Pretreatment with Pamidronate Decreases Bone Formation but Increases Callus Bone Volume in a Rat Closed Fracture Model.

Clinical concerns have been raised over prior exposure to bisphosphonates impairing fracture healing. To model this, groups of male Wistar rats were assigned to saline control or treatment groups receiving 0.15 mg/kg (low dose), 0.5 mg/kg (medium dose), and 5 mg/kg (high dose) Pamidronate (PAM) twice weekly for 4 weeks. At this point, closed fractures were made using an Einhorn apparatus, and bisphosphonate dosing was continued until the experimental endpoint. Specimens were analyzed at 2 and 6 weeks (N = 8 per group per time point). Twice weekly PAM dosing was found to have no effect on early soft callus remodeling at 2 weeks post fracture. At this time point, the highest dose PAM group gave significant increases in bone volume (+ 10%, p < 0.05), bone mineral content (+ 30%, p < 0.01), and bone mineral density (+ 10%, p < 0.01). This PAM dosing regimen showed more substantive effects on hard callus at 6 weeks post fracture, with PAM treatment groups showing + 46-79% increased bone volume. Dynamic bone labeling showed reduced calcein signal in the PAM-treated calluses (38-63%, p < 0.01) and reduced MAR (32-49%, p < 0.01), suggesting a compensatory reduction in bone anabolism. These data support the concept that bisphosphonates lead to profound decreases in bone turnover in fracture repair, however, this does not affect soft callus remodeling.

CSA-90 Promotes Bone Formation and Mitigates Methicillin-resistant Staphylococcus aureus Infection in a Rat Open Fracture Model.

BACKGROUND: Infection of open fractures remains a significant cause of morbidity and mortality to patients worldwide. Early administration of prophylactic antibiotics is known to improve outcomes; however, increasing concern regarding antimicrobial resistance makes finding new compounds for use in such cases a pressing area for further research. CSA-90, a synthetic peptidomimetic compound, has previously demonstrated promising antimicrobial action against Staphylococcus aureus in rat open fractures. However, its efficacy against antibiotic-resistant microorganisms, its potential as a therapeutic agent in addition to its prophylactic effects, and its proosteogenic properties all require further investigation. QUESTIONS/PURPOSES: (1) Does prophylactic treatment with CSA-90 reduce infection rates in a rat open fracture model inoculated with S aureus, methicillin-resistant S aureus (MRSA), and methicillin-resistant Staphylococcus epidermidis (MRSE) as measured by survival, radiographic union, and deep tissue swab cultures? (2) Does CSA-90 reduce infection rates when administered later in the management of an open fracture as measured by survival, radiographic union, and deep tissue swab cultures? (3) Does CSA-90 demonstrate a synergistic proosteogenic effect with bone morphogenetic protein 2 (BMP-2) in a noninfected rat ectopic bone formation assay as assessed by micro-CT bone volume measurement? (4) Can CSA-90 elute and retain its antimicrobial efficacy in vitro when delivered using clinically relevant agents measured using a Kirby-Bauer disc diffusion assay? METHODS: All in vivo studies were approved by the local animal ethics committee. In the open fracture studies, 12-week-old male Wistar rats underwent open midshaft femoral fractures stabilized with a 1.1-mm Kirschner wire and 10 µg BMP-2 ± 500 µg CSA-90 was applied to the fracture site using a collagen sponge along with 1 x 10 colony-forming units of bacteria (S aureus/MRSA/MRSE; n = 10 per group). In the delayed treatment study, débridement and treatment with 500 µg CSA-90 were performed at Day 1 and Day 5 after injury and bacterial insult (S aureus). All animals were reviewed daily for signs of local infection and/or sepsis. An independent, blinded veterinarian reviewed twice-weekly radiographs, and rats showing osteolysis and/or declining overall health were culled at his instruction. The primary outcome of both fracture studies was fracture infection, incorporating survival, radiographic union, and deep tissue swab cultures. For the ectopic bone formation assay, 0 to 10 µg BMP-2 and 0 to 500 µg CSA-90 were delivered on a collagen sponge into bilateral quadriceps muscle pouches of 8-week-old rats (n = 10 per group). Micro-CT quantification of bone volume and descriptive histologic analysis were performed for all in vivo studies. Modified Kirby-Bauer disc diffusion assays were used to quantify antimicrobial activity in vitro using four different delivery methods, including bone cement. RESULTS: Infection was observed in none of the MRSA inoculated open fractures treated with CSA-90 with 10 of 10 deep tissue swab cultures negative at the time of cull. Median survival was 43 days (range, 11-43 days) in the treated group versus 11 days (range, 8-11 days) in the untreated MRSA inoculated group (p < 0.001). However, delayed débridement and treatment of open fractures with CSA-90 at either Day 1 or Day 5 did not prevent infection, resulting in early culls by Day 21 with positive swab cultures (10 of 10 for each time point). Maximal ectopic bone formation was achieved with 500 µg CSA-90 and 10 µg BMP-2 (mean volume, 9.58 mm; SD, 7.83), creating larger bone nodules than formed with 250 µg CSA-90 and 10 µg BMP-2 (mean volume, 1.7 mm; SD, 1.07; p < 0.001). Disc diffusion assays showed that CSA-90 could successfully elute from four potential delivery agents including calcium sulphate (mean zone of inhibition, 11.35 mm; SD, 0.957) and bone cement (mean, 4.67 mm; SD, 0.516). CONCLUSIONS: CSA-90 shows antimicrobial action against antibiotic-resistant Staphylococcal strains in vitro and in an in vivo model of open fracture infection. CLINICAL RELEVANCE: The antimicrobial properties of CSA-90 combined with further evidence of its proosteogenic potential make it a promising compound to develop further for orthopaedic applications.

