VEGFA from osteoblasts is not required for lamellar bone formation following tibial loading.

The relationship between osteogenesis and angiogenesis is complex. Normal bone development requires angiogenesis, mediated by vascular endothelial growth factor A (VEGFA). Studies have demonstrated through systemic inhibition or genetic modification that VEGFA is indispensable for several types of bone repair, presumably via its role in supporting angiogenesis. But a direct role for VEGFA within osteoblasts, in the absence of angiogenesis, has also been suggested. To address the question of whether VEGFA from osteoblasts supports bone formation directly, we applied anabolic loading to induce lamellar bone formation in mice, a process shown to be independent of angiogenesis. We hypothesized that VEGFA from osteoblasts is required for lamellar bone formation. To test this hypothesis, we applied axial tibial compression to inducible Cre/LoxP mice from three lines. Vegfafl/fl mice were crossed with Ubiquitin C (UBC), Osterix (Osx) and Dentin-Matrix Protein 1 (DMP1) Cre-ERT2 mice to target all cells, (pre)osteoblast-lineage cells, and mature osteoblasts and osteocytes, respectively. Genotype effects were determined by comparing control (Vegfafl/fl) and Cre+ (VegfaΔ) mice for each line. At 5 months of age tamoxifen was injected for 5 days followed by a 3-week clearance prior to loading. Female and male mice (N = 100) were loaded for 5 days to peak forces to engender -3100 µÎµ peak compressive strain and processed for dynamic histomorphometry (day 12). Percent MS/BS increased 20-70 % as a result of loading, with no effect of genotype in Osx or Dmp1 lines. In contrast, the UBC groups had a significant decrease in relative periosteal BFR/BS in VegfaΔ vs. Vegfafl/fl mice. The UBC line did not have any cortical bone phenotype in non-loaded femurs. In summary, dynamic histomorphometry data confirmed that tibial loading induces lamellar bone formation. Contrary to our hypothesis, there was no decrease in loading-induced bone formation in the Osx or Dmp1 lines in the absence of VEGFA. There was a decrease in bone formation in the UBC line where all cells were targeted. This result indicates that VEGFA from a non-osteoblast cell source supports loading-induced lamellar bone formation, although osteoblast/osteocyte VEGFA is dispensable. These findings support a paracrine model whereby non-osteoblast VEGFA supports lamellar bone formation, independent of angiogenesis.

Cryogel Scaffold-Mediated Delivery of Adipose-Derived Stem Cells Promotes Healing in Murine Model of Atrophic Non-Union.

Non-union is defined as the permanent failure of a bone to heal and occurs clinically in 5% of fractures. Atrophic non-unions, characterized by absent/minimal callus formation, are poorly understood and difficult to treat. We recently demonstrated a novel murine model of atrophic non-union in the 3.6Col1A1-tk (Col1-tk) mouse, wherein dosing with the nucleoside analog ganciclovir (GCV) was used to deplete proliferating osteoprogenitor cells, leading to a radiographic and biomechanical non-union after the mid-shaft femur fracture. Using this Col1-tk atrophic non-union model, we hypothesized that the scaffold-mediated lentiviral delivery of doxycycline-inducible BMP-2 transgenes would induce osteogenesis at the fracture site. Cryogel scaffolds were used as a vehicle for GFP+ and BMP-2+ cell delivery to the site of non-union. Cryogel scaffolds were biofabricated through the cross-linking of a chitosan-gelatin polymer solution at subzero temperatures, which results in a macroporous, spongy structure that may be advantageous for a bone regeneration application. Murine adipose-derived stem cells were seeded onto the cryogel scaffolds, where they underwent lentiviral transduction. Following the establishment of atrophic non-unions in the femurs of Col1-tk mice (4 weeks post-fracture), transduced, seeded scaffolds were surgically placed around the site of non-union, and the animals were given doxycycline water to induce BMP-2 production. Controls included GFP+ cells on the cryogel scaffolds, acellular scaffolds, and sham (no scaffold). Weekly radiographs were taken, and endpoint analysis included micro-CT and histological staining. After 2 weeks of implantation, the BMP-2+ scaffolds were infiltrated with cartilage and woven bone at the non-union site, while GFP+ scaffolds had woven bone formation. Later, timepoints of 8 weeks had woven bone and vessel formation within the BMP-2+ and GFP + scaffolds with cortical bridging of the original fracture site in both groups. Overall, the cell-seeded cryogels promoted osseous healing. However, while the addition of BMP-2 promoted the endochondral ossification, it may provide a slower route to healing. This proof-of-concept study demonstrates the potential for cellularized cryogel scaffolds to enhance the healing of non-unions.

Postnatal Osterix but not DMP1 lineage cells significantly contribute to intramembranous ossification in three preclinical models of bone injury.

