Search

Although the functions of the peripheral nervous system in whole body homeostasis and sensation have been understood for many years, recent investigation has uncovered new roles for innervation in the musculoskeletal system. This review centers on advances regarding the function of nerves in the development and repair of two connected tissues: tendon and bone. Innervation in healthy tendons is generally confined to the tendon sheaths, and tendon-bone attachment units are typically aneural. In contrast to tendon, bone is an innervated and vascularized structure. Historically, the function of abundant peripheral nerves in bone has been limited to pain and some non-painful sensory perception in disease and injury. Indeed, much of our understanding of peripheral nerves in tendons, bones, and entheses is limited to the source and type of innervation in healthy and injured tissues. However, more recent studies have made important observations regarding the appearance, type, and innervation patterns of nerves during embryonic and postnatal development and in response to injury, which suggest a more expansive role for peripheral nerves in the formation of musculoskeletal tissues. Indeed, tendons and bones develop in a close spatiotemporal relationship in the embryonic mesoderm. Models of limb denervation have shed light on the importance of sensory innervation in bone and to a lesser extent, tendon development, and more recent work has unraveled key nerve signaling pathways. Furthermore, loss of sensory innervation also impairs healing of bone fractures and may contribute to chronic tendinopathy. However, more study is required to translate our knowledge of peripheral nerves to therapeutic strategies to combat bone and tendon diseases. more...

Bioprinting facilitates the generation of complex, three-dimensional (3D), cell-based constructs for various applications. Although multiple bioprinting technologies have been developed, extrusion-based systems have become the dominant technology due to the diversity of materials (bioinks) that can be utilized, either individually or in combination. However, each bioink has unique material properties and extrusion characteristics that affect bioprinting utility, accuracy, and precision. Here, we have extended our previous work to achieve high precision (i.e., repeatability) and printability across samples by optimizing bioink-specific printing parameters. Specifically, we hypothesized that a Fuzzy Inference System (FIS) could be used as a computational method to address the imprecision in 3D bioprinting test data and uncover the optimal printing parameters for a specific bioink that result in high accuracy and precision. To test this hypothesis, we have implemented a FIS model consisting of four inputs (bioink concentration, printing flowrate, speed, and temperature) and two outputs to quantify the precision (scaffold bioprinted linewidth variance) and printability. We validate our use of the bioprinting precision index (BPI) with both standard and normalized printability factors. Finally, we utilize optimized printing parameters to bioprint scaffolds containing up to 30 x 106 cells/mL with high printability and precision. In total, our results indicate that computational methods are a cost-efficient measure to improve the precision and robustness of extrusion 3D bioprinting with gelatin-based bioinks. more...

During skeletal development, expression of nerve growth factor (NGF) leads to the survival of afferent sensory nerves that express neurotrophic tyrosine kinase receptor type 1 (TrkA), the high affinity receptor for NGF. In adulthood, NGF is expressed by mature osteoblasts following mechanical loading and signals through TrkA receptors in resident sensory nerves to support load-induced bone formation. However, the regulation of NGF in osteoblasts following loading is not well understood. In this study, we sought to determine whether osteoblastic expression of toll-like receptor 4 (TLR4), a key receptor in the NF-κB signaling pathway, is required to initiate NGF-TrkA signaling to support skeletal adaptation following mechanical loading. First, we observed that NF-κB inhibition reduces NGF expression induced by axial forelimb compression. Moreover, we observed that TLR4+ periosteal cells are increased after mechanical loading. Therefore, we generated a novel mouse model in which Tlr4 is selectively removed from the mature osteoblast lineage. Although Tlr4 conditional knockout mice have normal skeletal mass and strength in adulthood, the loss of TLR4 signaling results in significant reductions in periosteal lamellar bone formation following axial forelimb compression. Furthermore, we demonstrated that the upregulation of Ngf following application of fluid shear stress to calvarial osteoblasts is significantly reduced by NF-κB and TLR4 inhibitors. Finally, RNA sequencing demonstrated that the deficits in load-induced bone formation in CKO mice can be attributed to dysregulated inflammatory signaling. In total, our study reveals a novel role for TLR4 in skeletal adaptation to mechanical loading in bone, which may enable new therapeutic strategies for diseases of low bone mass and provide new targets for musculoskeletal pain relief. more...

Bioprinting facilitates the generation of complex, three-dimensional (3D), cell-based constructs for various applications. Although multiple bioprinting technologies have been developed, extrusion-based systems have become the dominant technology due to the diversity of materials (bioinks) that can be utilized, either individually or in combination. However, each bioink has unique material properties and extrusion characteristics that affect bioprinting utility, accuracy, and precision. Here, we have extended our previous work to achieve high precision (i.e., repeatability) across samples by optimizing bioink-specific printing parameters. Specifically, we hypothesized that an adaptive neuro-fuzzy inference system (ANFIS) could be used as a computational method to address the imprecision in 3D bioprinting test data and uncover the optimal printing parameters for a specific bioink that result in high accuracy and precision. To test this hypothesis, we have implemented an ANFIS model consisting of four inputs (bioink concentration, printing pressure, speed, and temperature) and a single output to quantify the precision (scaffold bioprinted linewidth range). We validate our use of the bioprinting precision index (BPI) with both standard and normalized printability factors. In total, our results indicate that computational methods are a cost-efficient measure to improve the precision and robustness of extrusion 3D bioprinting with gelatin-based bioinks. more...

There is mounting evidence suggesting that the commonly used analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), may inhibit new bone formation with physical training and increase risk of stress fractures in physically active populations. Stress fractures are thought to occur when bones are subjected to repetitive mechanical loading, which can lead to a cycle of tissue microdamage, repair, and continued mechanical loading until fracture. Adaptive bone formation, particularly on the periosteal surface of long bones, is a concurrent adaptive response of bone to heightened mechanical loading that can improve the fatigue resistance of the skeletal structure, and therefore may play a critical role in offsetting the risk of stress fracture. Reports from animal studies suggest that NSAID administration may suppress this important adaptive response to mechanical loading. These observations have implications for populations such as endurance athletes and military recruits who are at risk of stress fracture and whose use of NSAIDs is widespread. However, results from human trials evaluating exercise and bone adaptation with NSAID consumption have been less conclusive. In this review, we identify knowledge gaps that must be addressed to further support NSAID-related guidelines intended for at-risk populations and individuals. more...

Objective: Dose optimization and pharmacokinetic evaluation of α-particle emitting radium-223 dichloride ((223)RaCl(2)) by planar γ-camera or single photon emission computed tomography (SPECT) imaging are hampered by the low photon abundance and injected activities. In this study, we demonstrate SPECT of (223)Ra using phantoms and small animal in vivo models. Methods: Line phantoms and mice bearing (223)Ra were imaged using a dedicated small animal SPECT by detecting the low-energy photon emissions from (223)Ra. Localization of the therapeutic agent was verified by whole-body and whole-limb autoradiography and its radiobiological effect confirmed by immunofluorescence. Results: A state-of-the-art commercial small animal SPECT system equipped with a highly sensitive collimator enables collection of sufficient counts for three-dimensional reconstruction at reasonable administered activities and acquisition times. Line sources of (223)Ra in both air and in a water scattering phantom gave a line spread function with a full-width-at-half-maximum of 1.45 mm. Early and late-phase imaging of the pharmacokinetics of the radiopharmaceutical were captured. Uptake at sites of active bone remodeling was correlated with DNA damage from the α particle emissions. Conclusions: This work demonstrates the capability to noninvasively define the distribution of (223)RaCl(2), a recently approved α-particle-emitting radionuclide. This approach allows quantitative assessment of (223)Ra distribution and may assist radiation-dose optimization strategies to improve therapeutic response and ultimately to enable personalized treatment planning. more...