Developmental dosing with a MEK inhibitor (PD0325901) rescues myopathic features of the muscle-specific but not limb-specific Nf1 knockout mouse.

Neurofibromatosis Type 1 (NF1) is a common autosomal dominant genetic disorder While NF1 is primarily associated with predisposition for tumor formation, muscle weakness has emerged as having a significant impact on quality of life. NF1 inactivation is linked with a canonical upregulation Ras-MEK-ERK signaling. This in this study we tested the capacity of the small molecule MEK inhibitor PD0325901 to influence the intramyocellular lipid accumulation associated with NF1 deficiency. Established murine models of tissue specific Nf1 deletion in skeletal muscle (Nf1MyoD-/-) and limb mesenchyme (Nf1Prx1-/-) were tested. Developmental PD0325901 dosing of dams pregnant with Nf1MyoD-/- progeny rescued the phenotype of day 3 pups including body weight and lipid accumulation by Oil Red O staining. In contrast, PD0325901 treatment of 4 week old Nf1Prx1-/- mice for 8 weeks had no impact on body weight, muscle wet weight, activity, or intramyocellular lipid. Examination of day 3 Nf1Prx1-/- pups showed differences between the two tissue-specific knockout strains, with lipid staining greatest in Nf1MyoD-/- mice, and fibrosis higher in Nf1Prx1-/- mice. These data show that a MEK/ERK dependent mechanism underlies NF1 muscle metabolism during development. However, crosstalk from Nf1-deficient non-muscle mesenchymal cells may impact upon muscle metabolism and fibrosis in neonatal and mature myofibers.

Sclerostin antibody enhances bone formation in a rat model of distraction osteogenesis.

