Bone and the Unfolded Protein Response: In Sickness and in Health.

Differentiation and optimal function of osteoblasts and osteoclasts are contingent on synthesis and maintenance of a healthy proteome. Impaired and/or altered secretory capacity of these skeletal cells is a primary driver of most skeletal diseases. The endoplasmic reticulum (ER) orchestrates the folding and maturation of membrane as well as secreted proteins at high rates within a calcium rich and oxidative organellar niche. Three ER membrane proteins monitor fidelity of protein processing in the ER and initiate an intricate signaling cascade known as the Unfolded Protein Response (UPR) to remediate accumulation of misfolded proteins in its lumen, a condition referred to as ER stress. The UPR aids in fine-tuning, expanding and/or modifying  the cellular proteome, especially in specialized secretory cells, to match everchanging physiologic cues and metabolic demands. Sustained activation of the UPR due to chronic ER stress, however, is known to hasten cell death and drive pathophysiology of several diseases. A growing body of evidence suggests that ER stress and an aberrant UPR may contribute to poor skeletal health and the development of osteoporosis. Small molecule therapeutics that target distinct components of the UPR may therefore have implications for developing novel treatment modalities relevant to the skeleton. This review summarizes the complexity of UPR actions in bone cells in the context of skeletal physiology and osteoporotic bone loss, and highlights the need for future mechanistic studies to develop novel UPR therapeutics that mitigate adverse skeletal outcomes.

Skeletal screening IMPC/KOMP using µCT and computer automated cryohistology: Application to the Efna4 KO mouse line.

The IMPC/KOMP program provides the opportunity to screen mice harboring well defined gene-inactivation mutations in a uniform genetic background. The program performs a global tissue phenotyping survey that includes skeletal x-rays and bone density measurements. Because of the relative insensitivity of the two screening tests for detecting variance in bone architecture, we initiated a secondary screen based on µCT and a cryohistolomorphological workflow that was performed on the femur and vertebral compartments on 220 randomly selected knockouts (KOs) and 36 control bone samples over a 2 1/2 year collection period provided by one of the production/phenotyping centers. The performance of the screening protocol was designed to balance throughput and cost versus sensitivity and informativeness such that the output would be of value to the skeletal biology community. Here we report the reliability of this screening protocol to establish criteria for control skeletal variance at the architectural, dynamic and cellular histomorphometric level. Unexpected properties of the control population include unusually high variance in BV/TV in male femurs and greater bone formation and bone turnover rates in the female femur and vertebral trabeculae bone compartments. However, the manner for maintaining bone formation differed between these two bone sites. The vertebral compartment relies on maintaining a greater number of bone forming surfaces while the femoral compartment utilized more matrix production per cell. The comparison of the architectural properties obtained by µCT and histomorphology revealed significant differences in values for BV/TV, Tb.Th and Tb.N which is attributable to sampling density of the two methods. However, as a screening tool, expressing the ratio of KO to the control line as obtained by either method was remarkably similar. It identified KOs with significant variance which led to a more detailed histological analysis. Our findings are exemplified by the Efna4 KO, in which a high BV/TV was identified by µCT and confirmed by histomorphometry in the femur but not in the vertebral compartment. Dynamic labeling showed a marked increase in BFR which was attributable to increased labeling surfaces. Cellular analysis confirmed partitioning of osteoblast to labeling surfaces and a marked decrease in osteoclastic activity on both labeling and quiescent surfaces. This pattern of increased bone modeling would not be expected based on prior studies of the Ephrin-Ephrin receptor signaling pathways between osteoblasts and osteoclasts. Overall, our findings underscore why unbiased screening is needed because it can reveal unknown or unanticipated genes that impact skeletal variation.

Surgical Induction of Posttraumatic Osteoarthritis in the Mouse.

