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Abstract Background Bone repair is dependent on the presence of osteocompetent progenitors that are able to differentiate and generate new bone. Muscle is found in close association with orthopaedic injury, however its capacity to make a cellular contribution to bone repair remains ambiguous. We hypothesized that myogenic cells of the MyoD-lineage are able to contribute to bone repair. Methods We employed a MyoD-Cre+:Z/AP+ conditional reporter mouse in which all cells of the MyoD-lineage are permanently labeled with a human alkaline phosphatase (hAP) reporter. We tracked the contribution of MyoD-lineage cells in mouse models of tibial bone healing. Results In the absence of musculoskeletal trauma, MyoD-expressing cells are limited to skeletal muscle and the presence of reporter-positive cells in non-muscle tissues is negligible. In a closed tibial fracture model, there was no significant contribution of hAP+ cells to the healing callus. In contrast, open tibial fractures featuring periosteal stripping and muscle fenestration had up to 50% of hAP+ cells detected in the open fracture callus. At early stages of repair, many hAP+ cells exhibited a chondrocyte morphology, with lesser numbers of osteoblast-like hAP+ cells present at the later stages. Serial sections stained for hAP and type II and type I collagen showed that MyoD-lineage cells were surrounded by cartilaginous or bony matrix, suggestive of a functional role in the repair process. To exclude the prospect that osteoprogenitors spontaneously express MyoD during bone repair, we created a metaphyseal drill hole defect in the tibia. No hAP+ staining was observed in this model suggesting that the expression of MyoD is not a normal event for endogenous osteoprogenitors. Conclusions These data document for the first time that muscle cells can play a significant secondary role in bone repair and this knowledge may lead to important translational applications in orthopaedic surgery. Please see related article: http://www.biomedcentral.com/1741-7015/9/136

Abstract Background Transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) utilize parallel and related signaling pathways, however the interaction between these pathways in bone remains unclear. TGF-β 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-β1, and the TGF-β 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 μg 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-β1 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 μg or 192 μg 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.

Bone marrow transplantation (BMT) of healthy donor cells has been postulated as a strategy for treating osteogenesis imperfecta (OI) and other bone fragility disorders. The effect of engraftment by tail vein injection and/or marrow ablation by 6 Gy whole body irradiation were tested in Col1a2+/G610C (OI) mice as a model of mild-moderate OI. Dual-emission X-ray absorptiometry, microCT, and 4-point bending were used to measure bone volume (BV), bone mineral density (BMD), and biomechanical strength. BV, BMD, and mechanical strength were reduced in OI mice compared to wild type (WT) controls. BMT with and without irradiation yielded no difference in BV and BMD outcomes for both OI and WT mice, at 3 weeks. Transplantation of OI cells into OI mice to test for paracrine effects of BMT also showed no difference with non-transplanted OI mice. In a parallel cell tracking study, donor marrow was taken from transgenic mice constitutively expressing tdTomato and transplanted into WT mice. Lineage tracking demonstrated that irradiation considerably enhanced engraftment of tdTomato+ cells. However, tdTomato+ cells predominantly expressed TRAP and not AP, indicating engrafted donor cells were chiefly from the hematopoietic lineages. These data show that whole marrow transplantation fails to rescue the bone phenotype of Col1a2+/G610C (OI) mice and that osteopoietic engraftment is not significantly enhanced by irradiation. These findings are highly relevant to modern approaches focused on the gene repair of patient cells ex vivo and their subsequent reintroduction into the osteopoietic compartment via the circulation. [ABSTRACT FROM AUTHOR]

ABSTRACT: Tibial pseudarthrosis associated with Neurofibromatosis type 1 (NF1) is an orthopedic condition with consistently poor clinical outcomes. Using a murine model that features localized double inactivation of the Nf1 gene in an experimental tibial fracture, we tested the effects of recombinant human bone morphogenetic protein‐2 (rhBMP‐2) and/or the bisphosphonate zoledronic acid (ZA). Tibiae were harvested at 3 weeks for analysis, at which time there was negligible healing in un‐treated control fractures (7% union). In contrast, rhBMP‐2 and rhBMP‐2/ZA groups showed significantly greater union (87% and 93%, p < 0.01 for both). Treatment with rhBMP‐2 led to a 12‐fold increase in callus bone volume and this was further increased in the rhBMP‐2/ZA group. Mechanical testing of the healed rhBMP‐2 and rhBMP‐2/ZA fractures showed that the latter group had significantly higher mechanical strength and was restored to that of the un‐fractured contralateral leg. Co‐treatment with rhBMP‐2/ZA also reduced fibrous tissue infiltration at the fracture site compared to rhBMP alone (p = 0.068). These data support the future clinical investigation of this combination of anabolic and anti‐resorptive agents for the treatment of NF1 pseudarthrosis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:930–936, 2018. [ABSTRACT FROM AUTHOR]

