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Anabolic agents, such as intermittent parathyroid hormone (PTH), exert their treatment efficacy through activation of two distinct bone formation processes, namely, remodeling-based bone formation (RBF, bone formation coupled with prior bone resorption) and modeling-based bone formation (MBF, bone formation without prior activation of bone resorption). However, if not followed by an antiresorptive agent, treatment benefit was quickly lost upon withdrawal from anabolic agents. By using in vivo micro-computed tomography imaging and multiplex cryohistology with sequential immunofluorescence staining, we investigated the temporal response of newly formed bone tissue from MBF and RBF and the preexisting bone tissue to withdrawal from PTH treatment and the associated cellular activity in an ovariectomized (OVX) rat model. We first demonstrated continued mineral apposition at both RBF and MBF sites following PTH discontinuation, resulting in an extended anabolic effect after 1-week withdrawal from PTH. It was further discovered that MBF sites had a greater contribution than RBF sites to the extended anabolic effect upon early withdrawal from PTH, evidenced by a higher percentage of alkaline phosphatase-positive (ALP+) surfaces and far greater bone formation activity at MBF versus RBF sites. Furthermore, significant bone loss occurred after 3 weeks of discontinuation from PTH, resulting from marked loss of newly formed bone tissue from RBF and preexisting bone tissue prior to treatment. In contrast, MBF surfaces had a delayed increase of tartrate-resistant acid phosphatase activity following PTH discontinuation. As a result, newly formed bone tissue from MBF had greater resistance to PTH discontinuation-induced bone loss than those from RBF and preexisting bone. Understanding various responses of two distinct bone formation types and preexisting bone to anabolic treatment discontinuation is critical to inform the design of follow-up treatment or cyclic treatment strategies to maximize treatment benefit of anabolic agents. © 2022 American Society for Bone and Mineral Research (ASBMR).

Despite the potent effect of intermittent parathyroid hormone (PTH) treatment on promoting new bone formation, bone mineral density (BMD) rapidly decreases upon discontinuation of PTH administration. To uncover the mechanisms behind this adverse phenomenon, we investigated the immediate responses in bone microstructure and bone cell activities to PTH treatment withdrawal and the associated long-term consequences. Unexpectedly, intact female and estrogen-deficient female rats had distinct responses to the discontinuation of PTH treatment. Significant tibial bone loss and bone microarchitecture deterioration occurred in estrogen-deficient rats, with the treatment benefits of PTH completely lost 9 weeks after discontinuation. In contrast, no adverse effect was observed in intact rats, with sustained treatment benefit 9 weeks after discontinuation. Intriguingly, there is an extended anabolic period during the first week of treatment withdrawal in estrogen-deficient rats, during which no significant change occurred in the number of osteoclasts, whereas the number of osteoblasts remained elevated compared with vehicle-treated rats. However, increases in number of osteoclasts and decreases in number of osteoblasts occurred 2 weeks after discontinuation of PTH treatment, leading to significant reduction in bone mass and bone microarchitecture. To leverage the extended anabolic period upon early withdrawal from PTH, a cyclic administration regimen with repeated cycles of on and off PTH treatment was explored. We demonstrated that the cyclic treatment regimen efficiently alleviated the PTH withdrawal-induced bone loss, improved bone mass, bone microarchitecture, and whole-bone mechanical properties, and extended the treatment duration. © 2021 American Society for Bone and Mineral Research (ASBMR).

OBJECTIVE To elucidate the role of decorin, a small leucine-rich proteoglycan, in the degradation of cartilage matrix during the progression of post-traumatic osteoarthritis (OA). METHODS Three-month-old decorin-null (Dcn-/- ) and inducible decorin-knockout (DcniKO ) mice were subjected to surgical destabilization of the medial meniscus (DMM) to induce post-traumatic OA. The OA phenotype that resulted was evaluated by assessing joint morphology and sulfated glycosaminoglycan (sGAG) staining via histological analysis (n = 6 mice per group), surface collagen fibril nanostructure via scanning electron microscopy (n = 4 mice per group), tissue modulus via atomic force microscopy-nanoindentation (n = 5 or more mice per group) and subchondral bone structure via micro-computed tomography (n = 5 mice per group). Femoral head cartilage explants from wild-type and Dcn-/- mice were stimulated with the inflammatory cytokine interleukin-1β (IL-1β) in vitro (n = 6 mice per group). The resulting chondrocyte response to IL-1β and release of sGAGs were quantified. RESULTS In both Dcn-/- and DcniKO mice, the absence of decorin resulted in accelerated sGAG loss and formation of highly aligned collagen fibrils on the cartilage surface relative to the control (P < 0.05). Also, Dcn-/- mice developed more salient osteophytes, illustrating more severe OA. In cartilage explants treated with IL-1β, loss of decorin did not alter the expression of either anabolic or catabolic genes. However, a greater proportion of sGAGs was released to the media from Dcn-/- mouse explants, in both live and devitalized conditions (P < 0.05). CONCLUSION In post-traumatic OA, decorin delays the loss of fragmented aggrecan and fibrillation of cartilage surface, and thus, plays a protective role in ameliorating cartilage degeneration.