Murine models of long-bone fracture, stress fracture, and cortical defect are used to discern the cellular and molecular mediators of intramembranous and endochondral bone healing. Previous work has shown that Osterix (Osx+) and Dentin Matrix Protein-1 (DMP1+) lineage cells and their progeny contribute to injury-induced woven bone formation during femoral fracture, ulnar stress fracture, and tibial cortical defect repair. However, the contribution of pre-existing versus newly-derived Osx+ and DMP1+ lineage cells in these murine models of bone injury is unclear. We addressed this knowledge gap by using male and female 12-week-old, tamoxifen-inducible Osx Cre_ERT2 and DMP1 Cre_ERT2 mice harboring the Ai9 TdTomato reporter allele. To trace pre-existing Osx+ and DMP1+ lineage cells, tamoxifen (TMX: 100 mg/kg gavage) was given in a pulse manner (three doses, 4 weeks before injury), while to label pre-existing and newly-derived lineage Osx+ and DMP1+ cells, TMX was first given 2 weeks before injury and continuously (twice weekly) throughout healing. TdTomato positive (TdT+) cell area and cell fraction were quantified from frozen histological sections of injured and uninjured contralateral samples at times corresponding with active woven bone formation in each model. We found that in uninjured cortical bone tissue, Osx Cre_ERT2 was more efficient than DMP1 Cre_ERT2 at labeling the periosteal and endosteal surfaces, as well as intracortical osteocytes. Pulse-labeling revealed that pre-existing Osx+ lineage and their progeny, but not pre-existing DMP1+ lineage cells and their progeny, significantly contributed to woven bone formation in all three injury models. In particular, these pre-existing Osx+ lineage cells mainly lined new woven bone surfaces and became embedded as osteocytes. In contrast, with continuous dosing, both Osx+ and DMP1+ lineage cells and their progeny contributed to intramembranous woven bone formation, with higher TdT+ tissue area and cell fraction in Osx+ lineage versus DMP1+ lineage calluses (femoral fracture and ulnar stress fracture). Similarly, Osx+ and DMP1+ lineage cells and their progeny significantly contributed to endochondral callus regions with continuous dosing only, with higher TdT+ chondrocyte fraction in Osx+ versus DMP1+ cell lineages. In summary, pre-existing Osx+ but not DMP1+ lineage cells and their progeny make up a significant amount of woven bone cells (particularly osteocytes) across three preclinical models of bone injury. Therefore, Osx+ cell lineage modulation may prove to be an effective therapy to enhance bone regeneration.

Ablation of Proliferating Osteoblast Lineage Cells After Fracture Leads to Atrophic Nonunion in a Mouse Model.

Nonunion is defined as the permanent failure of a fractured bone to heal, often necessitating surgical intervention. Atrophic nonunions are a subtype that are particularly difficult to treat. Animal models of atrophic nonunion are available; however, these require surgical or radiation-induced trauma to disrupt periosteal healing. These methods are invasive and not representative of many clinical nonunions where osseous regeneration has been arrested by a "failure of biology". We hypothesized that arresting osteoblast cell proliferation after fracture would lead to atrophic nonunion in mice. Using mice that express a thymidine kinase (tk) "suicide gene" driven by the 3.6Col1a1 promoter (Col1-tk), proliferating osteoblast lineage cells can be ablated upon exposure to the nucleoside analog ganciclovir (GCV). Wild-type (WT; control) and Col1-tk littermates were subjected to a full femur fracture and intramedullary fixation at 12 weeks age. We confirmed abundant tk+ cells in fracture callus of Col-tk mice dosed with water or GCV, specifically many osteoblasts, osteocytes, and chondrocytes at the cartilage-bone interface. Histologically, we observed altered callus composition in Col1-tk mice at 2 and 3 weeks postfracture, with significantly less bone and more fibrous tissue. Col1-tk mice, monitored for 12 weeks with in vivo radiographs and micro-computed tomography (µCT) scans, had delayed bone bridging and reduced callus size. After euthanasia, ex vivo µCT and histology showed failed union with residual bone fragments and fibrous tissue in Col1-tk mice. Biomechanical testing showed a failure to recover torsional strength in Col1-tk mice, in contrast to WT. Our data indicates that suppression of proliferating osteoblast-lineage cells for at least 2 weeks after fracture blunts the formation and remodeling of a mineralized callus leading to a functional nonunion. We propose this as a new murine model of atrophic nonunion. © 2021 American Society for Bone and Mineral Research (ASBMR).

Type 1 diabetic Akita mice have low bone mass and impaired fracture healing.