Background: Female runners are at increased risk of stress fractures (SFs) compared with men. Literature is lacking with regard to best practice for preventing and treating SFs in women. The purpose of the study was to compare physiological measures and running-related factors between women of various ages and running abilities with and without a history of running-related SFs. Hypothesis: Women with and without SF histories will differ with regard to medical and menstrual history, bone health, body composition, nutrition, and running history. Study Design: Prospective cohort study. Level of Evidence: Level 2. Methods: A total of 20 female runners with SF histories were matched based on age and running distance with 20 women without SF histories. Data included medical, menstrual, running, injury, and nutritional histories; blood histology related to nutritional, hormonal, and bone-related risk factors; and bone density, fat, and lean tissue using dual energy x-ray absorptiometry. Paired t tests were used to examine differences between women with and without SF histories, and Spearmen correlations were conducted to examine relationships between physiological factors. Results: Women with SF histories had lower hip bone mineral density compared with women without SF histories ( P < 0.05). SF history was moderately correlated with menstrual changes during increased training times ( r = 0.580; P < 0.0001) but was not correlated with any other physiological factor. There was a moderate correlation within the SF group ( r = 0.65; P = 0.004) for bone markers for resorption and formation both increasing, indicating increased bone turnover. Conclusion: Female runners with low hip bone mineral density, menstrual changes during peak training, and elevated bone turnover markers may be at increased risk of SF. Clinical Relevance: Female runners need routine screening for risks associated with SF occurrence. As bone mineral density and bone turnover markers are not routinely assessed in this population, important risk factors may be missed. more...

Chronic low back pain is a highly prevalent health condition intricately linked to intervertebral disc degeneration. One of the prominent features of disc degeneration that is commonly observed with aging is dystrophic calcification. ATP-binding cassette sub-family C member 6 (ABCC6), a presumed ATP efflux transporter, is a key regulator of systemic levels of the mineralization inhibitor pyrophosphate (PPi). Mutations in ABCC6 result in pseudoxanthoma elasticum (PXE), a progressive human metabolic disorder characterized by mineralization of the skin and elastic tissues. The implications of ABCC6 loss-of-function on pathological mineralization of structures in the spine, however, are unknown. Using the ABCC6-/- mouse model of PXE, we investigated age-dependent changes in the vertebral bone and intervertebral disc. ABCC6-/- mice exhibited diminished trabecular bone quality parameters at 7-months which remained significantly lower than the wild-type mice at 18 months-of-age. ABCC6-/- vertebrae showed increased TRAP staining along with decreased TNAP staining, suggesting an enhanced bone resorption as well as decreased bone formation. Surprisingly, however, loss of ABCC6 resulted only in a mild, aging disc phenotype without evidence of dystrophic mineralization. Finally, we tested the utility of oral K3Citrate to treat the vertebral phenotype since it is shown to regulate hydroxyapatite mechanical behavior. The treatment resulted in inhibition of osteoclastic response and an early improvement in mechanical properties of the bone underscoring the promise of potassium citrate as a therapeutic agent. Our data suggest that although ectopic mineralization is tightly regulated in the disc, loss of ABCC6 compromises vertebral bone quality and dysregulates osteoblast-osteoclast coupling.Author SummaryInherited mutations in the ABCC6 transporter gene results in mineralization, often in the form of hydroxyapatite, of connective tissues throughout the body, predominantly affecting the skin, eyes, and blood vessels. Functional loss of ABCC6 causes reduced levels of the potent mineralization inhibitor pyrophosphate (PPi) in blood resulting in these pathologies. Pathological mineralization is also a prominent feature of intervertebral disc degeneration, but the role of ABCC6 and systemic PPi levels and its correlation to disc mineralization and vertebral bone health has remained unexplored. In this study, we show for the first time that loss of ABCC6 in mice results in significant decline in vertebral bone quality and mild age-related disc degeneration without increased incidence of abnormal mineralization. Importantly, treatment of ABCC6 deficient mice with K3Citrate resulted in restoration of early cellular changes which drive bone loss and mechanical function of the vertebrae. In summary, our data reveal that ABCC6 is dispensable for mineralization prevention in the intervertebral disc. Unexpectedly, we found that vertebral bone quality and bone cell activities are linked to ABCC6 function. more...

The activation of the Wnt/β-catenin signaling pathway is critical for skeletal development but surprisingly little is known about the requirements for the specific frizzled (Fzd) receptors that recognize Wnt ligands. To define the contributions of individual Fzd proteins to osteoblast function, we profiled the expression of all 10 mammalian receptors during calvarial osteoblast differentiation. Expression of Fzd4 was highly upregulated during in vitro differentiation and therefore targeted for further study. Mice lacking Fzd4 in mature osteoblasts had normal cortical bone structure but reduced cortical tissue mineral density and also exhibited an impairment in the femoral trabecular bone acquisition that was secondary to a defect in the mineralization process. Consistent with this observation, matrix mineralization, markers of osteoblastic differentiation, and the ability of Wnt3a to stimulate the accumulation of β-catenin were reduced in cultures of calvarial osteoblasts deficient for Fzd4. Interestingly, Fzd4-deficient osteoblasts exhibited an increase in the expression of Fzd8 both in vitro and in vivo, which suggests that the two receptors may exhibit overlapping functions. Indeed, ablating a single Fzd8 allele in osteoblast-specific Fzd4 mutants produced a more severe effect on bone acquisition. Taken together, our data indicate that Fzd4 is required for normal bone development and mineralization despite compensation from Fzd8. more...

Debilitating stress fractures are surprisingly common in physically active individuals, including athletes, military recruits, and dancers. These individuals are overrepresented in the 30 million daily users of non-steroidal anti-inflammatory drugs (NSAIDs). We hypothesized that regular use of NSAIDs would predispose habitually loaded bones to stress fracture and delay the repair of these injuries. In this project, we used repetitive axial forelimb compression in mice as a model to test these hypotheses. First, adult mice were subjected to six bouts of forelimb compression over a period of two weeks, with aspirin, naproxen, or vehicle continuously administered through drinking water. Naproxen-treated mice had diminished load-induced bone formation as well as a significant loss in toughness in non-loaded bone, which were not observed in aspirin-treated mice. Furthermore, there were no differences in RANKL/OPG ratio or cortical bone parameters. Picrosirius red staining and second harmonic generation imaging revealed that alterations in bone collagen fibril size and organization were driving the loss of toughness in naproxen-treated mice. Separately, adult mice were subjected to an ulnar stress fracture generated by a single bout of fatigue loading, with NSAIDs provided 24 h before injury. Both aspirin-treated and naproxen-treated mice had normal forelimb use in the week after injury, whereas control mice favored the injured forelimb until day 7. However, woven bone volume was only significantly impaired by naproxen. Both NSAIDs were found to significantly inhibit Ptgs2 and Ngf expression following stress fracture, but only naproxen significantly affected serum PGE2 concentration. Overall, our results suggest that naproxen, but not aspirin, may increase the risk of stress fracture and extend the healing time of these injuries, warranting further clinical evaluation for patients at risk for fatigue injuries. more...