Given the prevalence and the scope of the personal and societal burden of osteoarthritis (OA), investigators continue to be deeply interested in understanding the pathogenic basis of disease and developing novel disease modifying OA therapies. Because joint trauma/injury is considered a leading predisposing factor in the development of OA, and since posttraumatic OA is one of the most common forms of OA in general, large animal and rodent models of knee injury that accurately recapitulate the OA disease process have become increasingly widespread over the past decade. To enable study in the context of defined genetic backgrounds, investigative teams have developed standardized protocols for injuring the mouse knee that aim to induce a reproducible degenerative process both in terms of severity and temporal pacing of disease progression. The destabilization of the medial meniscus (DMM) is one of the most commonly employed surgical procedure in rodents that reproducibly models posttraumatic OA and allows for the study of disease progression from initiation to end-stage disease. The description provided here sets the stage for both inexperienced and established investigators to employ the DMM procedure, or other similar surgical destabilization methods, to initiate the development of posttraumatic OA in the mouse. Successful application of this method provides a preclinical platform to study the mechanisms driving the pathogenesis of posttraumatic OA and for testing therapeutic strategies to treat it.

Modulation of Notch1 signaling regulates bone fracture healing.

Fracture healing involves interactions of different cell types, driven by various growth factors, and signaling cascades. Periosteal mesenchymal progenitor cells give rise to the majority of osteoblasts and chondrocytes in a fracture callus. Notch signaling has emerged as an important regulator of skeletal cell proliferation and differentiation. We investigated the effects of Notch signaling during the fracture healing process. Increased Notch signaling in osteochondroprogenitor cells driven by overexpression of Notch1 intracellular domain (NICD1) (αSMACreERT2 mice crossed with Rosa-NICD1) during fracture resulted in less cartilage, more mineralized callus tissue, and stronger and stiffer bones after 3 weeks. Periosteal cells overexpressing NICD1 showed increased proliferation and migration in vitro. In vivo data confirmed that increased Notch1 signaling caused expansion of alpha-smooth muscle actin (αSMA)-positive cells and their progeny including αSMA-derived osteoblasts in the callus without affecting osteoclast numbers. In contrast, anti-NRR1 antibody treatment to inhibit Notch1 signaling resulted in increased callus cartilage area, reduced callus bone mass, and reduced biomechanical strength. Our study shows a positive effect of induced Notch1 signaling on the fracture healing process, suggesting that stimulating the Notch pathway could be beneficial for fracture repair.

Cells from a GDF5 origin produce zonal tendon-to-bone attachments following anterior cruciate ligament reconstruction.

Following anterior cruciate ligament (ACL) reconstruction surgery, a staged repair response occurs where cells from outside the tendon graft participate in tunnel integration. The mechanisms that regulate this process, including the specific cellular origin, are poorly understood. Embryonic cells expressing growth and differentiation factor 5 (GDF5) give rise to several mesenchymal tissues in the joint and epiphyses. We hypothesized that cells from a GDF5 origin, even in the adult tissue, would give rise to cells that contribute to the stages of repair. ACLs were reconstructed in Gdf5-Cre;R26R-tdTomato lineage tracing mice to monitor the contribution of Gdf5-Cre;tdTom+ cells to the tunnel integration process. Anterior-posterior drawer tests demonstrated 58% restoration in anterior-posterior stability. Gdf5-Cre;tdTom+ cells within the epiphyseal bone marrow adjacent to tunnels expanded in response to the injury by 135-fold compared with intact controls to initiate tendon-to-bone attachments. They continued to mature the attachments yielding zonal insertion sites at 4 weeks with collagen fibers spanning across unmineralized and mineralized fibrocartilage and anchored to the adjacent bone. The zonal attachments possessed tidemarks with concentrated alkaline phosphatase activity similar to native entheses. This study established that mesenchymal cells from a GDF5 origin can contribute to zonal tendon-to-bone attachments within bone tunnels following ACL reconstruction.

CD55 Regulates Bone Mass in Mice by Modulating RANKL-Mediated Rac Signaling and Osteoclast Function.