Neutralizing monoclonal sclerostin antibodies are effective in promoting bone formation at a systemic level and in orthopedic scenarios including closed fracture repair. In this study we examined the effects of sclerostin antibody (Scl-Ab) treatment on regenerate volume, density, and strength in a rat model of distraction osteogenesis. Surgical osteotomy was performed on 179 Sprague Dawley rats. After 1 week, rats underwent distraction for 2 weeks, followed by 6 weeks for consolidation. Two treatment groups received biweekly subcutaneous Scl-AbIII (a rodent form of Scl-Ab; 25 mg/kg), either from the start of distraction onward or restricted to the consolidation phase. These groups were compared to controls receiving saline. Measurement modalities included longitudinal DXA, ex vivo QCT, and microCT, tissue histology, and biomechanical four-point bending tests. Bone volume was increased in both Scl-Ab treatments regimens by the end of consolidation (+26-38%, p < 0.05), as assessed by microCT. This was associated with increased mineral apposition. Importantly, Scl-Ab led to increased strength in united bones, and this reached statistical significance in animals receiving Scl-Ab during consolidation only (+177%, p < 0.01, maximum load to failure). These data demonstrate that Scl-Ab treatment increases bone formation, leading to regenerates with higher bone volume and improved strength. Our data also suggest that the optimal effects of Scl-Ab treatment are achieved in the latter stages of distraction osteogenesis. These findings support further investigation into the potential clinical application of sclerostin antibody to augment bone distraction, such as limb lengthening, particularly in the prevention of refracture. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1106-1113, 2018.

Dietary intervention rescues myopathy associated with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder with complex symptomology. In addition to a predisposition to tumors, children with NF1 can present with reduced muscle mass, global muscle weakness, and impaired motor skills, which can have a significant impact on quality of life. Genetic mouse models have shown a lipid storage disease phenotype may underlie muscle weakness in NF1. Herein we confirm that biopsy specimens from six individuals with NF1 similarly manifest features of a lipid storage myopathy, with marked accumulation of intramyocellular lipid, fibrosis, and mononuclear cell infiltrates. Intramyocellular lipid was also correlated with reductions in neurofibromin protein expression by western analysis. An RNASeq profile of Nf1null muscle from a muscle-specific Nf1 knockout mouse (Nf1MyoD-/-) revealed alterations in genes associated with glucose regulation and cell signaling. Comparison by lipid mass spectrometry demonstrated that Nf1null muscle specimens were enriched for long chain fatty acid (LCFA) containing neutral lipids, such as cholesterol esters and triacylglycerides, suggesting fundamentally impaired LCFA metabolism. The subsequent generation of a limb-specific Nf1 knockout mouse (Nf1Prx1-/-) recapitulated all observed features of human NF1 myopathy, including lipid storage, fibrosis, and muscle weakness. Collectively, these insights led to the evaluation of a dietary intervention of reduced LCFAs, and enrichment of medium-chain fatty acids (MCFAs) with L-carnitine. Following 8-weeks of dietary treatment, Nf1Prx1-/- mice showed a 45% increase in maximal grip strength, and a 71% reduction in intramyocellular lipid staining compared with littermates fed standard chow. These data link NF1 deficiency to fundamental shifts in muscle metabolism, and provide strong proof of principal that a dietary intervention can ameliorate symptoms.

Homozygous Dkk1 Knockout Mice Exhibit High Bone Mass Phenotype Due to Increased Bone Formation.

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.

Improved union and bone strength in a mouse model of NF1 pseudarthrosis treated with recombinant human bone morphogenetic protein-2 and zoledronic acid.

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.

Limitations of the Pax7-creERT2 transgene for driving deletion of Nf1 in adult mouse muscle.

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-CreER T2 strain and highlight a need to develop new tools for investigating the function of NF1 in mature muscle.

Combination sclerostin antibody and zoledronic acid treatment outperforms either treatment alone in a mouse model of osteogenesis imperfecta.