Purpose To summarize currently available data regarding the use of bone marrow aspirate concentrate (BMAC) for the treatment of focal chondral lesions of the knee in experimental animal models and human clinical studies. Methods A systematic review searching for the terms “(bone marrow)” AND “(aspirate OR concentrate)” AND “(cartilage OR chondral OR osteochondral)” was performed in the databases PubMed, Cochrane Central Register of Controlled Trials, and Google Scholar regarding the use of BMAC for the treatment of focal chondral lesions of the knee. The inclusion criteria were animal and clinical studies published in English that used autologous BMAC to treat focal chondral defects of the knee. We excluded studies that evaluated nonconcentrated preparations of bone marrow aspirate or preparations that were culture expanded. Results A total of 23 studies were included: 10 studies performed in animal models and 13 human clinical studies. Animal studies showed inconsistent outcomes regarding the efficacy of BMAC for the treatment of chondral or osteochondral lesions, assessed by gross morphology, second-look arthroscopy, magnetic resonance imaging, histology, immunohistochemistry, mechanical testing, and micro-tomography. Chondral defect filling was achieved with fibrocartilage or “hyaline-like” cartilage. Cells present in BMAC did not meet the criteria to be characterized as mesenchymal stem cells according to the International Society for Cell Therapy because freshly isolated cells failed to show tri-lineage differentiation. Overall, all clinical studies, independent of the study group or level of evidence, reported improved clinical outcomes and higher macroscopic, magnetic resonance imaging, and histology scores. Comparative trials favored BMAC over microfracture and reported equivalent outcomes between BMAC and matrix-induced autologous chondrocyte implantation. However, clinical studies were scant and showed low scientific rigor, poor methodologic quality, and low levels of evidence on average. Conclusions Although clinical success in short-term and midterm applications has been suggested for the application of BMAC for the restoration of cartilage defects in lesions of the knee, current study designs are generally of low scientific rigor. In addition, clinical applications of this technology in animal model investigations have shown inconsistent outcomes. Thus, clinicians should apply this technology cautiously. Level of Evidence Level IV, systematic review of Level II, III, and IV evidence studies. more...

3D bioprinting allows biocompatible materials and cells to be deposited in precise locations in three-dimensional space, enabling researchers to surpass the limitations of traditional 2D cell culture and to create innovative therapies. 3D bioprinting is one of the newest tools developed in the field of tissue engineering, which has traditionally utilized a paradigm revolving around scaffolds, cells, and signals. In this review, we discuss how new developments in each of these three research areas relates to bioprinting dental tissues - specifically teeth, periodontal ligament, and alveolar bone. Important considerations include how scaffold materials and geometry affect regeneration of dental tissues, the importance of using dental cells in these applications, and the role of signaling molecules for creating a clinically relevant bioengineered dental implant. We conclude with potential new directions for research that would allow the burgeoning field of regenerative dentistry to achieve its lofty goals. more...

Bioprinting facilitates the generation of complex, three-dimensional (3D), cell-based constructs for a variety of applications. Although multiple bioprinting technologies have been developed, extrusion-based systems have become the dominant technology due to the diversity of substrate materials (bioinks) that can be accommodated, either individually or in combination. However, each bioink has unique material properties and extrusion characteristics that limit bioprinting precision, particularly when generating constructs from different bioinks. Here, we aimed to achieve high precision (i.e. repeatability) across samples by generating bioink-specific printing parameters using a systematic approach. We hypothesized that a fuzzy system could be used as a “black box” method to tackle the inherent vagueness and imprecision in 3D bioprinting data and uncover the optimal printing parameters for a specific bioink that would result in high accuracy and precision. Our fuzzy model was used to approximate and quantify the precision and ease of printability for two common bioinks - type I collagen and Pluronic F127, with or without dilution in αMEM culture media. The model consisted of three inputs (pressure, speed, and dilution percent of bioink) and a single output (layer width). Using this system, we introduce the Bioink Precision Index (BPI), a metric that can be used to quantify and compare the precision of any bioink. Here, we show that printing with parameters optimized using BPI increases the precision for collagen (+15%) and Pluronic F127 (+29%) as compared to the manufacturer’s recommended printing parameters. more...

Bioprinting is an emerging tissue engineering method used to generate cell-laden scaffolds with high spatial resolution. Bioprinting parameters, such as pressure, nozzle size, and speed, have a large influence on the quality of the bioprinted construct. Moreover, cell suspension density, cell culture period, and other critical biological parameters directly impact the biological function of the final product. Therefore, an approximation model that can be used to find the values of bioprinting parameters that will result in optimal bioprinted constructs is highly desired. Here, we propose type-1 and type-2 fuzzy systems to handle the uncertainty and imprecision in optimizing the input values. Specifically, we focus on the biological parameters, such as culture period, that can be used to maximize the output value (mineralization volume). To achieve a more accurate approximation, we have compared a type-2 fuzzy system with a type-1 fuzzy system using two levels of uncertainty. We hypothesized that type-2 fuzzy systems may be preferred in biological systems, due to the inherent vagueness and imprecision of the input data. Here, our results demonstrate that the type-2 fuzzy system with a high uncertainty boundary (30%) is superior to type-1 and type-2 with low uncertainty boundary fuzzy systems in the overall output approximation error for bone bioprinting inputs. more...

Dupuytren's disease is a benign fibroproliferative disorder of the hand that results in disabling digital contractures that impair function and diminish the quality of life. The incidence of this disease has been correlated with chronic inflammatory states, but any direct association between inflammatory cytokines and Dupuytren's disease is not known. We hypothesized that advanced fibroproliferation is associated with increased levels of circulating inflammatory cytokines. Blood and fibrotic cord tissue were collected preoperatively from patients with severe contracture and control patients. Blood plasma concentrations of known inflammatory cytokines were evaluated using a multiplex immunoassay. Proteins from the cord tissue were analyzed by RNA sequencing and immunohistochemistry. Moreover, collagen-rich cords were analyzed using Fourier-transform infrared spectroscopy. The results indicate that patients exhibited significantly elevated circulating inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-2, and IL-12p70, as compared with controls. Similarly, IL-4 and IL-13 were detected significantly more frequently in Dupuytren's disease as compared with control. RNA sequencing revealed 5311 differentially expressed genes and distinct clustering between diseased and control samples. In addition to increased expression of genes associated with fibroproliferation, we also observed upregulation of transcripts activated by inflammatory cytokines, including prolactin inducible protein and keratin intermediate filaments. IL-2, but not TNF-α, was detected in fibrotic cord tissue by immunohistochemistry. Finally, spectroscopic assays revealed a significant reduction of the collagen content and alterations of collagen cross-linking within the Dupuytren's disease tissues. In total, our results illustrate that patients with severe Dupuytren's disease exhibit substantially elevated circulating inflammatory cytokines that may drive fibroproliferation. Clinical Significance: The results from this study establish the basis for a specific cytokine profile that may be useful for diagnostic testing and therapeutic intervention in Dupuytren's disease. more...