Over the past decades, synthetic musks have been widely used as fragrances for enhancing scent and covering odor in many personal care products (PCPs). The presence of synthetic musk is of potential concern since health effects, such as photo-allergic reactions and neurotoxicity due to the exposures have been observed. To assess the associate health risks from possible exposures of synthetic musks in Taiwan, headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography/tandem mass spectrometers (GC/MS/MS) in multiple reaction monitoring (MRM) mode was used to determine 10 synthetic musks in total 109 PCPs samples. The results showed that the higher levels of synthetic musks were found in perfume, body lotion and hair care products. Galaxolide (HHCB) and Tonalide (AHTN) were found in every category of the PCPs samples. The median concentrations in 109 samples measured were 958.19 μg g−1 and 674.03 μg g−1 for HHCB and AHTN, respectively. Cashmeran (DPMI) was also found in all the samples collected with a median concentration of 144.62 μg g−1, except for categories of facial essences. Musk ketone (MK) was found in several PCPs categories, including perfume, body lotion, hair care product, and shower bath products, with a median concentration of 693.27 μg g−1. In addition, dermal exposures of synthetic musks were also estimated for people in Taiwan. The daily exposure of total synthetic musks through applications of PCPs was estimated to be 22.54 μg kg−1 body weight day−1. The results indicate that the skin contact of perfume and body lotion were the major sources for human exposures to synthetic musks in Taiwan.

Postmenopausal osteoporosis affects a large number of women worldwide. Reduced estrogen levels during menopause lead to accelerated bone remodeling, resulting in low bone mass and increased fracture risk. Both peak bone mass and the rate of bone loss are important predictors of postmenopausal osteoporosis risk. However, whether peak bone mass and/or bone microstructure directly influence the rate of bone loss following menopause remains unclear. Our study aimed to establish the relationship between peak bone mass/microstructure and the rate of bone loss in response to estrogen deficiency following ovariectomy (OVX) surgery in rats of homogeneous background by tracking the skeletal changes using in vivo micro-computed tomography (μCT) and three-dimensional (3D) image registrations. Linear regression analyses demonstrated that the peak bone microstructure, but not peak bone mass, was highly predictive of the rate of OVX-induced bone loss. In particular, the baseline trabecular thickness was found to have the highest correlation with the degree of OVX-induced bone loss and trabecular stiffness reduction. Given the same bone mass, the rats with thicker baseline trabeculae had a lower rate of trabecular microstructure and stiffness deterioration after OVX. Moreover, further evaluation to track the changes within each individual trabecula via our novel individual trabecular dynamics (ITD) analysis suggested that a trabecular network with thicker trabeculae is less likely to disconnect or perforate in response to estrogen deficiency, resulting a lower degree of bone loss. Taken together, these findings indicate that the rate of estrogen-deficiency-induced bone loss could be predicted by peak bone microstructure, most notably the trabecular thickness. Given the same bone mass, a trabecular bone phenotype with thin trabeculae may be a risk factor toward accelerated postmenopausal bone loss.

PGC-1 (peroxisome-proliferator-activated receptor-γ coactivator-1) alpha is a potent transcriptional coactivator that coordinates the activation of numerous metabolic processes. Exercise strongly induces PGC-1alpha expression in muscle, and overexpression of PGC-1alpha in skeletal muscle activates mitochondrial oxidative metabolism and neovascularization, leading to markedly increased endurance. In light of these findings, PGC-1alpha has been proposed to protect from age-associated sarcopenia, bone loss, and whole-body metabolic dysfunction, although these findings have been controversial. We therefore comprehensively evaluated muscle and whole-body function and metabolism in 24-month-old transgenic mice that over-express PGC-1alpha in skeletal muscle. We find that the powerful effects of PGC-1alpha on promoting muscle oxidative capacity and protection from muscle fatigability persist in aged animals, although at the expense of muscle strength. However, skeletal muscle PGC-1alpha does not prevent bone loss and in fact accentuates it, nor does it have long-term benefit on whole-body metabolic composition or insulin sensitivity. Protection from sarcopenia is seen in male animals with overexpression of PGC-1alpha in skeletal muscle but not in female animals. In summary, muscle-specific expression of PGC-1alpha into old age has beneficial effects on muscle fatigability and may protect from sarcopenia in males, but does not improve whole-body metabolism and appears to worsen age-related trabecular bone loss.