Type 1 diabetes (T1DM) impairs bone formation and fracture healing in humans. Akita mice carry a mutation in one allele of the insulin-2 (Ins2) gene, which leads to pancreatic beta cell dysfunction and hyperglycemia by 5-6 weeks age. We hypothesized that T1DM in Akita mice is associated with decreased bone mass, weaker bones, and impaired fracture healing. Ins2 ± (Akita) and wildtype (WT) males were subjected to femur fracture at 18-weeks age and healing assessed 3-21 days post-fracture. Non-fractured left femurs were assessed for morphology (microCT) and strength (bending or torsion) at 19-21 weeks age. Fractured right femurs were assessed for callus mechanics (torsion), morphology and composition (microCT and histology) and gene expression (qPCR). Both Akita and WT mice gained weight from 3 to 18 weeks age, but Akita mice weighed less starting at 5 weeks (-5.2%, p < 0.05). At 18-20 weeks age Akita mice had reduced serum osteocalcin (-30%), cortical bone area (-16%), and thickness (-17%) compared to WT, as well as reduced cancellous BV/TV (-39%), trabecular thickness (-23%) and vBMD (-31%). Mechanical testing of non-fractured femurs showed decreased structural (stiffness, ultimate load) and material (ultimate stress) properties of Akita bones. At 14 and 21 days post fracture Akita mice had a significantly smaller callus than WT mice (~30%), with less cartilage and bone area. Assessment of torsional strength showed a weaker callus in Akita mice with lower stiffness (-42%), maximum torque (-44%) and work to fracture (-44%). In summary, cortical and cancellous bone mass were reduced in Akita mice, with lower bone mechanical properties. Fracture healing in Akita mice was impaired by T1DM, with a smaller, weaker fracture callus due to decreased cartilage and bone formation. In conclusion, the Akita mouse mimics some of the skeletal features of T1DM in humans, including osteopenia and impaired fracture healing, and may be useful to test interventions.

Interleukin-6 (IL-6) deficiency enhances intramembranous osteogenesis following stress fracture in mice.

Interleukin-6 (IL-6) is highly upregulated in response to skeletal injury, suggesting it plays a role in the inflammatory phase of fracture repair. However, the impact of IL-6 on successful repair remains incompletely defined. Therefore, we investigated the role of IL-6 in two models of fracture repair (full fracture and stress fracture) using 12-week old IL-6 global knockout mice (IL-6 KO) and wild type (WT) littermate controls. Callus morphology and mineral density 14 days after full femur fracture did not differ between IL-6 knockout mice and controls. In contrast, IL-6 KO mice had an enhanced bone response 7 days after ulnar stress fracture compared to WT, with increased total callus volume (p = 0.020) and callus bone volume (p = 0.045). IL-6 KO did not alter the recruitment of immune cells (Gr-1 or F4/80 positive) to the stress fracture callus. IL-6 KO also did not alter the number of osteoclasts in the stress fracture callus. Using RNA-seq, we identified differentially expressed genes in stress fracture vs. contralateral control ulnae, and observed that IL-6 KO resulted in only modest alterations to the gene expression response to stress fracture (SFx). Wnt1 was more highly upregulated in IL-6 KO SFx callus at both day 1 (fold change 12.5 in KO vs. 5.7 in WT) and day 3 (fold change 4.7 in KO vs. 1.9 in WT). Finally, using tibial compression to induce bone formation without bone injury, we found that IL-6 KO directly impacted osteoblast function, increasing the propensity for woven bone formation. In summary, we report that IL-6 knockout enhanced formation of callus and bone following stress fracture injury, likely through direct action on the osteoblast's ability to produce woven bone. This suggests a novel role of IL-6 as a suppressor of intramembranous bone formation.

Transcriptional profiling of intramembranous and endochondral ossification after fracture in mice.

Bone fracture repair represents an important clinical challenge with nearly 1 million non-union fractures occurring annually in the U.S. Gene expression differs between non-union and healthy repair, suggesting there is a pattern of gene expression that is indicative of optimal repair. Despite this, the gene expression profile of fracture repair remains incompletely understood. In this work, we used RNA-seq of two well-established murine fracture models to describe gene expression of intramembranous and endochondral bone formation. We used top differentially expressed genes, enriched gene ontology terms and pathways, callus cellular phenotyping, and histology to describe and contrast these bone formation processes across time. Intramembranous repair, as modeled by ulnar stress fracture, and endochondral repair, as modeled by femur full fracture, exhibited vastly different transcriptional profiles throughout repair. Stress fracture healing had enriched differentially expressed genes associated with bone repair and osteoblasts, highlighting the strong osteogenic repair process of this model. Interestingly, the PI3K-Akt signaling pathway was one of only a few pathways uniquely enriched in stress fracture repair. Full fracture repair involved a higher level of inflammatory and immune cell related genes than did stress fracture repair. Full fracture repair also differed from stress fracture in a robust downregulation of ion channel genes following injury, the role of which in fracture repair is unclear. This study offers a broad description of gene expression in intramembranous and endochondral ossification across several time points throughout repair and suggests several potentially intriguing genes, pathways, and cells whose role in fracture repair requires further study.