There are currently no pharmacological approaches in fracture healing designed to therapeutically stimulate endochondral ossification. In this study, we test nerve growth factor (NGF) as an understudied therapeutic for fracture repair. We first characterized endogenous expression of Ngf and its receptor tropomyosin receptor kinase A (TrkA) during tibial fracture repair, finding that they peak during the cartilaginous phase. We then tested two injection regimens and found that local β-NGF injections during the endochondral/cartilaginous phase promoted osteogenic marker expression. Gene expression data from β-NGF stimulated cartilage callus explants show a promotion in markers associated with endochondral ossification such as Ihh, Alpl, and Sdf-1. Gene ontology enrichment analysis revealed the promotion of genes associated with Wnt activation, PDGF- and integrin-binding. Subsequent histological analysis confirmed Wnt activation following local β-NGF injections. Finally, we demonstrate functional improvements to bone healing following local β-NGF injections which resulted in a decrease in cartilage and increase of bone volume. Moreover, the newly formed bone contained higher trabecular number, connective density, and bone mineral density. Collectively, we demonstrate β-NGF’s ability to promote endochondral repair in a murine model and uncover mechanisms that will serve to further understand the molecular switches that occur during cartilage to bone transformation. more...

Bioprinting is an emerging tissue engineering method used to generate cell-laden scaffolds with high spatial resolution. Bioprinted vascularized bone grafts are a potential application of this technology that would meet a critical clinical need, since current approaches to volumetric bone repair have significant limitations. However, generation of vascular networks suitable for bioprinting is challenging. Here, we propose a novel Q-learning approach to quickly generate 3D vascular networks within patient-specific bone geometry that are optimized for bioprinting. First, the inlet and outlet locations are specified and the scenario is modeled using a grid world for initial agent training. Next, the path planned in the grid world environment is converted to a Bezier curve, which is then used to generate the final 3D vascularized bone model. The vessels generated using this procedure have minimal tortuosity, which increases the likelihood of successful bioprinting. Furthermore, the ability to specify inlet and outlet position is necessary for both surgical feasibility as well as generation of more complex vascular networks. In total, this study demonstrates the reliability of our reinforcement learning method for automated generation of 3D vascular networks within patient-specific geometry that can be used for bioprinting vascularized bone grafts. more...

The periosteal and endosteal surfaces of mature bone are densely innervated by sensory nerves expressing TrkA, the high-affinity receptor for nerve growth factor (NGF). In previous work, we demonstrated that administration of exogenous NGF significantly increased load-induced bone formation through the activation of Wnt signaling. However, the translational potential of NGF is limited by the induction of substantial mechanical and thermal hyperalgesia in mice and humans. Here, we tested the effect of gambogic amide (GA), a recently identified robust small molecule agonist for TrkA, on hyperalgesia and load-induced bone formation. Behavioral analysis was used to assess pain up to one week after axial forelimb compression. Contrary to our expectations, GA treatment was not associated with diminished use of the loaded forelimb or sensitivity to thermal stimulus. Furthermore, dynamic histomorphometry revealed a significant increase in relative periosteal bone formation rate as compared to vehicle treatment. Additionally, we found that GA treatment was associated with an increase in the number of osteoblasts per bone surface in loaded limbs as well as a significant upregulation of Wnt1, Wnt7b, and Ngf in loaded bones. To determine if these effects were exclusively mediated by NGF-TrkA signaling in sensory nerves, we cultured MC3T3-E1 cells for 7 or 14 days in osteogenic differentiation media containing NGF (50 ng/mL), GA (5, 50, or 500 nM), or vehicle (DMSO). After 7 days of culture, we observed increases in osteoblastic differentiation markers Runx2, Bglap2, and Sp7 in response to GA, whereas treatment with NGF was not different than vehicle. Only cells treated with the highest dose of GA (500 nM) had significantly impaired cell proliferation. In conclusion, our study indicates GA may be useful for augmenting skeletal adaptation to mechanical forces without inducing hyperalgesia through actions on both sensory nerves and osteoblasts. more...

The membrane protein ANKH was known to prevent pathological mineralization of joints and was thought to export pyrophosphate (PPi) from cells. This did not explain, however, the presence of ANKH in tissues, such as brain, blood vessels and muscle. We now report that in cultured cells ANKH exports ATP, rather than PPi, and, unexpectedly, also citrate as a prominent metabolite. The extracellular ATP is rapidly converted into PPi, explaining the role of ANKH in preventing ankylosis. Mice lacking functional Ank (Ankank/ank mice) had plasma citrate concentrations that were 65% lower than those detected in wild type control animals. Consequently, citrate excretion via the urine was substantially reduced in Ankank/ank mice. Citrate was even undetectable in the urine of a human patient lacking functional ANKH. The hydroxyapatite of Ankank/ank mice contained dramatically reduced levels of both, citrate and PPi and displayed diminished strength. Our results show that ANKH is a critical contributor to extracellular citrate and PPi homeostasis and profoundly affects bone matrix composition and, consequently, bone quality., Author summary Biallelic inactivating mutations in the progressive ankylosis gene (ANKH/Ank, humans/mice) cause a severe form of arthritis, primarily affecting joints of hands, feet and the spine. Low extracellular concentrations of the mineralization inhibitor pyrophosphate underlie the joint calcification in mice and humans that are devoid of functional Ank or ANKH, respectively. It was previously thought that ANKH mediates the release of pyrophosphate from the cytosol to the extracellular environment. We here show, however, that PPi is formed in the extracellular environment by Ectonucleotide Pyrophosphatase Phosphodiesterase 1 (ENPP1), from ATP released via ANKH. Untargeted metabolomics further revealed that ANKH provides a pathway for the release of nucleoside triphosphates in general and, unexpectedly, the Krebs-cycle intermediate citrate. Mice lacking Ank activity (Ankank/ank mice), have substantially reduced concentrations of citrate in their plasma and urine. Citrate was even undetectable in urine of a human patient lacking functional ANKH. Bones of Ankank/ank mice have reduced mechanical strength and their bone matrix contains dramatically reduced levels of PPi and citrate. Our data demonstrate that ANKH/Ank crucially contributes to the disposition of citrate and PPi in vivo and profoundly affects bone quality. Extracellular citrate is ubiquitous, and additional phenotypes are therefore expected to be associated with absence of Ank activity. more...

Bone and skeletal muscle mass are highly correlated in mammals, suggesting the existence of common anabolic signaling networks that coordinate the development of these two anatomically adjacent tissues. The activin signaling pathway is an attractive candidate to fulfill such a role. Here, we generated mice with conditional deletion of activin receptor (ACVR) type 2A, ACVR2B, or both, in osteoblasts, to determine the contribution of activin receptor signaling in regulating bone mass. Immunohistochemistry localized ACVR2A and ACVR2B to osteoblasts and osteocytes. Primary osteoblasts expressed activin signaling components, including ACVR2A, ACVR2B, and ACVR1B (ALK4) and demonstrated increased levels of phosphorylated Smad2/3 upon exposure to activin ligands. Osteoblasts lacking ACVR2B did not show significant changes in vitro. However, osteoblasts deficient in ACVR2A exhibited enhanced differentiation indicated by alkaline phosphatase activity, mineral deposition, and transcriptional expression of osterix, osteocalcin, and dentin matrix acidic phosphoprotein 1. To investigate activin signaling in osteoblasts in vivo, we analyzed the skeletal phenotypes of mice lacking these receptors in osteoblasts and osteocytes (osteocalcin-Cre). Similar to the lack of effect in vitro, ACVR2B-deficient mice demonstrated no significant change in any bone parameter. By contrast, mice lacking ACVR2A had significantly increased femoral trabecular bone volume at 6 weeks of age. Moreover, mutant mice lacking both ACVR2A and ACVR2B demonstrated sustained increases in trabecular bone volume, similar to those in ACVR2A single mutants, at 6 and 12 weeks of age. Taken together, these results indicate that activin receptor signaling, predominantly through ACVR2A, directly and negatively regulates bone mass in osteoblasts. more...