Wnt antagonist Dkk1 is a negative regulator of bone formation and Dkk1 +/- heterozygous mice display a high bone mass phenotype. Complete loss of Dkk1 function disrupts embryonic head development. Homozygous Dkk1 -/- mice that were heterozygous for Wnt3 loss of function mutation (termed Dkk1 KO) are viable and allowed studying the effects of homozygous inactivation of Dkk1 on bone formation. Dkk1 KO mice showed a high bone mass phenotype exceeding that of heterozygous mice as well as a high incidence of polydactyly and kinky tails. Whole body bone density was increased in the Dkk1 KO mice as shown by longitudinal dual-energy X-ray absorptiometry. MicroCT analysis of the distal femur revealed up to 3-fold increases in trabecular bone volume and up to 2-fold increases in the vertebrae, compared to wild type controls. Cortical bone was increased in both the tibiae and vertebrae, which correlated with increased strength in tibial 4-point bending and vertebral compression tests. Dynamic histomorphometry identified increased bone formation as the mechanism underlying the high bone mass phenotype in Dkk1 KO mice, with no changes in bone resorption. Mice featuring only Wnt3 heterozygosity showed no evident bone phenotype. Our findings highlight a critical role for Dkk1 in the regulation of bone formation and a gene dose-dependent response to loss of DKK1 function. Targeting Dkk1 to enhance bone formation offers therapeutic potential for osteoporosis. [ABSTRACT FROM AUTHOR]

Neurofibromatosis Type 1 (NF1) is an autosomal dominant genetic disorder that results in a variety of characteristic manifestations. Prior studies have shown reduced muscle size and global skeletal muscle weakness in children with NF1. This associated weakness can lead to significant challenges impacting on quality of life. Pre-clinical studies using a muscle-specific NF1 knockout mouse have linked this weakness to an underlying primary metabolic deficiency in the muscle. However, the neonatal lethality of this strain prevents analysis of the role of NF1 in adult muscle. In this study, we present the characterization of an inducible muscle-specific NF1 knockout strain (Nf1Pax7i f/f ) produced by cross breeding the Pax7-CreERT2 strain with the conditional Nf1flox/flox line. Tamoxifen dosing of 8-week old Nf1Pax7i f/f mice led to recombination of the floxed allele in muscle, as detected by PCR. Detailed phenotypic analysis of treated adult mice over 8 weeks revealed no changes in bodyweight or muscle weight, no histological signs of myopathy, and no functional evidence of distress or impairment. Subsequent analysis using the Ai9 Cre-dependent tdTomato reporter strain was used to analyse labelling in embryos and in adult mice. Cell tracking studies identified a lower than expected rate of integration of recombined satellite cells into adult muscle. In contrast, a high persistent contribution of embryonic cells that were Pax7+ were found in adult muscle. These findings indicate important caveats with the use of the Pax7-CreERT2 strain and highlight a need to develop new tools for investigating the function of NF1 in mature muscle. [ABSTRACT FROM AUTHOR]

Cathepsin K inhibitors (CKIs) are an emerging class of drugs that are potent antagonists of osteoclastic activity. We speculated that they may be beneficial in bone tissue engineering, where a stress shielded environment can lead to rapid resorption of new bone. Most CKIs require frequent dosing, so to achieve a sustained release we manufactured polymer nanoparticles encapsulating the CKI L006235 (CKI/nP). CKI/nP and the collagen matrices that were used to deliver them were characterized by electron microscopy and fluorescent confocal microscopy, and data indicated that the particles were evenly distributed throughout the collagen. Elution studies indicated a linear release of the inhibitor from the CKI/nP, with approximately 2% of the drug being released per day. In an in vivo study, mice were implanted with collagen scaffolds containing rhBMP-2 that were loaded with the CKI/nP. Measurement of bone volume (BV) by microCT showed no significant increase with CKI/nP incorporation, and other parameters similarly showed no statistical differences. Cell culture studies confirmed the activity of the drug, even at low concentrations. These data indicate that polymer nanoparticles are an effective method for sustained drug delivery of a CKI, however, this may not be readily translatable to substantively improved bone tissue engineering outcomes. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 136-144, 2017. [ABSTRACT FROM AUTHOR]