The female skeleton undergoes substantial structural changes during the course of reproduction. Although bone mineral density recovers postweaning, reproduction may induce permanent alterations in maternal bone microarchitecture. However, epidemiological studies suggest that a history of pregnancy and/or lactation does not increase the risk of postmenopausal osteoporosis or fracture and may even have a protective effect. Our study aimed to explain this paradox by using a rat model, combined with in vivo micro-computed tomography (μCT) imaging and bone histomorphometry, to track the changes in bone structure and cellular activities in response to estrogen deficiency following ovariectomy (OVX) in rats with and without a reproductive history. Our results demonstrated that a history of reproduction results in an altered skeletal response to estrogen-deficiency-induced bone loss later in life. Prior to OVX, rats with a reproductive history had lower trabecular bone mass, altered trabecular microarchitecture, and more robust cortical structure at the proximal tibia when compared to virgins. After OVX, these rats underwent a lower rate of trabecular bone loss than virgins, with minimal structural deterioration. As a result, by 12 weeks post-OVX, rats with a reproductive history had similar trabecular bone mass, elevated trabecular thickness, and increased robustness of cortical bone when compared to virgins, resulting in greater bone stiffness. Further evaluation suggested that reproductive-history-induced differences in post-OVX trabecular bone loss were likely due to differences in baseline trabecular microarchitecture, particularly trabecular thickness. Rats with a reproductive history had a larger population of thick trabeculae, which may be protective against post-OVX trabecular connectivity deterioration and bone loss. Taken together, these findings indicate that reproduction-associated changes in bone microarchitecture appear to reduce the rate of bone loss induced by estrogen deficiency later in life, and thereby exert a long-term protective effect on bone strength. © 2018 American Society for Bone and Mineral Research.

Osteoporosis most commonly affects postmenopausal women. Although men are also affected, women over 65 are 6 times more likely to develop osteoporosis than men of the same age. This is largely due to accelerated bone remodeling after menopause; however, the peak bone mass attained during young adulthood also plays an important role in osteoporosis risk. Multiple studies have demonstrated sexual dimorphisms in peak bone mass, and additionally, the female skeleton is significantly altered during pregnancy/lactation. Although clinical studies suggest that a reproductive history does not increase the risk of developing postmenopausal osteoporosis, reproduction has been shown to induce long-lasting alterations in maternal bone structure and mechanics, and the effects of pregnancy and lactation on maternal peak bone quality are not well understood. This study compared the structural and mechanical properties of male, virgin female, and post-reproductive female rat bone at multiple skeletal sites and at three different ages. We found that virgin females had a larger quantity of trabecular bone with greater trabecular number and more plate-like morphology, and, relative to their body weight, had a greater cortical bone size and greater bone strength than males. Post-reproductive females had altered trabecular microarchitecture relative to virgins, which was highly similar to that of male rats, and showed similar cortical bone size and bone mechanics to virgin females. This suggests that, to compensate for future reproductive bone losses, females may start off with more trabecular bone than is mechanically necessary, which may explain the paradox that reproduction induces long-lasting changes in maternal bone without increasing postmenopausal fracture risk.

The maternal skeleton undergoes dramatic bone loss during pregnancy and lactation, and substantial bone recovery post-weaning. The structural adaptations of maternal bone during reproduction and lactation exert a better protection of the mechanical integrity at the critical load-bearing sites, suggesting the importance of physiological load-bearing in regulating reproduction-induced skeletal alterations. Although it is suggested that physical exercise during pregnancy and breastfeeding improves women's physical and psychological well-being, its effects on maternal bone health remain unclear. Therefore, the objective of this study was to investigate the maternal bone adaptations to external mechanical loading during pregnancy, lactation, and post-weaning recovery. By utilizing an in vivo dynamic tibial loading protocol in a rat model, we demonstrated improved maternal cortical bone structure in response to dynamic loading at tibial midshaft, regardless of reproductive status. Notably, despite the minimal loading responses detected in the trabecular bone in virgins, rat bone during lactation experienced enhanced mechano-responsiveness in both trabecular and cortical bone compartments when compared to rats at other reproductive stages or age-matched virgins. Furthermore, our study showed that the lactation-induced elevation in osteocyte peri-lacunar/canalicular remodeling (PLR) activities led to enlarged osteocyte lacunae. This may result in alterations in interstitial fluid flow-mediated mechanical stimulation on osteocytes and an elevation in solute transport through the lacunar-canalicular system (LCS) during high-frequency dynamic loading, thus enhancing mechano-responsiveness of maternal bone during lactation. Taken together, findings from this study provide important insights into the relationship between reproduction- and lactation-induced skeletal changes and external mechanical loading, emphasizing the importance of weight-bearing exercise on maternal bone health during reproduction and postpartum.