The coupling of osteoblasts and osteocytes by connexin43 (Cx43) gap junctions permits the sharing of second messengers that coordinate bone cell function and cortical bone acquisition. However, details of how Cx43 converts shared second messengers into signals that converge onto essential osteogenic processes are incomplete. Here, we use in vitro and in vivo methods to show that Cx43 and Runx2 functionally interact to regulate osteoblast gene expression and proliferation, ultimately affecting cortical bone properties. Using compound hemizygous mice for the Gja1 (Cx43) and Runx2 genes, we observed a skeletal phenotype not visible in wild-type or singly hemizygous animals. Cortical bone analysis by micro-computed tomography (μCT) revealed that 8-week-old male, compound Gja1+/- Runx2+/- mice have a marked increase in cross-sectional area, endosteal and periosteal bone perimeter, and an increase in porosity compared to controls. These compound Gja1+/- Runx2+/- mice closely approximate the cortical bone phenotypes seen in osteoblast-specific Gja1-conditional knockout models. Furthermore, μCT analysis of skulls revealed an altered interparietal bone geometry in compound hemizygotes. Consistent with this finding, Alizarin red/Alcian blue staining of 2-day-old Gja1+/- Runx2+/- neonates showed a hypomorphic interparietal bone, an exacerbation of the open fontanelles, and a further reduction in the hypoplastic clavicles compared to Runx2+/- neonates. Expression of osteoblast genes, including osteocalcin, osterix, periostin, and Hsp47, was markedly reduced in tibial RNA extracts from compound hemizygous mice, and osteoblasts from compound hemizygous mice exhibited increased proliferative capacity. Further, the reduced osteocalcin expression and hyperproliferative nature of osteoblasts from Cx43 deficient mice was rescued by Runx2 expression. In summary, these findings provide evidence that Cx43 and Runx2 functionally intersect in vivo to regulate cortical bone properties and affect osteoblast differentiation and proliferation, and likely contributes to aspects of the skeletal phenotype of Cx43 conditional knockout mice. © 2017 American Society for Bone and Mineral Research. more...

Adequate vascularization and innervation are required for proper bone development and homeostasis, and play important roles in bone health, repair of bone defects, and skeletal pain sensation in pathological conditions. Studies using genetically modified mice have started to shed light on the molecular regulation of the processes that drive blood vessel and nerve growth in the skeleton, particularly during endochondral bone development, growth, and remodeling. Prime roles in these processes are played by vascular endothelial growth factor and nerve growth factor, respectively. Here, we will summarize the current knowledge of the structure and regulation of the vascular and nerve compartments in bone and review their contributions to the controlled development, growth, maintenance, and repair of endochondral bones. more...

The membrane protein Ankylosis homologue (ANKH, mouse orthologue: ANK) prevents mineralization of joint-space and articular cartilage. The accepted view is that ANKH mediates cellular release of inorganic pyrophosphate (PPi), a strong physiological inhibitor of mineralization. Using global metabolite profiling, we identified citrate as the most prominent metabolite leaving HEK293 cells in an ANKH-dependent manner. Although PPi levels were increased in culture medium of HEK293-ANKH cells, PPi was formed extracellularly after release of ATP and other nucleoside triphosphates.Ankank/ankmice, which lack functional ANK, had substantially reduced concentrations of citrate in plasma and urine, while citrate was undetectable in urine of a human patient lacking functional ANKH. Bone hydroxyapatite ofAnkank/ankmice also contained markedly reduced levels of citrate and PPi and displayed diminished strength. Together, our data show that ANKH is a crucial factor in extracellular citrate and PPi homeostasis that is essential for normal bone development. more...

Background Increased tendon pain and tendon damage is a significant complication related to hyperlipidemia. Unlike the well-established pathogenesis associated with increased serum concentrations of total cholesterol, triglycerides, and low-density lipoprotein in atherosclerotic cardiovascular disease, the role of hyperlipidemia in promoting tendon damage remains controversial and requires mechanistic clarity. Methods In this study, we analyzed the consequences of hypercholesterolemia on the integrity of the collagen-based architecture of the Achilles tendon. The Achilles tendons from rabbits fed with normal-cholesterol (nCH) and high-cholesterol (hCH) diets were analyzed. We studied the morphology of tendons, distribution of lipids within their collagen-rich milieu, the relative amounts of fibrillar collagen I and collagen III, and selected biomechanical parameters of the tendons at the macroscale and the nanoscale. Results Histological assays of hCH tendons and tenosynovium demonstrated hypercellular areas with increased numbers of macrophages infiltrating the tendon structure as compared to the nCH tendons. While Oil Red staining revealed lipid-rich deposits in the hCH tendons, hybridization of tendon tissue with the collagen hybridizing peptide (CHP) demonstrated damage to the collagen fibers. Fourier-transform infrared (FTIR) spectra showed the presence of distinct peaks consistent with the presence of cholesterol ester. Additionally, the hCH tendons displayed regions of poor collagen content that overlapped with lipid-rich regions. The hCH tendons had a substantial fourfold increase in the collage III to collagen I ratio as compared to the nCH tendons. Tendons from the hCH rabbits showed poor biomechanical characteristics in comparison with control. The biomechanical changes were evident at the macrolevel and the nanolevel of tendon structure. Conclusions Our findings support the hypothesis that hypercholesterolemia coincides with the weakening of the tendons. It is likely that the intimate contact between collagen fibrils and cholesterol deposits contributes to the weakening of the fibrillar structure of the tendons. more...

Posttraumatic fibrotic scarring is a significant medical problem that alters the proper functioning of injured tissues. Current methods to reduce posttraumatic fibrosis rely on anti-inflammatory and anti-proliferative agents with broad intracellular targets. As a result, their use is not fully effective and may cause unwanted side effects. Our group previously demonstrated that extracellular collagen fibrillogenesis is a valid and specific target to reduce collagen-rich scar buildup. Our previous studies showed that a rationally designed antibody that binds the C-terminal telopeptide of the α2(I) chain involved in the aggregation of collagen molecules limits fibril assembly in vitro and reduces scar formation in vivo. Here, we have utilized a clinically relevant arthrofibrosis model to study the broad mechanisms of the anti-scarring activity of this antibody. Moreover, we analyzed the effects of targeting collagen fibril formation on the quality of healed joint tissues, including the posterior capsule, patellar tendon, and subchondral bone. Our results show that blocking collagen fibrillogenesis not only reduces collagen content in the scar, but also accelerates the remodeling of healing tissues and changes the collagen fibrils’ cross-linking. In total, this study demonstrated that targeting collagen fibrillogenesis to limit arthrofibrosis affects neither the quality of healing of the joint tissues nor disturbs vital tissues and organs. more...