In this study, we examined the therapeutic potential of anti-Sclerostin Antibody (Scl-Ab) and bisphosphonate treatments for the bone fragility disorder Osteogenesis Imperfecta (OI). Mice with the Amish OI mutation (Col1a2 G610C mice) and control wild type littermates (WT) were treated from week 5 to week 9 of life with (1) saline (control), (2) zoledronic acid given 0.025mg/kg s.c. weekly (ZA), (3) Scl-Ab given 50mg/kg IV weekly (Scl-Ab), or (4) a combination of both (Scl-Ab/ZA). Functional outcomes were prioritized and included bone mineral density (BMD), bone microarchitecture, long bone bending strength, and vertebral compression strength. By dual-energy absorptiometry, Scl-Ab treatment alone had no effect on tibial BMD, while ZA and Scl-Ab/ZA significantly enhanced BMD by week 4 (+16% and +27% respectively, P<0.05). Scl-Ab/ZA treatment also led to increases in cortical thickness and tissue mineral density, and restored the tibial 4-point bending strength to that of control WT mice. In the spine, all treatments increased compression strength over controls, but only the combined group reached the strength of WT controls. Scl-Ab showed greater anabolic effects in the trabecular bone than in cortical bone. In summary, the Scl-Ab/ZA intervention was superior to either treatment alone in this OI mouse model, however further studies are required to establish its efficacy in other preclinical and clinical scenarios.

Sclerostin Antibody Increases Callus Size and Strength but does not Improve Fracture Union in a Challenged Open Rat Fracture Model.

Open fractures remain a challenge in orthopedics. Current strategies to intervene are often inadequate, particularly in severe fractures or when treatment is delayed. Sclerostin is a negative regulator of bone growth and sclerostin-neutralizing antibodies (Scl-Ab) can increase bone mass and strength. The application of these antibodies to improve orthopedic repair has shown varied results, and may be dependent on the location and severity of the bony injury. We examined Scl-Ab treatment within an established rat osteotomy model with periosteal stripping analogous to open fracture repair. In one study, Scl-Ab was given 25 mg/kg bi-weekly, either from the time of fracture or from 3 weeks post-fracture up to an end-point of 12 weeks. A second study treated only delayed union open fractures that did not show radiographic union by week 6 post-fracture. Outcome measures included radiographic union, microCT analysis of bone volume and architecture, and histology. In the first study, Scl-Ab given from either 0 or 3 weeks significantly improved callus bone volume (+52%, p < 0.05 and +58%, p < 0.01) at 12 weeks, as well as strength (+48%, p < 0.05 and +70%, p < 0.05). Despite these improvements, union rate was not changed. In the second study treating only established delayed fractures, bony callus volume was similarly increased by Scl-Ab treatment; however, this did not translate to increased biomechanical strength or union improvement. Sclerostin antibody treatment has limited effects on the healing of challenging open fractures with periosteal stripping, but shows the greatest benefits on callus size and strength with earlier intervention.

Local co-delivery of rhBMP-2 and cathepsin K inhibitor L006235 in poly(d,l-lactide-co-glycolide) nanospheres.

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.

RAP-011 augments callus formation in closed fractures in rats.

ACE-011 is a bone anabolic agent generated by fusing the extracellular domain of the Activin Type 2A receptor (ActRIIA) to an IgG-Fc. The orthopedic utility of ACE-011 was investigated using a murine analogue, RAP-011. Initially, a rat closed fracture model was tested using bi-weekly (biw) 10 mg/kg RAP-011. RAP-011 significantly increased callus length and callus bone volume (BV, +43% at 6w, p < 0.01). The polar moment of inertia was calculated to be substantively increased (+80%, p < 0.01), however mechanical bending tests showed a more modest increase in maximum load to failure (+24%, p < 0.05). Histology indicated enhanced appositional bone growth, but it was hypothesized that reduced remodeling, evidenced by decreased serum CTX (-16% at 6w, p < 0.01), could be compromising bone quality in the callus. A second closed fracture study was performed to examine lower "pulse" [RAP-011(p)] and "sustained" [RAP-011(s)] regimens of biw 0.6mg/kg × 2, 0.35mg/kg × 3 and 0.18mg/kg × 2, 0.1mg/kg × 7 respectively, compared with PTH(1-34) (25 µg/kg/d) and vehicle controls. RAP-011 treatments gave modest increases in callus length and callus BV at 6w (p < 0.01), but did not achieve an increase in maximum load over vehicle. In summary, RAP-011 is effective in promoting bone formation during repair, but optimizing callus bone quality will require further investigation.