VEGFA From Early Osteoblast Lineage Cells (Osterix+) Is Required in Mice for Fracture Healing.

Bone formation via intramembranous and endochondral ossification is necessary for successful healing after a wide range of bone injuries. The pleiotropic cytokine, vascular endothelial growth factor A (VEGFA) has been shown, via nonspecific pharmacologic inhibition, to be indispensable for angiogenesis and ossification following bone fracture and cortical defect repair. However, the importance of VEGFA expression by different cell types during bone healing is not well understood. We sought to determine the role of VEGFA from different osteoblast cell subsets following clinically relevant models of bone fracture and cortical defect. Ubiquitin C (UBC), Osterix (Osx), or Dentin matrix protein 1 (Dmp1) Cre-ERT2 mice (male and female) containing floxed VEGFA alleles (VEGFAfl/fl ) were either given a femur full fracture, ulna stress fracture, or tibia cortical defect at 12 weeks of age. All mice received tamoxifen continuously starting 2 weeks before bone injury and throughout healing. UBC Cre-ERT2 VEGFAfl/fl (UBC cKO) mice, which were used to mimic nonspecific inhibition, had minimal bone formation and impaired angiogenesis across all bone injury models. UBC cKO mice also exhibited impaired periosteal cell proliferation during full fracture, but not stress fracture repair. Osx Cre-ERT2 VEGFAfl/fl (Osx cKO) mice, but not Dmp1 Cre-ERT2 VEGFAfl/fl (Dmp1 cKO) mice, showed impaired periosteal bone formation and angiogenesis in models of full fracture and stress fracture. Neither Osx cKO nor Dmp1 cKO mice demonstrated significant impairments in intramedullary bone formation and angiogenesis following cortical defect. These data suggest that VEGFA from early osteolineage cells (Osx+), but not mature osteoblasts/osteocytes (Dmp1+), is critical at the time of bone injury for rapid periosteal angiogenesis and woven bone formation during fracture repair. Whereas VEGFA from another cell source, not from the osteoblast cell lineage, is necessary at the time of injury for maximum cortical defect intramedullary angiogenesis and osteogenesis. © 2019 American Society for Bone and Mineral Research.

Activation of hedgehog signaling by systemic agonist improves fracture healing in aged mice.

Fracture healing is a complex process of many coordinated biological pathways. This system can go awry resulting in nonunion, which leads to significant patient morbidity. The Hedgehog (Hh) signaling pathway is upregulated in fracture healing. We hypothesized that the Hh signaling pathway can be pharmacologically modulated to positively affect fracture healing. Diaphyseal femur fractures were created in elderly mice (18 months, C57BL/6 females), which have a blunted and delayed healing response compared to younger mice, and were stabilized with intramedullary pins. To activate the Hh pathway we targeted the receptor Smoothened using an agonist (Hh-Ag1.5 [Hh-Ag]) and compared this to a vehicle control. Expression of Hh target genes were significantly increased in the fracture callus of the agonist group compared to controls, indicating pathway activation. Expression of osteogenic and chondrogenic-related genes was greatly upregulated in fracture callus versus intact femora, although Hh agonist treatment did not consistently enhance this response. Blindly graded, radiographic callus healing scores were significantly higher in the Hh-Ag groups at post operative day (POD) 14, indicating earlier callus bridging. On microCT, Hh-Ag treatment led to greater callus volume (+40%) and bone volume (+25%) at POD21. By day 14, callus vascularity, as assessed by 3D microCT angiography vessel volume, was 85% greater in the Hh-Ag group. Finally, mechanical strength of the calluses in the Hh-Ag groups was significantly greater than in the control groups at POD21. In conclusion, systemic administration of a Hh agonist appears to improve the osseous and vascular healing responses in a mouse fracture healing-impaired model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Gli1 identifies osteogenic progenitors for bone formation and fracture repair.

Bone formation in mammals requires continuous production of osteoblasts throughout life. A common molecular marker for all osteogenic mesenchymal progenitors has not been identified. Here, by lineage-tracing experiments in fetal or postnatal mice, we discover that Gli1+ cells progressively produce osteoblasts in all skeletal sites. Most notably, in postnatal growing mice, the Gli1+ cells residing immediately beneath the growth plate, termed here "metaphyseal mesenchymal progenitors" (MMPs), are essential for cancellous bone formation. Besides osteoblasts, MMPs also give rise to bone marrow adipocytes and stromal cells in vivo. RNA-seq reveals that MMPs express a number of marker genes previously assigned to mesenchymal stem/progenitor cells, including CD146/Mcam, CD44, CD106/Vcam1, Pdgfra, and Lepr. Genetic disruption of Hh signaling impairs proliferation and osteoblast differentiation of MMPs. Removal of ß-catenin causes MMPs to favor adipogenesis, resulting in osteopenia coupled with increased marrow adiposity. Finally, postnatal Gli1+ cells contribute to both chondrocytes and osteoblasts during bone fracture healing. Thus Gli1 marks mesenchymal progenitors responsible for both normal bone formation and fracture repair.