The periosteal and endosteal surfaces of mature bone are densely innervated by sensory nerves expressing TrkA, the high-affinity receptor for nerve growth factor (NGF). In previous work, we demonstrated that administration of exogenous NGF significantly increased load-induced bone formation through the activation of Wnt signaling. However, the translational potential of NGF is limited by the induction of substantial mechanical and thermal hyperalgesia in mice and humans. Here, we tested the effect of gambogic amide (GA), a recently identified robust small molecule agonist for TrkA, on hyperalgesia and load-induced bone formation. Behavioral analysis was used to assess pain up to one week after axial forelimb compression. Contrary to our expectations, GA treatment was not associated with diminished use of the loaded forelimb or sensitivity to thermal stimulus. Furthermore, dynamic histomorphometry revealed a significant increase in relative periosteal bone formation rate as compared to vehicle treatment. Additionally, we found that GA treatment was associated with an increase in the number of osteoblasts per bone surface in loaded limbs as well as a significant increase in the fold change of Ngf, Wnt7b, and Axin2 mRNA expression as compared to vehicle (control). To test the effect of GA on osteoblasts directly, we cultured MC3T3-E1 cells for up to 21 days in osteogenic differentiation media containing NGF, GA, or vehicle (control). Media containing GA induced the significant upregulation of the osteoblastic differentiation markers Runx2, Bglap2, and Sp7 in a dose-dependent manner, whereas treatment with NGF was not associated with any significant increases in these markers. Furthermore, consistent with our in vivo findings, we observed that administration of 50 nM of GA upregulated expression of Ngf at both Day 3 and Day 7. However, cells treated with the highest dose of GA (500 nM) had significantly increased apoptosis and impaired cell proliferation. In conclusion, our study indicates GA may be useful for augmenting skeletal adaptation to mechanical forces without inducing hyperalgesia. more...

SUMMARYDebilitating stress fractures are surprisingly common in physically active individuals, including athletes, military recruits, and dancers. These individuals are overrepresented in the 30 million daily users of non-steroidal anti-inflammatory drugs (NSAIDs). We hypothesized that regular use of NSAIDs would predispose habitually loaded bones to stress fracture and delay the repair of these injuries. To test this hypothesis, adult mice were subjected to six bouts of axial forelimb compression over two weeks. Aspirin, naproxen, or vehicle was administered 24 hours before loading. Naproxen-treated mice had diminished load-induced bone formation as well as a significant loss in toughness in non-loaded bone, which were not observed in aspirin-treated mice. Furthermore, there were no differences in RANKL/OPG ratio or cortical bone parameters. Picrosirius red staining and second harmonic generation imaging revealed that alterations in bone collagen fibril size and organization were driving the loss of toughness in naproxen-treated mice. Separately, adult mice were subjected to an ulnar stress fracture generated by a single bout of fatigue loading, with NSAIDs provided 24 hours before injury. Both aspirin-treated and naproxen-treated mice had normal forelimb use in the week after injury, whereas control mice favored the injured forelimb until day 7. However, woven bone volume was only significantly impaired by naproxen. Both NSAIDs were found to significantly inhibit Cox2 and Ngf expression following stress fracture, but only naproxen significantly affected serum PGE2 concentration. Overall, our results suggest that naproxen, but not aspirin, may increase the risk of stress fracture and extend the healing time of these injuries, warranting further clinical evaluation for patients at risk for fatigue injuries. more...

Objective: Dose optimization and pharmacokinetic evaluation of alpha emitting Radium-223 dichloride (223RaCl2) by planar gamma camera or single photon emission computed tomographic (SPECT) imaging are hampered by the low photon abundance and injection activities. Here, we demonstrate SPECT of 223Ra using phantoms and small animal in vivo models. Methods: Line phantoms and mice bearing 223Ra were imaged using a next generation dedicated small animal SPECT by detecting the low energy photon emissions from 223Ra. Localization of the therapeutic agent was verified by whole body and whole limb autoradiography and its effect determined by immunofluorescence. Results: A state-of-the-art commercial small animal SPECT system equipped with a highly sensitive collimator enables collection of sufficient counts for three-dimensional reconstruction. Line sources of 223Ra in both air and in a water scattering phantom gave linear response functions with provide full-width-at-half-maximum of 1.45 mm. Early and late phase imaging of the pharmacokinetics of the radiopharmaceutical were captured. Uptake at sites of active bone remodeling were correlated with DNA damage from the alpha particle emissions. Conclusions: This work demonstrates the capability to noninvasively define the distribution of 223Ra, a recently approved alpha emitting radionuclide. This approach allows quantitative assessment of 223Ra distribution and may provide radiation dose optimization strategies to improve therapeutic response and ultimately to enable personalized treatment planning. more...

Loeys-Dietz syndrome (LDS) is a connective tissue disorder characterized by vascular and skeletal abnormalities resembling Marfan syndrome, including a predisposition for pathologic fracture. LDS is caused by heterozygous mutations in the genes encoding transforming growth factor-β (TGF-β) type 1 and type 2 receptors. In this study, we characterized the skeletal phenotype of mice carrying a mutation in the TGF-β type 2 receptor associated with severe LDS in humans. Cortical bone in LDS mice showed significantly reduced tissue area, bone area, and cortical thickness with increased eccentricity. However, no significant differences in trabecular bone volume were observed. Dynamic histomorphometry performed in calcein-labeled mice showed decreased mineral apposition rates in cortical and trabecular bone with normal numbers of osteoblasts and osteoclasts. Mechanical testing of femurs by three-point bending revealed reduced femoral strength and fracture resistance. In vitro, osteoblasts from LDS mice demonstrated increased mineralization with enhanced expression of osteoblast differentiation markers compared with control cells. These changes were associated with impaired TGF-β1-induced Smad2 and Erk1/2 phosphorylation and upregulated TGF-β1 ligand mRNA expression, compatible with G357W as a loss-of-function mutation in the TGF-β type 2 receptor. Paradoxically, phosphorylated Smad2/3 in cortical osteocytes measured by immunohistochemistry was increased relative to controls, possibly suggesting the cross-activation of TGF-β-related receptors. The skeletal phenotype observed in the LDS mouse closely resembles the principal structural features of bone in humans with LDS and establishes this mouse as a valid in vivo model for further investigation of TGF-β receptor signaling in bone. more...

HIF-1 is a transcription factor typically associated with angiogenic gene transcription under hypoxic conditions. In this study, mice with HIF-1α deleted in the osteoblast lineage (ΔHIF-1α) were subjected to damaging or non-damaging mechanical loading known to produce woven or lamellar bone, respectively, at the ulnar diaphysis. By microCT, ΔHIF-1α mice produced significantly less woven bone than wild type (WT) mice 7days after damaging loading. This decrease in woven bone volume and extent was accompanied by a significant decrease in vascularity measured by immunohistochemistry against vWF. Additionally, osteocytes, rather than osteoblasts, appear to be the main bone cell expressing HIF-1α following damaging loading. In contrast, 10days after non-damaging mechanical loading, dynamic histomorphometry measurements demonstrated no impairment in loading-induced lamellar bone formation in ΔHIF-1α mice. In fact, both non-loaded and loaded ulnae from ΔHIF-1α mice had increased bone formation compared with WT ulnae. When comparing the relative increase in periosteal bone formation in loaded vs. non-loaded ulnae, it was not different between ΔHIF-1α mice and controls. There were no significant differences observed between WT and ΔHIF-1α mice in endosteal bone formation parameters. The increases in periosteal lamellar bone formation in ΔHIF-1α mice are attributed to non-angiogenic effects of the knockout. In conclusion, these results demonstrate that HIF-1α is a pro-osteogenic factor for woven bone formation after damaging loading, but an anti-osteogenic factor for lamellar bone formation under basal conditions and after non-damaging loading. more...

