Sclerostin is a negative regulator of osteoblast differentiation and bone formation. Expressed by osteocytes, it acts through antagonising the Wnt/â-catenin pathway and/or BMP activity. Distraction osteogenesis, used for limb lengthening and reconstruction, can be complicated by disuse osteopenia and poor healing response, both of which would benefit from pro anabolic therapy.

We examined the effects of Sclerostin Antibody (Scl-AbIII, Amgen Inc.,) in a rat model of distraction osteogenesis. A femoral osteotomy was stabilized with an external fixator in male Sprague Dawley rats. After a week of latency, the gap was distracted twice daily for 14 days to a total of 7 mm. Saline or Scl-Ab was administered twice weekly throughout the distraction period and up to 4, 6 or 8 weeks post commencement of distraction. Three groups were examined: Saline, Continuous Scl-Ab throughout the study (C Scl-Ab), and Delayed Scl-Ab with commencement of Scl-Ab after distraction (D Scl-Ab).

Regenerate bone mineral content (BMC), determined by DEXA, was increased 36% at 4 weeks and 86% at 6 weeks with C Scl-Ab, resulting in a 65% increase in bone mineral density (BMD) at 6 weeks, compared with Saline (p<0.01). D Scl-Ab treatment showed a 41% increase in BMC and a 31% increase in BMD compared with Saline at 6 weeks (p<0.05). At 8 weeks, C Scl-Ab remained significantly increased over Saline (72% in BMC; 60% in BMD).

Micro-CT scans of the regenerate revealed increases in bone volume of 88% with C Scl Ab and 65% with D Scl-Ab compared with Saline at 6 weeks (p<0.05). By 8 weeks, these increases were 36% for C Scl-Ab (p<0.05) and 37% for D Scl-Ab compared with Saline (p<0.01). Importantly, mean moment of inertia was increased over two-fold in both Scl-Ab groups at 6 weeks compared with Saline (p<0.05). Histology at 6 weeks confirmed micro-CT data with 85–88% increases in bone volume/tissue volume (BV/TV) in the regenerate with both C Scl-Ab and D Scl-Ab compared with Saline (p<0.05). Analysis of bone formation at 6 weeks revealed increases in mineral apposition rate of 56% in C Scl-Ab and 52% in D Scl-Ab compared with Saline (p<0.05).

Scl-Ab treatment increased bone formation in this model of distraction osteogenesis, resulting in a larger regenerate callus (increased BMC and BV/TV). We expect further studies to reveal increases in mechanical strength. Scl-Ab may hold promise as a therapeutic to accelerate regenerate formation and consolidation in distraction osteogenesis for limb reconstruction.

Bone morphogenetic proteins (BMPs) are able to induce osteogenic differentiation in many cells, including muscle cells. However, the actual contribution of muscle cells to bone formation and repair is unclear. Our objective was to examine the capacity of myogenic cells to contribute to BMP-induced ectopic bone formation and fracture repair.

Osteogenic gene expression was measured by quantitative PCR in osteoprogenitors, myoblasts, and fibroblasts following BMP-2 treatment. The MyoD-Cre x ROSA26R and MyoD-Cre x Z/AP mouse strains were used to track the fate of MyoD+ cells in vivo. In these double-transgenic mice, MyoD+ progenitors undergo a permanent recombination event to induce reporter gene expression. Ectopic bone was produced by the intramuscular implantation of BMP-7. Closed tibial fractures and open tibial fractures with periosteal stripping were also performed. Cellular contribution was tracked at one, two and three week time points by histological staining.

Osteoprogenitors and myoblasts exhibited comparable expression of early and late bone markers; in contrast bone marker expression was considerably less in fibroblasts. The sensitivity of cells to BMP-2 correlated with the expression of BMP receptor-1a (Bmpr1a). Pilot experiments using the MyoD-Cre x Rosa26R mice identified a contribution by MyoD expressing cells in BMP-induced ectopic bone formation. However, false positive LacZ staining in osteoclasts led us to seek alternative systems such as the MyoD-cre x Z/AP mice that have negligible background staining. Initially, a minor contribution from MyoD expressing cells was noted in the ectopic bones in the MyoD-cre x Z/AP mice, but without false positive osteoclast staining. Soft tissue trauma usually precedes the formation of ectopic bone. Hence, to mimic the clinical condition more precisely, physical injury to the muscle was performed. Traumatising the muscle two days prior to BMP-7 implantation: (1) induced MyoD expression in quiescent satellite cells; (2) increased ectopic bone formation; and (3) greatly enhanced the number of MyoD positive cells in the ectopic bone. In open tibial fractures the majority of the initial callus was MyoD+ indicating a significant contribution by myogenic cells. In contrast, closed fractures with the periosteum intact had a negligible myogenic contribution.

Myoblasts but not fibroblasts were highly responsive to BMP stimulation and this was associated with BMP receptor expression. Our transgenic mouse models demonstrate for the first time that muscle progenitors can significantly contribute to ectopic bone formation and fracture repair. This may have translational applications for clinical orthopaedic therapies.