Exogenous hedgehog antagonist delays but does not prevent fracture healing in young mice.

Fracture healing recapitulates many aspects of developmental osteogenesis. The hedgehog (Hh) signaling pathway, essential to skeletal development, is upregulated during fracture healing, although its importance is unclear. Our goal was to assess the functional importance of Hh signaling in endochondral fracture healing. We created closed, transverse diaphyseal femur fractures in mice, stabilized with an intramedullary pin, and administered a systemic Hh inhibitor or vehicle. Because Hh pathway activation is mediated by the receptor Smoothened (Smo), we used the Smo antagonist GDC-0449 (GDC, 50mg/kg, twice daily) to target the pathway. First, in vehicle-treated 10-wk. female C57BL/6 mice we confirmed that Hh signaling was increased in fracture callus compared to intact bone, with >5-fold upregulation of target genes Ptch1 and Gli1. Additionally, using 10-wk. male and female Gli1 reporter mice, we saw a strong activation of the reporter in the osseous regions of the fracture callus 7-10days after fracture. GDC treatment significantly blunted these responses, indicating effective inhibition of fracture-induced Hh signaling in bone. Moreover, microCT analysis revealed that GDC treatment significantly reduced cancellous and cortical bone volume at non-fracture sites (tibial metaphysis and diaphysis), suggesting that the drug inhibited normal bone formation. GDC treatment had a modest effect on fracture healing, with evidence of delayed callus mineralization radiographically (significantly lower Goldberg score at day 14) and by microCT (reduced callus vBMD at 14days), and a delay in the recovery of torsional rotation to normal (elevated rotation-at-peak torque at 21days). On the other hand, GDC treatment did not inhibit qPCR or morphological measures of chondrogenesis or angiogenesis, and did not impair the recovery of failure torque (at day 14 or 21), a measure of biomechanical competence. In summary, GDC treatment inhibited Hh signaling, which delayed but did not prevent fracture healing in young mice. We conclude that Hh signaling is strongly induced after fracture and may play a role in early callus mineralization, although it does not appear to be required for eventual healing.

Loss of scleraxis in mice leads to geometric and structural changes in cortical bone, as well as asymmetry in fracture healing.

Scleraxis (Scx) is a known regulator of tendon development, and recent work has identified the role of Scx in bone modeling. However, the role of Scx in fracture healing has not yet been explored. This study was conducted to identify the role of Scx in cortical bone development and fracture healing. Scx green fluorescent protein-labeled (ScxGFP) reporter and Scx-knockout (Scx-mutant) mice were used to assess bone morphometry and the effects of fracture healing on Scx localization and gene expression, as well as callus healing response. Botulinum toxin (BTX) was used to investigate muscle unloading effects on callus shape. Scx-mutant long bones had structural and mechanical defects. Scx gene expression was elevated and bmp4 was decreased at 24 h after fracture. ScxGFP+ cells were localized throughout the healing callus after fracture. Scx-mutant mice demonstrated disrupted callus healing and asymmetry. Asymmetry of Scx-mutant callus was not due to muscle unloading. Wild-type littermates (age matched) served as controls. This is the first study to explore the role of Scx in cortical bone mechanics and fracture healing. Deletion of Scx during development led to altered long bone properties and callus healing. This study also demonstrated that Scx may play a role in the periosteal response during fracture healing.-McKenzie, J. A., Buettmann, E., Abraham, A. C., Gardner, M. J., Silva, M. J., Killian, M. L. Loss of scleraxis in mice leads to geometric and structural changes in cortical bone, as well as asymmetry in fracture healing.

Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice.