Activation of modeling-based bone formation (MBF - bone formation without prior activation of bone resorption), has been identified as an important mechanism by which anabolic agents, such as intermittent parathyroid hormone (PTH), rapidly elicit new bone formation. Using a novel cryohistology imaging platform, coupled with sequential multicolor fluorochrome injections, we demonstrated that MBF and remodeling-based bone formation (RBF) in the adult rat tibia model have similar contributions to trabecular bone homeostasis. PTH treatment resulted in a 2.4–4.9 fold greater bone formation rate over bone surface (BFR/BS) by RBF and a 4.3–8.5 fold greater BFR/BS by MBF in male, intact female, and ovariectomized female rats. Moreover, regardless of bone formation type, once a formation site is activated by PTH, mineral deposition continues throughout the entire treatment duration. Furthermore, by tracking the sequence of multicolor fluorochrome labels, we discovered that MBF, a highly efficient but often overlooked regenerative mechanism, is activated more rapidly but attenuated faster than RBF in response to PTH. This suggests that MBF and RBF contribute differently to PTH’s anabolic effect in rats: MBF has a greater contribution to the acute elevation in bone mass at the early stage of treatment while RBF contributes to the sustained treatment effect.

Interleukin-15 receptor alpha (IL15RA) is an important component of interleukin-15 (IL15) pro-inflammatory signaling. In addition, IL15 and IL15RA are present in the circulation and are detected in a variety of tissues where they influence physiological functions such as muscle contractility and overall metabolism. In the skeletal system, IL15RA was previously shown to be important for osteoclastogenesis. Little is known, however, about its role in osteoblast function and bone mineralization. In this study, we evaluated bone structural and mechanical properties of an Il15ra whole-body knockout mouse (Il15ra−/−) and used in vitro and bioinformatic analyses to understand the role IL15/IL15RA signaling on osteoblast function. We show that lack of IL15RA decreased bone mineralization in vivo and in isolated primary osteogenic cultures, suggesting a cell-autonomous effect. Il15ra−/− osteogenic cultures also had reduced Rankl/Opg mRNA ratio, indicating defective osteoblast/osteoclast coupling. We analyzed the transcriptome of primary pre-osteoblasts from normal and Il15ra−/− mice and identified 1150 genes that were differentially expressed at a FDR of 5%. Of these, 844 transcripts were upregulated and 306 were downregulated in Il15ra−/− cells. The largest functional clusters, highlighted using DAVID analysis, were related to metabolism, immune response, bone mineralization and morphogenesis. The transcriptome analysis was validated by qPCR of some of the most significant hits. Using bioinformatic approaches, we identified candidate genes, including Cd200 and Enpp1, that could contribute to the reduced mineralization. Silencing Il15ra using shRNA in the calvarial osteoblast MC3T3-E1 cell line decreased ENPP1 activity. Taken together, these data support that IL15RA plays a cell-autonomous role in osteoblast function and bone mineralization.

Postmenopausal osteoporosis is often treated with bisphosphonates (eg, alendronate, [ALN]), but oversuppression of bone turnover by long-term bisphosphonate treatment may decrease bone tissue heterogeneity. Thus, alternate treatment strategies after long-term bisphosphonates are of great clinical interest. The objective of the current study was to determine the effect of intermittent parathyroid hormone (PTH) following 12 weeks of ALN (a bisphosphonate) treatment in 6-month-old, ovariectomized (OVX) rats on bone microarchitecture, bone remodeling dynamics, and bone mechanical properties at multiple length scales. By using in vivo μCT and 3D in vivo dynamic bone histomorphometry techniques, we demonstrated the efficacy of PTH following ALN therapy for stimulating new bone formation, and increasing trabecular thickness and bone volume fraction. In healthy bone, resorption and formation are coupled and balanced to sustain bone mass. OVX results in resorption outpacing formation, and subsequent bone loss and reduction in bone tissue modulus and tissue heterogeneity. We showed that ALN treatment effectively reduced bone resorption activity and regained the balance with bone formation, preventing additional bone loss. However, ALN treatment also resulted in significant reductions in the heterogeneity of bone tissue mineral density and tissue modulus. On the other hand, PTH treatment was able to shift the bone remodeling balance in favor of formation, with or without a prior treatment with ALN. Moreover, by altering the tissue mineralization, PTH alleviated the reduction in heterogeneity of tissue material properties induced by prolonged ALN treatment. Furthermore, switching to PTH treatment from ALN improved bone's postyield mechanical properties at both the whole bone and apparent level compared to ALN alone. The current findings suggest that intermittent PTH treatment should be considered as a viable treatment option for patients with prior treatment with bisphosphonates. © 2017 American Society for Bone and Mineral Research.