Lineage tracking of mesenchymal and endothelial progenitors in BMP-induced bone formation.

To better understand the relative contributions of mesenchymal and endothelial progenitor cells to rhBMP-2 induced bone formation, we examined the distribution of lineage-labeled cells in Tie2-Cre:Ai9 and αSMA-creERT2:Col2.3-GFP:Ai9 reporter mice. Established orthopedic models of ectopic bone formation in the hind limb and spine fusion were employed. Tie2-lineage cells were found extensively in the ectopic bone and spine fusion masses, but co-staining was only seen with tartrate-resistant acid phosphatase (TRAP) activity (osteoclasts) and CD31 immunohistochemistry (vascular endothelial cells), and not alkaline phosphatase (AP) activity (osteoblasts). To further confirm the lack of a functional contribution of Tie2-lineage cells to BMP-induced bone, we developed conditional knockout mice where Tie2-lineage cells are rendered null for key bone transcription factor osterix (Tie2-cre:Osx(fx/fx) mice). Conditional knockout mice showed no difference in BMP-induced bone formation compared to littermate controls. Pulse labeling of mesenchymal cells with Tamoxifen in mice undergoing spine fusion revealed that αSMA-lineage cells contributed to the osteoblastic lineage (Col2.3-GFP), but not to endothelial cells or osteoclast populations. These data indicate that the αSMA+ and Tie2+ progenitor lineages make distinct cellular contributions to bone formation, angiogenesis, and resorption/remodeling.

Local delivery of the cationic steroid antibiotic CSA-90 enables osseous union in a rat open fracture model of Staphylococcus aureus infection.

BACKGROUND: Treatment of infected open fractures remains a major clinical challenge. In this study, we investigated the novel broad-spectrum antibiotic CSA-90 (cationic steroid antibiotic-90) as an antimicrobial agent. METHODS: CSA-90 was screened in an osteoblast cell culture model for effects on differentiation and mineralization. Local delivery of CSA-90 was then tested alone and in combination with recombinant human bone morphogenetic protein-2 (rhBMP-2) in a mouse ectopic bone formation model (n=40 mice) and in a rat open fracture model inoculated with pathogenic Staphylococcus aureus (n=84 rats). RESULTS: CSA-90 enhanced matrix mineralization in cultured osteoblasts and increased rhBMP-2-induced bone formation in vivo. All animals in which an open fracture had been inoculated with Staphylococcus aureus and not treated with local CSA-90, including those treated with rhBMP-2, had to be culled prior to the experimental end point (six weeks) because of localized osteolysis and deterioration of overall health, whereas CSA-90 prevented establishment of infection in all open fractures in which it was used (p≤0.012). Increased union rates were seen for the fractures treated with rhBMP-2 or with the combination of rhBMP-2 and CSA-90 compared with that observed for the fractures treated with CSA-90 alone (p=0.04). CONCLUSIONS: CSA-90 can promote osteogenesis and be used for prevention of Staphylococcus aureus infection in preclinical models. CLINICAL RELEVANCE: Local delivery of CSA-90 represents a novel strategy for prevention of infection and may have specific benefits in the context of orthopaedic injuries.

Inducible cell labeling and lineage tracking during fracture repair.