Sensory nerves emanating from the dorsal root extensively innervate the surfaces of mammalian bone, a privileged location for the regulation of biomechanical signaling. Here, we show that NGF-TrkA signaling in skeletal sensory nerves is an early response to mechanical loading of bone and is required to achieve maximal load-induced bone formation. First, the elimination of TrkA signaling in mice harboring mutant TrkA(F592A) alleles was found to greatly attenuate load-induced bone formation induced by axial forelimb compression. Next, both in vivo mechanical loading and in vitro mechanical stretch were shown to induce the profound up-regulation of NGF in osteoblasts within 1 h of loading. Furthermore, inhibition of TrkA signaling following axial forelimb compression was observed to reduce measures of Wnt/β-catenin activity in osteocytes in the loaded bone. Finally, the administration of exogenous NGF to wild-type mice was found to significantly increase load-induced bone formation and Wnt/β-catenin activity in osteocytes. In summary, these findings demonstrate that communication between osteoblasts and sensory nerves through NGF-TrkA signaling is essential for load-induced bone formation in mice. more...

Biallelic mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), a disease characterized by calcification in the skin, eyes, and blood vessels. The function of ATP-binding cassette C6 (ABCC6) and the pathogenesis of PXE remain unclear. We used mouse models and patient fibroblasts to demonstrate genetic interaction and shared biochemical and cellular mechanisms underlying ectopic calcification in PXE and related disorders caused by defined perturbations in extracellular adenosine 5′-triphosphate catabolism. Under osteogenic culture conditions, ABCC6 mutant cells calcified, suggesting a provoked cell-autonomous defect. Using a conditional Abcc6 knockout mouse model, we excluded the prevailing pathogenic hypothesis that singularly invokes failure of hepatic secretion of an endocrine inhibitor of calcification. Instead, deficiency of Abcc6 in both local and distant cells was necessary to achieve the early onset and penetrant ectopic calcification observed upon constitutive gene targeting. ABCC6 mutant cells additionally had increased expression and activity of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme that degrades pyrophosphate, a major inhibitor of calcification. A selective and orally bioavailable TNAP inhibitor prevented calcification in ABCC6 mutant cells in vitro and attenuated both the development and progression of calcification in Abcc6 −/− mice in vivo, without the deleterious effects on bone associated with other proposed treatment strategies. more...

Angiogenesis and osteogenesis are critically linked, although the role of angiogenesis is not well understood in osteogenic mechanical loading. In this study, either damaging or non-damaging cyclic axial compression was used to generate woven bone formation (WBF) or lamellar bone formation (LBF), respectively, at the mid-diaphysis of the adult rat forelimb. αvβ3 integrin–targeted nanoparticles or vehicle was injected intravenously after mechanical loading. β3 integrin subunit expression on vasculature was maximal 7 days after damaging mechanical loading, but was still robustly expressed 14 days after loading. Accordingly, targeted nanoparticle delivery in WBF-loaded limbs was increased compared with non-loaded limbs. Vascularity was dramatically increased after WBF loading (+700% on day 14) and modestly increased after LBF loading (+50% on day 14). This increase in vascularity was inhibited by nanoparticle treatment in both WBF- and LBF-loaded limbs at days 7 and 14 after loading. Decreased vascularity led to diminished woven, but not lamellar, bone formation. Decreased woven bone formation resulted in impaired structural properties of the skeletal repair, particularly in post-yield behavior. These results demonstrate that αvβ3 integrin–mediated angiogenesis is critical for recovering fracture resistance after bone injury but is not required for bone modeling after modest mechanical strain. © 2014 American Society for Bone and Mineral Research. more...

Insulin signaling in osteoblasts regulates global energy balance by stimulating the production of osteocalcin, a bone-derived protein that promotes insulin production and action. To identify the signaling pathways in osteoblasts that mediate insulin's effects on bone and energy metabolism, we examined the function of the tuberous sclerosis 2 (Tsc2) protein, a key target important in coordinating nutrient signaling. Here, we show that loss of Tsc2 in osteoblasts constitutively activates mTOR and destabilizes Irs1, causing osteoblasts to differentiate poorly and become resistant to insulin. Young Tsc2 mutant mice demonstrate hypoglycemia with increased levels of insulin and undercarboxylated osteocalcin. However, with age, Tsc2 mutants develop metabolic features similar to mice lacking the insulin receptor in the osteoblast, including peripheral adiposity, hyperglycemia, and decreased pancreatic β cell mass. These metabolic abnormalities appear to result from chronic elevations in undercarboxylated osteocalcin that lead to downregulation of the osteocalcin receptor and desensitization of the β cell to this hormone. Removal of a single mTOR allele from the Tsc2 mutant mice largely normalizes the bone and metabolic abnormalities. Together, these findings suggest that Tsc2 serves as a key checkpoint in the osteoblast that is required for proper insulin signaling and acts to ensure normal bone acquisition and energy homeostasis. more...

Bone is a highly vascularized tissue, although this aspect of bone is often overlooked. In this article, the importance of blood flow in bone repair and regeneration will be reviewed. First, the skeletal vascular anatomy, with an emphasis on long bones, the distinct mechanisms for vascularizing bone tissue, and methods for remodeling existing vasculature are discussed. Next, techniques for quantifying bone blood flow are briefly summarized. Finally, the body of experimental work that demonstrates the role of bone blood flow in fracture healing, distraction osteogenesis, osteoporosis, disuse osteopenia, and bone grafting is examined. These results illustrate that adequate bone blood flow is an important clinical consideration, particularly during bone regeneration and in at-risk patient groups. more...

Alterations in resorption cavities and bone remodeling events during anti-resorptive treatment are believed to contribute to reductions in fracture risk. Here, we examine changes in the size of individual remodeling events associated with treatment with a selective estrogen receptor modulator (raloxifene) or a bisphosphonate (risedronate). Adult female rats (6 months of age) were submitted to ovariectomy (n = 17) or sham surgery (SHAM, n = 5). One month after surgery, the ovariectomized animals were separated into three groups: untreated (OVX, n = 5), raloxifene treated (OVX+Ral, n = 6) and risedronate treated (OVX+Ris, n = 6). At 10 months of age, the lumbar vertebrae were submitted to three-dimensional dynamic bone histomorphometry to examine the size (depth, breadth, volume) of individual resorption cavities and formation events. Maximum resorption cavity depth did not differ between the SHAM (23.66 ± 1.87 µm, mean ± SD) and OVX (22.88 ± 3.69 µm) groups but was smaller in the OVX+Ral (14.96 ± 2.30 µm) and OVX+Ris (14.94 ± 2.70 µm) groups (p < 0.01). Anti-resorptive treatment was associated with reductions in the surface area of resorption cavities and the volume occupied by each resorption cavity (p < 0.01 each). The surface area and volume of individual formation events (double-labeled events) in the OVX+Ris group were reduced as compared to other groups (p < 0.02). Raloxifene treated animals showed similar amounts of bone remodeling (ES/BS and dLS/BS) compared to sham-operated controls but smaller cavity size (depth, breadth and volume). The current study shows that anti-resorptive agents influence the size of resorption cavities and individual remodeling events and that the effect of anti-resorptives on individual remodeling events may not always be directly related to the degree of suppression of bone remodeling. more...