Post-natal osteogenesis after mechanical trauma or stimulus occurs through either endochondral healing, intramembranous healing or lamellar bone formation. Bone morphogenetic protein 2 (BMP2) is up-regulated in each of these osteogenic processes and is expressed by a variety of cells including osteoblasts and vascular cells. It is known that genetic knockout of Bmp2 in all cells or in osteo-chondroprogenitor cells completely abrogates endochondral healing after full fracture. However, the importance of BMP2 from differentiated osteoblasts and endothelial cells is not known. Moreover, the importance of BMP2 in non-endochondral bone formation such as intramembranous healing or lamellar bone formation is not known. Using inducible and tissue-specific Cre-lox mediated targeting of Bmp2 in adult (10-24 week old) mice, we assessed the role of BMP2 expression globally, by osteoblasts, and by vascular endothelial cells in endochondral healing, intramembranous healing and lamellar bone formation. These three osteogenic processes were modeled using full femur fracture, ulnar stress fracture, and ulnar non-damaging cyclic loading, respectively. Our results confirmed the requirement of BMP2 for endochondral fracture healing, as mice in which Bmp2 was knocked out in all cells prior to fracture failed to form a callus. Targeted deletion of Bmp2 in osteoblasts (osterix-expressing) or vascular endothelial cells (vascular endothelial cadherin-expressing) did not impact fracture healing in any way. Regarding non-endochondral bone formation, we found that BMP2 is largely dispensable for intramembranous bone formation after stress fracture and also not required for lamellar bone formation induced by mechanical loading. Taken together our results indicate that osteoblasts and endothelial cells are not a critical source of BMP2 in endochondral fracture healing, and that non-endochondral bone formation in the adult mouse is not as critically dependent on BMP2.

Hedgehog signaling mediates woven bone formation and vascularization during stress fracture healing.

Hedgehog (Hh) signaling is critical in developmental osteogenesis, and recent studies suggest it may also play a role in regulating osteogenic gene expression in the post-natal setting. However, there is a void of studies directly assessing the effect of Hh inhibition on post-natal osteogenesis. This study utilized a cyclic loading-induced ulnar stress fracture model to evaluate the hypothesis that Hh signaling contributes to osteogenesis and angiogenesis during stress fracture healing. Immediately prior to loading, adult rats were given GDC-0449 (Vismodegib - a selective Hh pathway inhibitor; 50mg/kg orally twice daily), or vehicle. Hh signaling was upregulated in response to stress fracture at 3 days (Ptch1, Gli1 expression), and was markedly inhibited by GDC-0449 at 1 day and 3 days in the loaded and non-loaded ulnae. GDC-0449 did not affect Hh ligand expression (Shh, Ihh, Dhh) at 1 day, but decreased Shh expression by 37% at 3 days. GDC-0449 decreased woven bone volume (-37%) and mineral density (-17%) at 7 days. Dynamic histomorphometry revealed that the 7 day callus was composed predominantly of woven bone in both groups. The observed reduction in woven bone occurred concomitantly with decreased expression of Alpl and Ibsp, but was not associated with differences in early cellular proliferation (as determined by callus PCNA staining at 3 days), osteoblastic differentiation (Osx expression at 1 day and 3 days), chondrogenic gene expression (Acan, Sox9, and Col2α1 expression at 1 day and 3 days), or bone resorption metrics (callus TRAP staining at 3 days, Rankl and Opg expression at 1 day and 3 days). To evaluate angiogenesis, vWF immunohistochemistry showed that GDC-0449 reduced fracture callus blood vessel density by 55% at 3 days, which was associated with increased Hif1α gene expression (+30%). Dynamic histomorphometric analysis demonstrated that GDC-0449 also inhibited lamellar bone formation. Lamellar bone analysis of the loaded limb (directly adjacent to the woven bone callus) showed that GDC-0449 significantly decreased mineral apposition rate (MAR) and bone formation rate (BFR/BS) (-17% and -20%, respectively). Lamellar BFR/BS in the non-loaded ulna was also significantly decreased (-37%), indicating that Hh signaling was required for normal bone modeling. In conclusion, Hh signaling plays an important role in post-natal osteogenesis in the setting of stress fracture healing, mediating its effects directly through regulation of bone formation and angiogenesis.

Long bone structure and strength depend on BMP2 from osteoblasts and osteocytes, but not vascular endothelial cells.

The importance of bone morphogenetic protein 2 (BMP2) in the skeleton is well known. BMP2 is expressed in a variety of tissues during development, growth and healing. In this study we sought to better identify the role of tissue-specific BMP2 during post-natal growth and to determine if BMP2 knockout affects the ability of terminally differentiated cells to create high quality bone material. We targeted BMP2 knockout to two differentiated cell types known to express BMP2 during growth and healing, early-stage osteoblasts and their progeny (osterix promoted Cre) and vascular endothelial cells (vascular-endothelial-cadherin promoted Cre). Our objectives were to assess post-natal bone growth, structure and strength. We hypothesized that removal of BMP2 from osteogenic and vascular cells (separately) would result in smaller skeletons with inferior bone material properties. At 12 and 24 weeks of age the osteoblast knockout of BMP2 reduced body weight by 20%, but the vascular knockout had no effect. Analysis of bone in the tibia revealed reductions in cortical and cancellous bone size and volume in the osteoblast knockout, but not in the vascular endothelial knockout. Furthermore, forelimb strength testing revealed a 30% reduction in ultimate force at both 12 and 24 weeks in the osteoblast knockout of BMP2, but no change in the vascular endothelial knockout. Moreover, mechanical strength testing of femurs from osteoblast knockout mice demonstrated an increased Young's modulus (greater than 35%) but decreased post-yield displacement (greater than 50%) at both 12 and 24 weeks of age. In summary, the osteoblast knockout of BMP2 reduced bone size and altered mechanical properties at the whole-bone and material levels. Osteoblast-derived BMP2 has an important role in post-natal skeletal growth, structure and strength, while vascular endothelial-derived BMP2 does not.