CD55 is a glycosylphosphatidylinositol (GPI)-anchored protein that regulates complement-mediated and innate and adaptive immune responses. Although CD55 is expressed in various cell types in the bone marrow, its role in bone has not been investigated. In the current study, trabecular bone volume measured by µCT in the femurs of CD55KO female mice was increased compared to wild type (WT). Paradoxically, osteoclast number was increased in CD55KO with no differences in osteoblast parameters. Osteoclasts from CD55KO mice exhibited abnormal actin-ring formation and reduced bone-resorbing activity. Moreover, macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) treatment failed to activate Rac guanosine triphosphatase (GTPase) in CD55KO bone marrow macrophage (BMM) cells. In addition, apoptotic caspases activity was enhanced in CD55KO, which led to the poor survival of mature osteoclasts. Our results imply that CD55KO mice have increased bone mass due to defective osteoclast resorbing activity resulting from reduced Rac activity in osteoclasts. We conclude that CD55 plays an important role in the survival and bone-resorption activity of osteoclasts through regulation of Rac activity. © 2019 American Society for Bone and Mineral Research.

Author Correction: An atlas of genetic influences on osteoporosis in humans and mice.

In the version of this article initially published, in Fig. 5a, the data in the right column of 'DAAM2 gRNA1' were incorrectly plotted as circles indicating 'untreated' rather than as squares indicating 'treated'. The error has been corrected in the HTML and PDF versions of the article.

An atlas of genetic influences on osteoporosis in humans and mice.

Osteoporosis is a common aging-related disease diagnosed primarily using bone mineral density (BMD). We assessed genetic determinants of BMD as estimated by heel quantitative ultrasound in 426,824 individuals, identifying 518 genome-wide significant loci (301 novel), explaining 20% of its variance. We identified 13 bone fracture loci, all associated with estimated BMD (eBMD), in ~1.2 million individuals. We then identified target genes enriched for genes known to influence bone density and strength (maximum odds ratio (OR) = 58, P = 1 × 10-75) from cell-specific features, including chromatin conformation and accessible chromatin sites. We next performed rapid-throughput skeletal phenotyping of 126 knockout mice with disruptions in predicted target genes and found an increased abnormal skeletal phenotype frequency compared to 526 unselected lines (P < 0.0001). In-depth analysis of one gene, DAAM2, showed a disproportionate decrease in bone strength relative to mineralization. This genetic atlas provides evidence linking associated SNPs to causal genes, offers new insight into osteoporosis pathophysiology, and highlights opportunities for drug development.

Responses to spaceflight of mouse mandibular bone and teeth.

OBJECTIVE: To determine if spaceflight and microgravity affect non-weight bearing bones and development and mineralization of teeth, reasoning that combining an organ and a cellular level approach can lead to greater insights about these effects. DESIGN: Mandibles and incisors of mice flown on the US STS-135 space shuttle mission and the Russian Bion-M1 satellite were studied using micro-computed tomography and immunohistochemistry. Ground controls were mice housed in standard vivarium cages and flight habitats. RESULTS: Incisor length was greater in the 13-day STS-135 flight mice than in either control group. Initial incisor mineralization occurred more posteriorly, and incisor, enamel and dentin volumes and enamel and dentin thicknesses were greater in the 30-day Bion-M1 flight and habitat control mice than in vivarium control mice. Mandibular bone volume (BV) was increased in STS-135 flight and habitat groups and decreased in Bion-M1 flight and habitat groups compared to vivarium controls. No significant histological alterations occurred, but changes were seen in the bone and tooth proteins dentin sialoprotein, amelogenin and the type II regulatory subunit of protein kinase A. The percentage of sclerostin positive osteocytes was greatest in flight mice, and greater in STS-135 flight and habitat control mice than in the corresponding Bion-M1 groups. TRAP staining, representing osteoclastic bone remodeling, differed between the two flights and corresponded with changes in BV. Interpretation of the findings was limited by a small number of flight mice, different sex and ages of the mice in the two missions, and different habitats and diets. CONCLUSIONS: Microgravity has measurable effects on mandibular bone physiology and incisor development and mineralization. The results also showed that the habitat had an effect either in flight or ground control samples, as demonstrated by the changes in BV and apparent slowing of incisor eruption. Therefore, developing appropriate habitats is critical for future spaceflight missions.

Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A.

Increased bone resorption is considered to explain why intermittent PTH is anabolic for bone but continuous PTH is catabolic. However, when cyclooxygenase-2 (COX2) is absent in mice, continuous PTH becomes anabolic without decreased resorption. In murine bone marrow stromal cells (BMSCs), serum amyloid A (SAA)3, induced in the hematopoietic lineage by the combination of COX2-produced prostaglandin and receptor activator of nuclear factor κB ligand (RANKL), suppresses PTH-stimulated osteoblast differentiation. To determine whether SAA3 inhibits the anabolic effects of PTH in vivo, wild-type (WT) and SAA3 knockout (KO) mice were infused with PTH. In WT mice, continuous PTH induced SAA3 and was catabolic for bone. In KO mice, PTH was anabolic, increasing trabecular bone, serum markers of bone formation, and osteogenic gene expression. In contrast, PTH increased all measurements associated with bone resorption, as well as COX2 gene expression, similarly in KO and WT mice. SAA1 and SAA2 in humans are likely to have analogous functions to SAA3 in mice. RANKL induced both SAA1 and SAA2 in human bone marrow macrophages in a COX2-dependent manner. PTH stimulated osteogenesis in human BMSCs only when COX2 or RANKL was inhibited. Addition of recombinant SAA1 or SAA2 blocked PTH-stimulated osteogenesis. In summary, SAA3 suppresses the bone formation responses but not the bone resorption responses to PTH in mice, and in the absence of SAA3, continuous PTH is anabolic. In vitro studies in human bone marrow suggest that SAA may be a target for enhancing the therapeutic effects of PTH in treating osteoporosis.

Screening Gene Knockout Mice for Variation in Bone Mass: Analysis by µCT and Histomorphometry.

PURPOSE OF REVIEW: The international mouse phenotyping consortium (IMPC) is producing defined gene knockout mouse lines. Here, a phenotyping program is presented that is based on micro-computed tomography (µCT) assessment of distal femur and vertebra. Lines with significant variation undergo a computer-based bone histomorphometric analysis. RECENT FINDINGS: Of the 220 lines examined to date, approximately 15% have a significant variation (high or low) by µCT, most of which are not identified by the IMPC screen. Significant dimorphism between the sexes and bone compartments adds to the complexity of the skeletal findings. The µCT information that is posted at www.bonebase.org can group KOMP lines with similar morphological features. The histological data is presented in a graphic form that associates the cellular features with a specific anatomic group. The web portal presents a bone-centric view appropriate for the skeletal biologist/clinician to organize and understand the large number of genes that can influence skeletal health. Cataloging the relative severity of each variant is the first step towards compiling the dataset necessary to appreciate the full polygenic basis of degenerative bone disease.

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues.

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

Genetics of aging bone.

With aging, the skeleton experiences a number of changes, which include reductions in mass and changes in matrix composition, leading to fragility and ultimately an increase of fracture risk. A number of aspects of bone physiology are controlled by genetic factors, including peak bone mass, bone shape, and composition; however, forward genetic studies in humans have largely concentrated on clinically available measures such as bone mineral density (BMD). Forward genetic studies in rodents have also heavily focused on BMD; however, investigations of direct measures of bone strength, size, and shape have also been conducted. Overwhelmingly, these studies of the genetics of bone strength have identified loci that modulate strength via influencing bone size, and may not impact the matrix material properties of bone. Many of the rodent forward genetic studies lacked sufficient mapping resolution for candidate gene identification; however, newer studies using genetic mapping populations such as Advanced Intercrosses and the Collaborative Cross appear to have overcome this issue and show promise for future studies. The majority of the genetic mapping studies conducted to date have focused on younger animals and thus an understanding of the genetic control of age-related bone loss represents a key gap in knowledge.