In skeletal tissue, loss or mutation of the gap junction protein connexin 43 (Cx43, also known as GJA1) in cells of the osteoblast lineage leads to a profound cortical bone phenotype and defective tissue remodeling. There is mounting evidence in bone cells that the C-terminus (CT) of Cx43 is a docking platform for signaling effectors and is required for efficient downstream signaling. Here, we examined this function, using a mouse model of Cx43 CT-truncation (Gja1 K258Stop). Relative to Gja1+/− controls, male Gja1−/K258Stop mice have a cortical bone phenotype that is remarkably similar to those reported for deletion of the entire Cx43 gene in osteoblasts. Furthermore, we show that the Cx43 CT binds several signaling proteins that are required for optimal osteoblast function, including PKCδ, ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) and β-catenin. Deletion of the Cx43 CT domain affects these signaling cascades, impacting osteoblast proliferation, differentiation, and collagen processing and organization. These data imply that, at least in bone, Cx43 gap junctions not only exchange signals, but also recruit the appropriate effector molecules to the Cx43 CT in order to efficiently activate signaling cascades that affect cell function and bone acquisition. more...

The coupling of osteoblasts and osteocytes by connexin43 (Cx43) gap junctions permits the sharing of second messengers that coordinate bone cell function and cortical bone acquisition. However, details of how Cx43 converts shared second messengers into signals that converge onto essential osteogenic processes are incomplete. Here, we use in vitro and in vivo methods to show that Cx43 and Runx2 functionally interact to regulate osteoblast gene expression and proliferation, ultimately affecting cortical bone properties. Using compound hemizygous mice for the Gja1 (Cx43) and Runx2 genes, we observed a skeletal phenotype not visible in wild-type or singly hemizygous animals. Cortical bone analysis by microCT revealed that 8-week-old male, compound Gja1+/− Runx2+/− mice have a marked increase in cross-sectional area, endosteal and periosteal bone perimeter, and an increase in porosity compared to controls. These compound Gja1+/− Runx2+/− mice closely approximate the cortical bone phenotypes seen in osteoblast-specific Gja1-conditional knockout models. Furthermore, microCT analysis of skulls revealed an altered interparietal bone geometry in compound hemizygotes. Consistent with this finding, Alizarin red/ Alcian blue staining of 2 day-old Gja1+/− Runx2+/− neonates showed a hypomorphic interparietal bone, an exacerbation of the open fontanelles, and a further reduction in the hypoplastic clavicles compared to Runx2+/− neonates. Expression of osteoblast genes, including osteocalcin, osterix, periostin, and Hsp47, was markedly reduced in tibial RNA extracts from compound hemizygous mice, and osteoblasts from compound hemizygous mice exhibited increased proliferative capacity. Further, the reduced osteocalcin expression and hyperproliferative nature of osteoblasts from Cx43 deficient mice was rescued by Runx2 expression. In summary, these findings provide evidence that Cx43 and Runx2 functionally intersect in vivo to regulate cortical bone properties and affect osteoblast differentiation and proliferation, and likely contributes to aspects of the skeletal phenotype of Cx43 conditional knockout mice. more...

SummaryDeveloping tissues dictate the amount and type of innervation they require by secreting neurotrophins, which promote neuronal survival by activating distinct tyrosine kinase receptors. Here, we show that nerve growth factor (NGF) signaling through neurotrophic tyrosine kinase receptor type 1 (TrkA) directs innervation of the developing mouse femur to promote vascularization and osteoprogenitor lineage progression. At the start of primary ossification, TrkA-positive axons were observed at perichondrial bone surfaces, coincident with NGF expression in cells adjacent to centers of incipient ossification. Inactivation of TrkA signaling during embryogenesis in TrkAF592A mice impaired innervation, delayed vascular invasion of the primary and secondary ossification centers, decreased numbers of Osx-expressing osteoprogenitors, and decreased femoral length and volume. These same phenotypic abnormalities were observed in mice following tamoxifen-induced disruption of NGF in Col2-expressing perichondrial osteochondral progenitors. We conclude that NGF serves as a skeletal neurotrophin to promote sensory innervation of developing long bones, a process critical for normal primary and secondary ossification. more...

Blood flow is an important factor in bone production and repair, but its role in osteogenesis induced by mechanical loading is unknown. Here, we present techniques for evaluating blood flow and fluoride metabolism in a pre-clinical stress fracture model of osteogenesis in rats.Bone formation was induced by forelimb compression in adult rats. (15)O water and (18)F fluoride PET imaging were used to evaluate blood flow and fluoride kinetics 7 days after loading. (15)O water was modeled using a one-compartment, two-parameter model, while a two-compartment, three-parameter model was used to model (18)F fluoride. Input functions were created from the heart, and a stochastic search algorithm was implemented to provide initial parameter values in conjunction with a Levenberg-Marquardt optimization algorithm.Loaded limbs are shown to have a 26% increase in blood flow rate, 113% increase in fluoride flow rate, 133% increase in fluoride flux, and 13% increase in fluoride incorporation into bone as compared to non-loaded limbs (p 0.05 for all results).The results shown here are consistent with previous studies, confirming this technique is suitable for evaluating the vascular response and mineral kinetics of osteogenic mechanical loading. more...

Loeys-Dietz syndrome (LDS) is a connective tissue disorder characterized by vascular and skeletal abnormalities resembling Marfan syndrome, including a predisposition for pathologic fracture. LDS is caused by heterozygous mutations in the genes encoding transforming growth factor-β (TGF-β) type 1 and type 2 receptors. In this study, we characterized the skeletal phenotype of mice carrying a mutation in the TGF-β type 2 receptor associated with severe LDS in humans. Cortical bone in LDS mice showed significantly reduced tissue area, bone area, and cortical thickness with increased eccentricity. However, no significant differences in trabecular bone volume were observed. Dynamic histomorphometry performed in calcein-labeled mice showed decreased mineral apposition rates in cortical and trabecular bone with normal numbers of osteoblasts and osteoclasts. Mechanical testing of femurs by three-point bending revealed reduced femoral strength and fracture resistance. In vitro, osteoblasts from LDS mice demonstrated increased mineralization with enhanced expression of osteoblast differentiation markers compared with control cells. These changes were associated with impaired TGF-β1-induced Smad2 and Erk1/2 phosphorylation and upregulated TGF-β1 ligand mRNA expression, compatible with G357W as a loss-of-function mutation in the TGF-β type 2 receptor. Paradoxically, phosphorylated Smad2/3 in cortical osteocytes measured by immunohistochemistry was increased relative to controls, possibly suggesting the cross-activation of TGF-β-related receptors. The skeletal phenotype observed in the LDS mouse closely resembles the principal structural features of bone in humans with LDS and establishes this mouse as a valid in vivo model for further investigation of TGF-β receptor signaling in bone. more...