Endochondral fracture healing with external fixation in the Sost knockout mouse results in earlier fibrocartilage callus removal and increased bone volume fraction and strength.

Sclerostin deficiency, via genetic knockout or anti-Sclerostin antibody treatment, has been shown to cause increased bone volume, density and strength of calluses following endochondral bone healing. However, there is limited data on the effect of Sclerostin deficiency on the formative early stage of fibrocartilage (non-bony tissue) formation and removal. In this study we extensively investigate the early fibrocartilage callus. Closed tibial fractures were performed on Sost(-/-) mice and age-matched wild type (C57Bl/6J) controls and assessed at multiple early time points (7, 10 and 14days), as well as at 28days post-fracture after bony union. External fixation was utilized, avoiding internal pinning and minimizing differences in stability stiffness, a variable that has confounded previous research in this area. Normal endochondral ossification progressed in wild type and Sost(-/-) mice with equivalent volumes of fibrocartilage formed at early day 7 and day 10 time points, and bony union in both genotypes by day 28. There were no significant differences in rate of bony union; however there were significant increases in fibrocartilage removal from the Sost(-/-) fracture calluses at day 14 suggesting earlier progression of endochondral healing. Earlier bone formation was seen in Sost(-/-) calluses over wild type with greater bone volume at day 10 (221%, p<0.01). The resultant Sost(-/-) united bony calluses at day 28 had increased bone volume fraction compared to wild type calluses (24%, p<0.05), and the strength of the fractured Sost(-/-) tibiae was greater than that that of wild type fractured tibiae. In summary, bony union was not altered by Sclerostin deficiency in externally-fixed closed tibial fractures, but fibrocartilage removal was enhanced and the resultant united bony calluses had increased bone fraction and increased strength.

A Combination of rhBMP-2 (Recombinant Human Bone Morphogenetic Protein-2) and MEK (MAP Kinase/ERK Kinase) Inhibitor PD0325901 Increases Bone Formation in a Murine Model of Neurofibromatosis Type I Pseudarthrosis.

BACKGROUND: Congenital tibial dysplasia is a severe pediatric condition that classically results in a persistent pseudarthrosis. A majority of these cases are associated with neurofibromatosis type I (NF1), a genetic disorder in which inactivation of the NF1 gene leads to overactivity of the Ras-MEK-MAPK (mitogen-activated protein kinase) signaling pathway. We therefore hypothesized that pharmaceutical inhibition of MEK-MAPK may be a beneficial therapeutic strategy. METHODS: In vitro methods were used to demonstrate a role for the MEK inhibitor PD0325901 in promoting osteogenic differentiation in Nf1-/- calvarial osteoblasts. Local applications of rhBMP-2 and/or PD0325901 were then tested in a mouse model of NF1 tibial pseudarthrosis featuring localized double inactivation of the Nf1 gene in a fracture. Mice received no treatment, PD0325901 (10 mg/kg/day from two days before fracture to ten days after fracture), rhBMP-2 (10 µg), or a combination of rhBMP-2 and PD0325901. RESULTS: Animals treated with the delivery vehicle alone, PD0325901, rhBMP-2, or the PD0325901 + rhBMP-2 combination showed union rates of 0%, 8%, 69% (p < 0.01), or 80% (p < 0.01), respectively, at twenty-one days after fracture. Mice treated with the rhBMP-2 + PD0325901 combination displayed a callus volume sixfold greater than the vehicle controls and twofold greater than the group receiving rhBMP-2 alone. Although MEK inhibition combined with rhBMP-2 led to increases in bone formation and union, the proportion of fibrous tissue in the callus was not significantly reduced. CONCLUSIONS: The data suggest that MEK inhibition can promote bone formation in combination with rhBMP-2 in the context of an NF1 pseudarthrosis. However, PD0325901 did not promote substantive bone anabolism in the absence of an exogenous anabolic stimulus and did not suppress fibrosis. CLINICAL RELEVANCE: This study examines a signaling pathway-based approach to treating poor bone healing in a model of NF1 pseudarthrosis.