Angiogenesis is required for stress fracture healing in rats.

Although angiogenesis and osteogenesis are critically linked, the importance of angiogenesis for stress fracture healing is unknown. In this study, mechanical loading was used to create a non-displaced stress fracture in the adult rat forelimb. Fumagillin, an anti-angiogenic agent, was used as the water soluble analogue TNP-470 (25mg/kg) as well as incorporated into lipid-encapsulated α(v)ß(3) integrin targeted nanoparticles (0.25mg/kg). In the first experiment, TNP-470 was administered daily for 5 days following mechanical loading, and changes in gene expression, vascularity, and woven bone formation were quantified. Although no changes in vascularity were detected 3 days after loading, treatment-related downregulation of angiogenic (Pecam1) and osteogenic (Bsp, Osx) genes was observed at this early time point. On day 7, microCT imaging of loaded limbs revealed diminished woven bone formation in treated limbs compared to vehicle treated limbs. In the second experiment, α(v)ß(3) integrin targeted fumagillin nanoparticles were administered as before, albeit with a 100-fold lower dose, and changes in vascularity and woven bone formation were determined. There were no treatment-related changes in vessel count or volume 3 days after loading, although fewer angiogenic (CD105 positive) blood vessels were present in treated limbs compared to vehicle treated limbs. This result manifested on day 7 as a reduction in total vascularity, as measured by histology (vessel count) and microCT (vessel volume). Similar to the first experiment, treated limbs had diminished woven bone formation on day 7 compared to vehicle treated limbs. These results indicate that angiogenesis is required for stress fracture healing, and may have implications for inducing rapid repair of stress fractures.

Sniff nasal inspiratory pressure and sleep disordered breathing in childhood neuromuscular disorders.

The ease of sniff nasal inspiratory pressure testing may extend application of respiratory muscle assessment to younger and cognitively-impaired children. We sought to quantify sniff nasal inspiratory pressure in childhood neuromuscular disorders, and to correlate this measure with conventional pulmonary function tests and overnight polysomnography. Thirty children (mean 9.7 ± 3.8 years, range 4.3-16.5 years) with diagnosed neuromuscular disorders (Duchenne muscular dystrophy, spinal muscular atrophy, Becker muscular dystrophy, congenital myopathy, facioscapulohumeral muscular dystrophy, myotonic dystrophy, multi-minicore disease) underwent assessment. Thirty-seven percent displayed cognitive impairment. Those with neuromuscular disorders were then compared with 32 volunteer age- and gender-matched controls (mean 10.9 ± 2.9 years, range 6.6-17.2 years) with normal respiratory function. Twenty-three children with neuromuscular disorders also underwent overnight polysomnography. Children with neuromuscular disorders demonstrated significantly impaired sniff nasal inspiratory pressure, maximal inspiratory pressure, FEV(1) and FVC (p<0.05). A positive correlation was identified between daytime sniff nasal inspiratory pressure and maximal inspiratory pressure (r=0.58), FEV(1) (r=0.55) and FVC (r=0.46), though not with polysomnography variables (respiratory disturbance index, nadir SpO(2), peak CO(2)). Moderate prevalence of nocturnal hypoxia was observed, and 32% of children demonstrated sleep disordered breathing. Sniff nasal inspiratory pressure assessment was well tolerated, representing a promising surrogate measure for assessment of respiratory function in childhood neuromuscular disorders.

Plasma fetuin-A concentrations in young and older high- and low-active men.

Fetuin-A is a liver-derived factor that may play a role in insulin resistance and age-related chronic diseases (eg, type 2 diabetes mellitus and cardiovascular [CV] disease). Regular exercise improves CV risk and insulin sensitivity; however, it is unknown whether chronic exercise training is related to circulating levels of fetuin-A. Therefore, this study examined whether plasma fetuin-A levels were related to age and chronic physical activity in men. We hypothesized that chronic physical activity would be related to lower plasma fetuin-A levels in younger and older men. In healthy high-active (HI) and low-active (LO) young (HI, n = 7; LO, n = 8) and older (HI, n = 12, LO, n = 11) men, we determined cardiorespiratory fitness (maximal oxygen uptake), plasma fetuin-A levels, plasma insulin, insulin resistance (homeostasis model assessment of insulin resistance), and the standard risk factors for CV disease. Groups were matched for body mass index. Fetuin-A was significantly higher (~20%) in both young and older LO men compared with their HI counterparts, and fetuin-A was inversely related to maximal oxygen uptake (r = -0.40, P = .014). Plasma fetuin-A levels showed trends to be significantly correlated with insulin (r = -0.34, P = .052) and homeostasis model assessment of insulin resistance (r = 0.33, P = .058) in the older individuals. In younger participants, fetuin-A was related to blood pressure and cholesterol measures. These results indicate that low levels of fetuin-A are related to cardiorespiratory fitness and a number of conventional CV and metabolic disease risk factors independent of age and body mass index. Therefore, the maintenance of low levels of circulating fetuin-A may be a novel mechanism contributing to enhanced insulin sensitivity with regular physical activity.