Pregnancy, lactation, and weaning result in dramatic changes in maternal calcium metabolism. In particular, the increased calcium demand during lactation causes a substantial degree of maternal bone loss. This reproductive bone loss has been suggested to be largely reversible, as multiple clinical studies have found that parity and lactation history have no adverse effect on postmenopausal fracture risk. However, the precise effects of pregnancy, lactation, and post-weaning recovery on maternal bone structure are not well understood. Our study aimed to address this question by longitudinally tracking changes in trabecular and cortical bone microarchitecture at the proximal tibia in rats throughout three cycles of pregnancy, lactation, and post-weaning using in vivo μCT. We found that the trabecular thickness underwent a reversible deterioration during pregnancy and lactation, which was fully recovered after weaning, whereas other parameters of trabecular microarchitecture (including trabecular number, spacing, connectivity density, and structure model index) underwent a more permanent deterioration, which recovered minimally. Thus, pregnancy and lactation resulted in both transient and long-lasting alterations in trabecular microstructure. In the meantime, multiple reproductive cycles appeared to improve the robustness of cortical bone (resulting in an elevated cortical area and polar moment of inertia), as well as increase the proportion of the total load carried by the cortical bone at the proximal tibia. Taken together, changes in the cortical and trabecular compartments suggest that whereas rat tibial trabecular bone appears to be highly involved in maintaining calcium homeostasis during female reproduction, cortical bone adapts to increase its load-bearing capacity, allowing the overall mechanical function of the tibia to be maintained. © 2017 American Society for Bone and Mineral Research.

A moderate radiation dose, in vivo μCT scanning protocol was developed and validated for long-term monitoring of multiple skeletal sites (femur, tibia, vertebra) in mice. A customized, 3D printed mouse holder was designed and utilized to minimize error associated with animal repositioning, resulting in good to excellent reproducibility in most cortical and trabecular bone microarchitecture and density parameters except for connectivity density. Repeated in vivo μCT scans of mice were performed at the right distal femur and the 4(th) lumbar vertebra every 3 weeks until sacrifice at 9 weeks after the baseline scan. Comparing to the non-radiated counterparts, no radiation effect was found on trabecular bone volume fraction, osteoblast and osteoblast number/surface, or bone formation rate at any skeletal site. However, trabecular number, thickness, and separation, and structure model index were sensitive to ionizing radiation associated with the μCT scans, resulting in subtle but significant changes over multiple scans. Although the extent of radiation damage on most trabecular bone microarchitecture measures are comparable or far less than the age-related changes during the monitoring period, additional considerations need to be taken to minimize the confounding radiation factors when designing experiments using in vivo μCT imaging for long-term monitoring of mouse bone.

Focal radiotherapy is frequently associated with skeletal damage within the radiation field. Our previous in vitro study showed that activation of Wnt/β-catenin pathway can overcome radiation-induced DNA damage and apoptosis of osteoblastic cells. Neutralization of circulating sclerostin with a monoclonal antibody (Scl-Ab) is an innovative approach for treating osteoporosis by enhancing Wnt/β-catenin signaling in bone. Together with the fact that focal radiation increases sclerostin amount in bone, we sought to determine whether weekly treatment with Scl-Ab would prevent focal radiotherapy-induced osteoporosis in mice. Micro-CT and histomorphometric analyses demonstrated that Scl-Ab blocked trabecular bone structural deterioration after radiation by partially preserving osteoblast number and activity. Consistently, trabecular bone in sclerostin null mice was resistant to radiation via the same mechanism. Scl-Ab accelerated DNA repair in osteoblasts after radiation by reducing the number of γ-H2AX foci, a DNA double-strand break marker, and increasing the amount of Ku70, a DNA repair protein, thus protecting osteoblasts from radiation-induced apoptosis. In osteocytes, apart from using similar DNA repair mechanism to rescue osteocyte apoptosis, Scl-Ab restored the osteocyte canaliculi structure that was otherwise damaged by radiation. Using a lineage tracing approach that labels all mesenchymal lineage cells in the endosteal bone marrow, we demonstrated that radiation damage to mesenchymal progenitors mainly involves shifting their fate to adipocytes and arresting their proliferation ability but not inducing apoptosis, which are different mechanisms from radiation damage to mature bone forming cells. Scl-Ab treatment partially blocked the lineage shift but had no effect on the loss of proliferation potential. Taken together, our studies provide proof-of-principle evidence for a novel use of Scl-Ab as a therapeutic treatment for radiation-induced osteoporosis and establish molecular and cellular mechanisms that support such treatment. © 2016 American Society for Bone and Mineral Research.