Mouse models incorporating inducible Cre-ERT2/LoxP recombination coupled with sensitive fluorescent reporter lines are being increasingly used to track cell lineages in vivo. In this study we use two inducible reporter strains, Ai9iCol2a1 (Ai9×Col2a1-creERT2) to track contribution of chondrogenic progenitors during bone regeneration in a closed fracture model and Ai9i UBC (Ai9×UBC-creERT2) to examine methods for inducing localized recombination. By comparing with Ai9 littermate controls as well as inducible reporter mice not dosed with tamoxifen, we revealed significant leakiness of the CreERT2 system, particularly in the bone marrow of both lines. These studies highlight the challenges associated with highly sensitive reporters that may be activated without induction in tissues where the CreERT2 fusion is expressed. Examination of the growth plate in the Ai9iCol2a1 strain showed cells of the osteochondral lineage (cell co-staining with chondrocyte and osteoblast markers) labeled with the tdTom reporter. However, no such labeling was noted in healing fractures of Ai9iCol2a1 mice. Attempts to label a single limb using intramuscular injection of 4-hydroxytamoxifen in the Ai9i UBC strain resulted in complete labeling of the entire animal, comparable to intraperitoneal injection. While a challenge to interpret, these data are nonetheless informative regarding the limitations of these inducible reporter models, and justify caution and expansive controls in future studies using such models.

PTH(1-34) Treatment Increases Bisphosphonate Turnover in Fracture Repair in Rats.

Bisphosphonates (BP) are antiresorptive drugs with a high affinity for bone. Despite the therapeutic success in treating osteoporosis and metabolic bone diseases, chronic BP usage has been associated with reduced repair of microdamage and atypical femoral fracture (AFF). The latter has a poor prognosis, and although anabolic interventions such as teriparatide (PTH(1-34) ) have been suggested as treatment options, there is a limited evidence base in support of their efficacy. Because PTH(1-34) acts to increase bone turnover, we hypothesized that it may be able to increase BP in turnover in the skeleton, which, in turn, may improve bone healing. To test this, we employed a mixture of fluorescent Alexa647-labelled pamidronate (Pam) and radiolabeled (14) C-ZA (zoledronic acid). These traceable BPs were dosed to Wistar rats in models of normal growth and closed fracture repair. Rats were cotreated with saline or 25 µg/kg/d PTH(1-34) , and the effects on BP liberation and bone healing were examined by X-ray, micro-CT, autoradiography, and fluorescent confocal microscopy. Consistent with increased BP remobilization with PTH(1-34) , there was a significant decrease in fluorescence in both the long bones and in the fracture callus in treated animals compared with controls. This was further confirmed by autoradiography for (14) C-ZA. In this model of acute BP treatment, callus bone volume (BV) was significantly increased in fractured limbs, and although we noted significant decreases in callus-bound BP with PTH(1-34) , these were not sufficient to alter this BV. However, increased intracellular BP was noted in resorbing osteoclasts, confirming that, in principle, PTH(1-34) increases bone turnover as well as BP turnover.

Activated protein C (APC) can increase bone anabolism via a protease-activated receptor (PAR)1/2 dependent mechanism.

Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP-2) induced ectopic bone formation model. Local co-administration of 10 µg rhBMP-2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (p<0.01) compared to rhBMP-2 alone. This was associated with a significant increase in CD31+ and TRAP+ cells in tissue sections of ectopic bone, consistent with enhanced vascularity and bone turnover. The actions of APC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease-activated receptors (PARs). Cultured pre-osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre-osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP-2 for bone repair.

Local delivery of recombinant human bone morphogenetic proteins and bisphosphonate via sucrose acetate isobutyrate can prevent femoral head collapse in Legg-Calve-Perthes disease: a pilot study in pigs.