Loss of Bone in Sickle Cell Trait and Sickle Cell Disease Female Mice Is Associated With Reduced IGF-1 in Bone and Serum.

Characterization of the bone phenotype of 24-week-old female transgenic sickle cell disease (SCD), sickle cell trait (SCT) revealed significant reductions in bone mineral density and bone mineral content relative to control with a further significant decreased in SCD compared with SCT. By microcomputed tomography, femur middiaphyseal cortical area was significantly reduced in SCT and SCD. Cortical thickness was significantly decreased in SCD vs control. Diaphysis structural stiffness and strength were significantly reduced in SCT and SCD. Histomorphometry showed a significant increase in osteoclast perimeter in SCD and significantly decreased bone formation in SCD and SCT compared with control with a further significant decrease in SCD compared with SCT. Collagen-I mRNA was significantly decreased in tibiae from SCT and SCD and osterix, Runx2, osteoclacin, and Dmp-1 mRNA were significantly decreased in tibiae of SCD compared with control. Serum osteocalcin was significantly decreased and ferritin was significantly increased in SCD compared with control. Igf1 mRNA and serum IGF1 were significantly decreased in SCD and SCT. IGF1 protein was decreased in bone marrow stromal cells from SCT and SCD cultured in osteogenic media. Crystal violet staining revealed fewer cells and significantly reduced alkaline phosphatase positive mineralized nodules in SCT and SCD that was rescued by IGF1 treatment. We conclude that reduced bone mass in SCD and SCT mice carries architectural consequences that are detrimental to the mechanical integrity of femoral diaphysis. Furthermore reduced IGF1 and osteoblast terminal differentiation contributed to reduced bone formation in SCT and SCD mice.

Combination therapy with BMP-2 and a systemic RANKL inhibitor enhances bone healing in a mouse critical-sized femoral defect.

Recombinant human BMP-2 (rhBMP-2) is a potent osteoinductive agent, but has been associated not only with bone formation, but also osteoclastogenesis and bone resorption. Osteoprotegerin (OPG) is a RANKL inhibitor that blocks differentiation and function of osteoclasts. We hypothesized that the combination of local BMP-2 (recombinant protein or a product of gene therapy) plus systemic OPG-Fc is more effective than BMP-2 alone in promoting bone repair. To test this hypothesis we used a mouse critical-sized femoral defect model. Col2.3eGFP (osteoblastic marker) male mice were treated with rhBMP-2 (group I), rhBMP-2 and systemic OPG (group II), rhBMP-2 and delayed administration of OPG (group III), mouse BM cells transduced with a lentiviral vector containing the BMP-2 gene (LV-BMP-2; group IV), LV-BMP-2 and systemic OPG (group V), a carrier alone (group VI) and administration of OPG alone (group VII). All bone defects treated with BMP-2 (alone or combined with OPG) healed, whereas minimal bone formation was noted in animals treated with the carrier alone or OPG alone. MicroCT analysis showed that bone volume (BV) in rhBMP-2+OPG and LV-BMP-2+OPG groups was significantly higher compared to rhBMP-2 alone (p<0.01) and LV-BMP-2 alone (p<0.001). Similar results were observed in histomorphometry, with rhBMP-2 alone defects exhibiting significantly lower bone area (B.Ar) compared to rhBMP-2+OPG defects (p<0.005) and LV-BMP-2 defects having a significantly lower B.Ar compared to all BMP-2+OPG treated groups (p≤0.01). TRAP staining demonstrated a major osteoclast response in the groups that did not receive OPG (rhBMP-2, LV-BMP-2 and sponge alone) beginning as early as 7days post-operatively. In conclusion, we demonstrated that locally delivered BMP-2 (recombinant protein or gene therapy) in combination with systemically administered OPG improved bone healing compared to BMP-2 alone in a mouse critical-sized bone defect. These data indicate that osteoclasts can diminish healing responses to BMP-2 and that RANKL inhibition may thus accentuate BMP-2 efficacy.