In vivo μCT imaging allows for high-resolution, longitudinal evaluation of bone properties. Based on this technology, several recent studies have developed in vivo dynamic bone histomorphometry techniques that utilize registered μCT images to identify regions of bone formation and resorption, allowing for longitudinal assessment of bone remodeling. However, this analysis requires a direct voxel-by-voxel subtraction between image pairs, necessitating rotation of the images into the same coordinate system, which introduces interpolation errors. We developed a novel image transformation scheme, matched-angle transformation (MAT), whereby the interpolation errors are minimized by equally rotating both the follow-up and baseline images instead of the standard of rotating one image while the other remains fixed. This new method greatly reduced interpolation biases caused by the standard transformation. Additionally, our study evaluated the reproducibility and precision of bone remodeling measurements made via in vivo dynamic bone histomorphometry. Although bone remodeling measurements showed moderate baseline noise, precision was adequate to measure physiologically relevant changes in bone remodeling, and measurements had relatively good reproducibility, with intra-class correlation coefficients of 0.75-0.95. This indicates that, when used in conjunction with MAT, in vivo dynamic histomorphometry provides a reliable assessment of bone remodeling.

In this study we established an image analysis scheme for the investigation of cortical and trabecular bone development during skeletal growth and tested this concept on in vivo µCT images of rats. To evaluate its efficacy, we applied the technique to young (1-month-old) and adult (3-month-old) rat tibiae with vehicle (Veh) or intermittent parathyroid hormone (PTH) treatment. By overlaying 2 sequential scans based on their distinct trabecular microarchitecure, we calculated the linear growth rate of young rats to be 0.31 mm/day at the proximal tibia. Due to rapid growth (3.7 mm in 12 days), the scanned bone region at day 12 had no overlap with the bone tissue scanned at day 0. Instead, the imaged bone region at day 12 represented newly generated bone tissue from the growth plate. The new bone of the PTH-treated rats had significantly greater trabecular bone volume fraction, number, and thickness than those of the Veh-treated rats, indicating PTH’s anabolic effect on bone modeling. In contrast, the effect of PTH on adult rat trabecular bone was found to be caused by PTH’s anabolic effect on bone remodeling. The cortical bone at the proximal tibia of young rats also thickened more in the PTH group (23%) than the Veh group (14%). This was primarily driven by endosteal bone formation and coalescence of trabecular bone into the cortex. This process can be visualized by aligning the local bone structural changes using image registration. As a result, the cortex after PTH treatment was 31% less porous, and had a 22% greater polar moment of inertia compared to the Veh group. Lastly, we monitored the longitudinal bone growth in adult rats by measuring the distance of bone flow away from the proximal tibial growth plate from 3 months to 19 months of age and discovered a total of 3.5 mm growth in 16 months. It was demonstrated that this image analysis scheme can efficiently evaluate bone growth, bone modeling, and bone remodeling, and is ready to be translated into a clinical imaging platform.

Each year, 33% of US citizens suffer from a musculoskeletal condition that requires medical intervention, with direct medical costs approaching $1 trillion USD per year. Despite the ubiquity of skeletal dysfunction, there are currently limited safe and efficacious bone growth factors in clinical use. Notch is a cell–cell communication pathway that regulates self-renewal and differentiation within the mesenchymal/osteoblast lineage. The principal Notch ligand in bone, Jagged-1, is a potent osteoinductive protein that positively regulates post-traumatic bone healing in animals. This report describes the temporal regulation of Notch during intramembranous bone formation using marrow ablation as a model system and demonstrates decreased bone formation following disruption of Jagged-1 in mesenchymal progenitor cells. Notch gain-of-function using recombinant Jagged-1 protein on collagen scaffolds promotes healing of craniofacial (calvarial) and appendicular (femoral) surgical defects in both mice and rats. Localized delivery of Jagged-1 promotes bone apposition and defect healing, while avoiding the diffuse bone hypertrophy characteristic of the clinically problematic bone morphogenetic proteins. It is concluded that Jagged-1 is a bone-anabolic agent with therapeutic potential for regenerating traumatic or congenital bone defects., Jagged-1 helps heal fractures faster Localized and temporary delivery of the protein Jagged-1 promotes bone regeneration in rodents. Despite the large incidence of bone injuries in humans, current therapies to stimulate bone growth can have serious side effects. Kurt Hankenson at the University of Michigan, US, and colleagues have been investigating ways to stimulate Notch receptors, key regulators of bone formation during development. They found that levels of the Notch receptor’s binding partner Jagged-1 were significantly increased after bone injury and that disruption of Jagged-1 prevented bone healing in mice. Bone repair in both mice and rats was significantly improved following delivery of Jagged-1 on a collagen scaffold to the site of injury compared with controls. These findings could pave the way for safer and more efficient therapies to repair bone damage due to injury or inherited bone diseases.