Spatial control of bone formation using a porous polymer scaffold co-delivering anabolic rhBMP-2 and anti-resorptive agents.

Current clinical delivery of recombinant human bone morphogenetic proteins (rhBMPs) utilises freeze-dried collagen. Despite effective new bone generation, rhBMP via collagen can be limited by significant complications due to inflammation and uncontrolled bone formation. This study aimed to produce an alternative rhBMP local delivery system to permit more controllable and superior rhBMP-induced bone formation. Cylindrical porous poly(lactic-co-glycolic acid) (PLGA) scaffolds were manufactured by thermally-induced phase separation. Scaffolds were encapsulated with anabolic rhBMP-2 (20 µg) ± anti-resorptive agents: zoledronic acid (5 µg ZA), ZA pre-adsorbed onto hydroxyapatite microparticles, (5 µg ZA/2% HA) or IkappaB kinase (IKK) inhibitor (10 µg PS-1145). Scaffolds were inserted in a 6-mm critical-sized femoral defect in Wistar rats, and compared against rhBMP-2 via collagen. The regenerate region was examined at 6 weeks by 3D microCT and descriptive histology. MicroCT and histology revealed rhBMP-induced bone was more restricted in the PLGA scaffolds than collagen scaffolds (-92.3% TV, p < 0.01). The regenerate formed by PLGA + rhBMP-2/ZA/HA showed comparable bone volume to rhBMP-2 via collagen, and bone mineral density was +9.1% higher (p < 0.01). Local adjunct ZA/HA or PS-1145 significantly enhanced PLGA + rhBMP-induced bone formation by +78.2% and +52.0%, respectively (p ≤ 0.01). Mechanistically, MG-63 human osteoblast-like cells showed cellular invasion and proliferation within PLGA scaffolds. In conclusion, PLGA scaffolds enabled superior spatial control of rhBMP-induced bone formation over clinically-used collagen. The PLGA scaffold has the potential to avoid uncontrollable bone formation-related safety issues and to customise bone shape by scaffold design. Moreover, local treatment with anti-resorptive agents incorporated within the scaffold further augmented rhBMP-induced bone formation.

Modulation of endochondral ossification by MEK inhibitors PD0325901 and AZD6244 (Selumetinib).

MEK inhibitors (MEKi) PD0325901 and AZD6244 (Selumetinib) are drugs currently under clinical investigation for cancer treatment, however the Ras-MAPK pathway is also an important mediator of normal bone cell differentiation and function. In this study we examined the effects of these compounds on endochondral processes using both in vitro and in vivo models. Treatment with PD0325901 or AZD6244 significantly increased Runx2 and Alkaline phosphate gene expression in calvarial osteoblasts and decreased TRAP+ cells in induced osteoclast cultures. To test the effects of these drugs on bone healing, C57/Bl6 mice underwent a closed tibial fracture and were treated with PD0325901 or AZD6244 at 10mg/kg/day. Animals were culled at day 10 and at day 21 post-fracture for analysis of the fracture callus and the femoral growth plate in the contralateral leg. MEKi treatment markedly increased cartilage volume in the soft callus at day 10 post-fracture (+60% PD0325901, +20% AZD6244) and continued treatment led to a delay in cartilage remodeling. At the growth plate, we observed an increase in the height of the hypertrophic zone relative to the proliferative zone of +78% in PD0325901 treated mice. Osteoclast surface was significantly decreased both at the terminal end of the growth plate and within the fracture calluses of MEKi treated animals. The mechanistic effects of MEKi on genes encoding cartilage matrix proteins and catabolic enzymes were examined in articular chondrocyte cultures. PD0325901 or AZD6244 led to increased matrix protein expression (Col2a1 and Acan) and decreased expression of catabolic factors (Mmp13 and Adamts-5). Taken together, these data support the hypothesis that MEKi treatment can impact chondrocyte hypertrophy, matrix resorption, and fracture healing. These compounds can also affect bone architecture by expanding the hypertrophic zone of the growth plate and reducing osteoclast surface systemically.