Accelerated fracture healing in transgenic mice overexpressing an anabolic isoform of fibroblast growth factor 2.

The effect of targeted expression of an anabolic isoform of basic fibroblast growth factor (FGF2) in osteoblastic lineage on tibial fracture healing was assessed in mice. Closed fracture of the tibiae was performed in Col3.6-18 kDaFgf2-IRES-GFPsaph mice in which a 3.6 kb fragment of type I collagen promoter (Col3.6) drives the expression of only the 18 kD isoform of FGF2 (18 kDaFgf2/LMW) with green fluorescent protein-sapphire (GFPsaph) as well as Vector mice (Col3.6-IRES-GFPsaph, Vector) that did not harbor the FGF2 transgene. Radiographic, micro-CT, DEXA, and histologic analysis of fracture healing of tibiae harvested at 3, 10 and 20 days showed a smaller fracture callus but accelerated fracture healing in LMWTg compared with Vector mice. At post fracture day 3, FGF receptor 3 and Sox 9 mRNA were significantly increased in LMWTg compared with Vector. Accelerated fracture healing was associated with higher FGF receptor 1, platelet derived growth factors B, C, and D, type X collagen, vascular endothelial cell growth factor, matrix metalloproteinase 9, tartrate resistant acid phosphatase, cathepsin K, runt-related transcription factor-2, Osterix and Osteocalcin and lower Sox9, and type II collagen expression at 10 days post fracture. We postulate that overexpression of LMW FGF2 accelerated the fracture healing process due to its effects on factors that are important in chondrocyte and osteoblast differentiation and vascular invasion.

Evaluation of the donor cell contribution in rhBMP-2 mediated bone formation with chitosan thermogels using fluorescent protein reporter mice.

Our current understanding regarding the contribution of donor cells in growth factor and cell based tissue regeneration strategies is limited. The present study attempts to utilize fluorescent protein reporter mice [Col3.6Topaz (enhanced yellow fluorescent protein, EYFP) as host and Col3.6Cyan (enhanced cyan fluorescent protein, ECFP) as donor] to determine donor cell contribution in bone regeneration using a bilateral calvarial defect model. Thermogelling chitosan hydrogels (Chi-AHP) were used as bone marrow stromal cell (BMSC) delivery vehicle in the presence and absence of recombinant human bone morphogenetic protein-2 (rhBMP-2). Co-delivery of rhBMP-2 and donor BMSCs led to a significant increase in bone formation indicating the potential of using the combination approach for improved regeneration. On a cellular level, presence of rhBMP-2 resulted in an increased host cell derived osteoblast infiltration at 8 weeks. However, the new mineralized tissue presented two distinct morphological features based on its cellular origin. Regenerated tissue associated with ECFP positive donor cells showed a woven bone-like structure with diffuse alizarin complexone (AC) label and minimal tartrate resistant acid phosphatase (TRAP) activity, indicating the presence of immature early osteoblast cells depositing mineralized tissue without progressing to a mature lamellar bone. Host derived bone showed sharp mineralization line (AC), strong alkaline phosphatase (ALP) and TRAP activity, indicating the presence of actively mineralizing and remodeling, mature lamellar bone matrix. The study demonstrated the remarkable potential of transgenic reporters to improve our understanding of donor cell contribution during bone formation.

Systemic Administration of Sclerostin Antibody Enhances Bone Morphogenetic Protein-Induced Femoral Defect Repair in a Rat Model.