Current osteoporosis treatments improve bone mass by increasing net bone formation: anti-resorptive drugs such as bisphosphonates block osteoclast activity, while anabolic agents such as parathyroid hormone (PTH) increase bone remodeling, with a greater effect on formation. Although these drugs are widely used, their role in modulating formation and resorption is not fully understood, due in part to technical limitations in the ability to longitudinally assess bone remodeling. Importantly, it is not known whether or not PTH-induced bone formation is independent of resorption, resulting in controversy over the effectiveness of combination therapies that use both PTH and an anti-resorptive. In this study, we developed a μCT-based, in vivo dynamic bone histomorphometry technique for rat tibiae, and applied this method to longitudinally track changes in bone resorption and formation as a result of treatment with alendronate (ALN), PTH, or combination therapy of both PTH and ALN (PTH+ALN). Correlations between our μCT-based measures of bone formation and measures of bone formation based on calcein-labeled histology (r = 0.72 - 0.83) confirm the accuracy of this method. Bone remodeling parameters measured through μCT-based in vivo dynamic bone histomorphometry indicate an increased rate of bone formation in rats treated with PTH and PTH+ALN, together with a decrease in bone resorption measures in rats treated with ALN and PTH+ALN. These results were further supported by traditional histology-based measurements, suggesting that PTH was able to induce bone formation while bone resorption was suppressed.

During pregnancy and lactation, the maternal skeleton provides calcium for fetal/infant growth, resulting in substantial bone loss, which partially recovers after weaning. However, the amount of bone that is lost and the extent of post-weaning recovery are highly variable among different skeletal sites, and, despite persistent alterations in bone structure at some locations, reproductive history does not increase postmenopausal fracture risk. To explain this phenomenon, we hypothesized that the degree of reproductive bone loss/recovery at trabecular sites may vary depending on the extent to which the trabecular compartment is involved in the bone’s load-bearing function. Using a rat model, we quantified the proportion of the load carried by the trabeculae, as well as the extent of reproductive bone loss and recovery, at two distinct skeletal sites: the tibia and lumbar vertebra. Both sites underwent significant bone loss during pregnancy and lactation, which was partially recovered post-weaning. However, the extent of the deterioration and the resumption of trabecular load-bearing capacity after weaning varied substantially. Tibial trabecular bone, which bore a low proportion of the total applied load, underwent dramatic and irreversible microstructural deterioration during reproduction. Meanwhile, vertebral trabecular bone bore a greater fraction of the load, underwent minimal deterioration in microarchitecture, and resumed its full load-bearing capacity after weaning. Because pregnancy and lactation are physiological processes, the distinctive responses to these natural events among different skeletal sites may help to elucidate the extent of the trabecular bone’s structural versus metabolic functions.

Bone atrophy and its related fragility fractures are frequent, late side effects of radiotherapy in cancer survivors and have a detrimental impact on their quality of life. In another study, we showed that parathyroid hormone 1-34 and anti-sclerostin antibody attenuates radiation-induced bone damage by accelerating DNA repair in osteoblasts. DNA damage responses are partially regulated by the ubiquitin proteasome pathway. In the current study, we examined whether proteasome inhibitors have similar bone-protective effects against radiation damage. MG132 treatment greatly reduced radiation-induced apoptosis in cultured osteoblastic cells. This survival effect was owing to accelerated DNA repair as revealed by γH2AX foci and comet assays and to the up-regulation of Ku70 and DNA-dependent protein kinase, catalytic subunit, essential DNA repair proteins in the nonhomologous end-joining pathway. Administration of bortezomib (Bzb) reversed the loss of trabecular bone structure and strength in mice at 4 wk after focal radiation. Histomorphometry revealed that Bzb significantly increased the number of osteoblasts and activity in the irradiated area and suppressed the number and activity of osteoclasts, regardless of irradiation. Two weeks of Bzb treatment accelerated DNA repair in bone-lining osteoblasts and thus promoted their survival. Meanwhile, it also inhibited bone marrow adiposity. Taken together, we demonstrate a novel role of proteasome inhibitors in treating radiation-induced osteoporosis.-Chandra, A., Wang, L., Young, T., Zhong, L., Tseng, W.-J., Levine, M. A., Cengel, K., Liu, X. S., Zhang, Y., Pignolo, R. J., Qin, L. Proteasome inhibitor bortezomib is a novel therapeutic agent for focal radiation-induced osteoporosis.