Comparing histological, vascular and molecular responses associated with woven and lamellar bone formation induced by mechanical loading in the rat ulna.

Osteogenesis occurs by formation of woven or lamellar bone. Little is known about the molecular regulation of these two distinct processes. We stimulated periosteal bone formation at the ulnar mid-diaphysis of adult rats using a single bout of forelimb compression. We hypothesized that loading that stimulates woven bone formation induces higher over-expression of genes associated with cell proliferation, angiogenesis and osteogenesis compared to loading that stimulates lamellar bone formation. We first confirmed that a single bout of 100 cycles of loading using either a rest-inserted (0.1 Hz) or haversine (2 Hz) waveform (15 N peak force) was non-damaging and increased lamellar bone formation (LBF loading). Woven bone formation (WBF loading) was stimulated using a previously described, damaging fatigue loading protocol (2 Hz, 1.3 mm disp., 18 N peak force). There were dramatic differences in gene expression levels (based on qRT-PCR) between loading protocols that produced woven and lamellar bone. In contrast, gene expression levels were not different between LBF loading protocols using a rest-inserted or haversine waveform. Cell proliferation markers Hist4 and Ccnd1 were strongly upregulated (5- to 17-fold) 1 and 3 days after WBF loading, prior to woven bone formation, but not after LBF loading. The angiogenic genes Vegf and Hif1a were upregulated within 1 h after WBF loading and were strongly up on days 1-3 (3- to 15-fold). In sharp contrast, we observed only a modest increase (<2-fold) in Vegfa and Hif1a expression on day 3 following LBF loading. Consistent with these relative differences in gene expression, vascular perfusion 3 days after loading revealed significant increases in vessel number and volume following WBF loading, but not after LBF loading. Lastly, bone formation markers (Runx2, Osx, Bsp) were more strongly upregulated for woven (4- to 89-fold) than for lamellar bone (2-fold), consistent with the differences in new bone volume observed 10 days after loading. In summary, robust early increases both molecularly and histologically for cell proliferation and angiogenesis precede woven bone formation, whereas lamellar bone formation is associated with only a modest upregulation of molecular signals at later timepoints.

AKT1 G205T genotype influences obesity-related metabolic phenotypes and their responses to aerobic exercise training in older Caucasians.

As part of the insulin signalling pathway, Akt influences growth and metabolism. The AKT1 gene G205T (rs1130214) polymorphism has potential functional effects. Thus, we determined whether the G205T polymorphism influences metabolic variables and their responses to aerobic exercise training. Following dietary stabilization, healthy, sedentary, 50- to 75-year-old Caucasian men (n = 51) and women (n = 58) underwent 6 months of aerobic exercise training. Before and after completing the intervention, dual-energy X-ray absorptiometry was used to measure percentage body fat, computed tomography to measure visceral and subcutaneous fat, and oral glucose tolerance testing to measure glucose total area under the curve (AUC), insulin AUC and insulin sensitivity. Taqman assay was used to determine AKT1 G205T genotypes. At baseline, men with the GG genotype (n = 29) had lower maximal oxygen consumption (VO2 max) values (P = 0.026) and higher percentage body fat (P = 0.046), subcutaneous fat (P = 0.021) and insulin AUC (P = 0.003) values than T allele carriers (n = 22). Despite their rather disadvantageous starting values, men with the GG genotype seemed to respond to exercise training more robustly than men with the T allele, highlighted by significantly greater fold change improvements in insulin AUC (P = 0.012) and glucose AUC (P = 0.035). Although the GG group also significantly improved VO2 max with training, the change in VO2 max was not as great as that of the T allele carriers (P = 0.037). In contrast, after accounting for hormone replacement therapy use, none of the variables differed in the women at baseline. As a result of exercise training, women with the T allele (n = 20) had greater fold change improvements in fasting glucose (P = 0.011), glucose AUC (P = 0.017) and insulin sensitivity (P = 0.044) than GG genotype women (n = 38). Our results suggest that the AKT1 G205T polymorphism influences metabolic variables and their responses to aerobic exercise training in older, previously sedentary individuals.