BACKGROUND: Recombinant human bone morphogenetic protein (rhBMP)-2 is a potent osteoinductive agent; however, its clinical use has been reduced because of safety and efficacy concerns. In preclinical studies involving a critical-sized defect in a rat model, sclerostin antibody (Scl-Ab) treatment increased bone formation within the defect but did not result in reliable healing. The purpose of the current study was to evaluate bone repair of a critical-sized femoral defect in a rat model with use of local implantation of rhBMP-2 combined with systemic administration of Scl-Ab. METHODS: A critical-sized femoral defect was created in rats randomized into three treatment groups: local rhBMP-2 and systemic Scl-Ab (Scl + BMP), local rhBMP-2 alone, and collagen sponge alone (operative control). The Scl + BMP group received local rhBMP-2 (10 µg) on a collagen sponge placed within the defect intraoperatively and then twice weekly injections of Scl-Ab (25 mg/kg) administered postoperatively. The femora were evaluated at twelve weeks with use of radiography, microcomputed tomography (microCT), histomorphometric analysis, and biomechanical testing. RESULTS: At twelve weeks, all Scl + BMP and rhBMP-2 only samples were healed. No femora healed in the operative control group. Histomorphometric analysis demonstrated more bone in the Scl + BMP samples than in the samples treated with rhBMP-2 alone (p = 0.029) and the control samples (p = 0.003). MicroCT revealed that the Scl + BMP group had a 90% greater bone volume within the defect region compared with the rhBMP-2 group and a 350% greater bone volume compared with the operative control group (p < 0.001). Biomechanical testing showed that the group treated with Scl + BMP had greater torsional strength and rigidity compared with the rhBMP-2 group (p < 0.001 and p = 0.047) and the intact femoral control group (p < 0.001). Torque to failure was lower in the rhBMP-2 group compared with the intact femoral control group (p < 0.002). Mean energy to failure was higher in the Scl + BMP samples compared with the rhBMP-2 only samples (p = 0.001). CONCLUSIONS: In a critical-sized femoral defect in a rat model, local rhBMP-2 combined with systemic administration of Scl-Ab resulted in more robust healing that was stronger and more rigid than results for rhBMP-2 alone and intact nonoperative femora. CLINICAL RELEVANCE: Our study demonstrated that combining an osteoinductive agent with a systemically administered antibody that promotes bone formation can enhance bone repair and has potential as a therapeutic regimen in humans.

Genetic regulation of bone strength: a review of animal model studies.

Population- and family-based studies have established that fragility fracture risk is heritable; yet, the genome-wide association studies published to date have only accounted for a small fraction of the known variation for fracture risk of either the femur or the lumbar spine. Much work has been carried out using animal models toward finding genetic loci that are associated with bone strength. Studies using animal models overcome some of the issues associated with using patient data, but caution is needed when interpreting the results. In this review, we examine the types of tests that have been used for forward genetics mapping in animal models to identify loci and/or genes that regulate bone strength and discuss the limitations of these test methods. In addition, we present a summary of the quantitative trait loci that have been mapped for bone strength in mice, rats and chickens. The majority of these loci co-map with loci for bone size and/or geometry and thus likely dictate strength via modulating bone size. Differences in bone matrix composition have been demonstrated when comparing inbred strains of mice, and these matrix differences may be associated with differences in bone strength. However, additional work is needed to identify loci that act on bone strength at the materials level.

Fixation stability dictates the differentiation pathway of periosteal progenitor cells in fracture repair.

This study compared fracture repair stabilized by intramedullary pin (IMP) or external fixation (EF) in GFP reporter mice. A modified IMP was used as control while EF utilized six needles inserted transversely through the tibia and into a segment of a syringe barrel. X-rays taken at days 0, 14, and 35 showed that IMP resulted in significant three-dimensional deformity with a large callus while EF showed minimal deformity and callus formation. Cryohistological analysis of IMP at day 14 confirmed a large ColX-RFPchry+ callus surrounded by woven bone (Col3.6-GFPcyan) and TRAP+ osteoclasts with mature bone (hOC-GFPtpz) at the base. By day 35, cartilaginous components had been resorbed and an outer cortical shell (OCS) showed evidence of inward modeling. In contrast, the EF at day 14 showed no evidence of cartilage formation. Instead, periosteal-derived osteoblasts (Col3.6-GFPcyan) entered the fracture cleft and formed woven bone that spanned the marrow space. By day 35, mature bone had formed that was contiguous with the opposing cortical bone. Fracture site stability greatly affects the cellular response during repair and must be considered in the preclinical models that test therapies for improving fracture healing.