PURPOSE: Legg-Calve-Perthes disease is a paediatric condition encompassing idiopathic osteonecrosis of the femoral head (ONFH). Preventing collapse and the need for subsequent joint replacement remains the major goal of clinical management. This exploratory study utilises a porcine model of surgically induced ONFH. METHODS: rhBMP-2 with and without zoledronic acid (ZA) was delivered by intra-osseous injection in the phase-transitioning sucrose acetate isobutyrate (SAIB) in an attempt to prevent femoral head collapse. Epiphyseal quotient (EQ) at eight weeks post-surgery was the primary outcome measure. Heterotopic ossification in the joint capsule and bisphosphonate retention in the femoral head were key secondary outcomes. RESULTS: Femoral heads with ONFH and no treatment all collapsed (3/3, EQ < 0.4, P < 0.05 compared to no ONFH). Local delivery of rhBMP-2/SAIB into the femoral head prevented collapse by EQ measurement one of four samples; however, this specimen still showed evidence of significant collapse. In contrast, the combination of local rhBMP-2 and local ZA prevented collapse in two of four samples. Confocal fluorescence microscopy showed locally dosed bisphosphonate entered and was retained in the femoral head. This group also showed strong Calcein signal, indicating new bone formation. Treatment with rhBMP-2 was associated with a limited amount of heterotrophic ossification in the joint capsules in some specimens. CONCLUSIONS: Operators reported SAIB to be an efficient way to deliver rhBMP-2 to the femoral head. These data suggest that rhBMP-2 is ineffective for preventing femoral head collapse without the addition of bisphosphonate. Further research will be required to validate the clinical efficacy of a combined local rhBMP-2/bisphosphonate approach.

A collagen-hydroxyapatite scaffold allows for binding and co-delivery of recombinant bone morphogenetic proteins and bisphosphonates.

An emerging paradigm in orthopedics is that a bone-healing outcome is the product of the anabolic (bone-forming) and catabolic (bone-resorbing) outcomes. Recently, surgical and tissue engineering strategies have emerged that combine recombinant human bone morphogenetic proteins (rhBMPs) and bisphosphonates (BPs) in order to maximize anabolism and minimize catabolism. Collagen-based scaffolds that are the current surgical standard can bind rhBMPs, but not BPs. We hypothesized that a biomimetic collagen-hydroxyapatite (CHA) scaffold would bind both agents and produce superior in vivo outcomes. Consistent with this concept, in vitro elution studies utilizing rhBMP-2 ELISA assays and scintillation counting of (14)C-radiolabeled zoledronic acid (ZA) confirmed delayed release of both agents from the CHA scaffold. Next, scaffolds were tested for their capacity to form ectopic bone after surgical implantation into the rat hind limb. Using CHA, a significant 6-fold increase in bone volume was seen in rhBMP-2/ZA groups compared to rhBMP-2 alone, confirming the ability of ZA to enhance rhBMP-2 bone formation. CHA scaffolds were found to be capable of generating mineralized tissue in the absence of rhBMP-2. This study has implications for future clinical treatments of critical bone defects. It demonstrates the relative advantages of co-delivering anabolic and anti-catabolic agents using a multicomponent scaffold system.

Neuropeptide Y modulates fracture healing through Y1 receptor signaling.

Neuropeptide Y acting via it's Y1 receptor represents a powerful pathway in the control of bone mass. The global or osteoblast-specific Y1 receptor deletion induces pronounced bone anabolic effects in mice. However, the contribution of Y1 receptor deletion in bone repair/healing remained to be clarified. Therefore, in this study we characterized the role of Y1 receptor deletion in fracture healing. Closed tibial fractures were generated in germline (Y1 (-/-) ) and osteoblastic-specific Y1 receptor knockout mice. The progression of tibial repair monitored from 1- until 6-weeks post-fracture demonstrated that in Y1 (-/-) mice there is a delay in fracture repair, as seen by a decrease in bone callus volume and callus strength. Moreover, the histological features included elevated avascular and cartilage area and consequently delayed cartilage removal, and hence impaired union. Interestingly, this delay in bone repair was not related directly to Y1 receptors expressed by mature osteoblasts. These findings suggest that the global absence of the Y1 receptor delays fracture healing, through impairing the early phases of fracture repair to achieve bony union. The data acquired on the role of Y1 receptor signaling disruption in bone regeneration is critical for the design of future therapeutic strategies.