1

Ectopic bone induction at four weeks by SVAK-12. There was a dose-dependent increase in the rhBMP-2-induced bone formation as evidenced by the increasing mean bone scores with increasing doses of SVAK-12. Significant differences compared with implants treated with the vehicle (DMSO)—i.e., the control group—were found at the 100, 250, and 500-μg doses of SVAK-12 (*p < 0.02). Typical radiographs of the ectopic ossicles in the various treatment groups are shown below their respective bone scores. The radiopaque areas of the SVAK-12-treated groups (25, 50, 100, 250, 500, and 750 μg/100 μL) were larger and more dense than those in the control group.

Fig. 2

Representative histological image of a collagen disk containing rhBMP-2 and SVAK-12 four weeks after subcutaneous transplantation, showing ectopic trabecular bone formation and mineralized osteoid. This photomicrograph demonstrates that the bone formed has been deposited by osteoblasts and is not a chemical deposition of mineral (Gomori one-step trichrome stain, ×33).

Fig. 3

Radiographic healing scoring system. The radiographs of the femora were graded by four blinded observers independently using this scoring system. The three components of the scoring system included gap size, bone in the gap, and mineralized callus size. The composite score ranged from 0 to 6.

Fig. 4

The composite radiographic healing scores, which differed significantly between the vehicle-treated group and the SVAK-12-treated group (*p = 0.028).

Fig. 5-A

Fig. 5-B

Fig. 6-A

Fig. 6-B

Fig. 7

Effect of SVAK-12 injection on the healing fracture callus. A single percutaneous injection of SVAK-12 twenty-four hours after the fracture resulted in a thicker fracture callus (area defined by arrows) after five weeks (b) compared with that in the vehicle-injection group (a) (methylene blue/acid fuchsin stain, ×4).