Daily injections of parathyroid hormone (PTH) are the only FDA-approved anabolic treatment for osteoporosis; however PTH is only clinically approved for treatment periods of up to 24 months. To enhance its anabolic effect, combining PTH with anti-resorptive therapy was proposed and expected to maximize the effectiveness of PTH. The current study aimed to elucidate structural mechanisms through which combination therapy can further improve bone strength over a limited treatment window of 12 days, to more closely examine the early phase of the anabolic window. We examined 30 female rats treated with either Vehicle (Veh), Alendronate (ALN), PTH, or both PTH and ALN (PTH+ALN). Standard and Individual Trabecula Segmentation (ITS)-based microstructural analyses were performed using in vivo micro computed tomography. We found an increase in BV/TV in all treatments with the highest in the PTH+ALN group. Tb.Th* increased in both PTH and PTH+ALN groups well beyond that of the Veh or ALN groups. SMI decreased in all treatments with PTH+ALN having the greatest tendency towards plate-like structures. ITS confirmed the trend towards more plate-like structures with increased plate Tb.N and increased plate-to-rod ratio that was most pronounced in the PTH+ALN group. Using image-based finite element analysis, we demonstrated that stiffness increased in all treatment groups, again with the largest increase in the PTH+ALN group, indicating the resulting structural implications of increased plate-like structure. Static and dynamic bone histomorphometry and a serum resorption marker confirmed that PTH+ALN significantly increased bone formation activities and suppressed bone resorption activities. Overall the results indicate that PTH+ALN treatment has an additive effect due to a preferential increase in plate-like structures.

The pericellular matrix (PCM), a thin coating surrounding nearly all mammalian cells, plays a critical role in many cell-surface phenomena. In osteocytes, the PCM is believed to control both “outside-in” (mechanosensing) and “inside-out” (signaling molecule transport) processes. However, the osteocytic PCM is challenging to study in situ because it is thin (∼100 nm) and enclosed in mineralized matrix. To this end, we recently developed a novel tracer velocimetry approach that combined fluorescence recovery after photobleaching (FRAP) imaging with hydrodynamic modeling to quantify the osteocytic PCM in young murine bone. In this study, we applied the technique to older mice expressing or deficient for perlecan/HSPG2, a large heparan-sulfate proteoglycan normally secreted in osteocytic PCM. The objectives were (1) to characterize transport within an altered PCM; (2) to test the sensitivity of our approach in detecting the PCM alterations; and (3) to dissect the roles of the PCM in osteocyte mechanosensing. We found that: (1) solute transport increases in the perlecan-deficient (hypomorphic [Hypo]) mice compared with control mice; (2) PCM fiber density decreases with aging and perlecan deficiency; (3) osteocytes in the Hypo bones are predicted to experience higher shear stress (+34%), but decreased fluid drag force (−35%) under 3-N peak tibial loading; and (4) when subjected to tibial loading in a preliminary in vivo experiment, the Hypo mice did not respond to the anabolic stimuli as the CTL mice did. These findings support the hypothesis that the PCM fibers act as osteocyte's sensing antennae, regulating load-induced cellular stimulations and thus bone's sensitivity and in vivo bone adaptation. If this hypothesis is further confirmed, osteocytic PCM could be new targets to develop osteoporosis treatments by modulating bone's intrinsic sensitivity to mechanical loading and be used to design patient-specific exercise regimens to promote bone formation. © 2014 American Society for Bone and Mineral Research.

This paper describes the use of micro-computed tomography for determining in vitro degradation of calcium-phosphate ceramic materials. Samples were immersed in a simulated body fluid and scanned at the initial and final timepoints. Analysis indicates degradation of material density as well as changes in mineral content and mineral density. Findings are confirmed with known methods of materials characterization (SEM, mass analysis, and Archimedes’s density calculations), indicating changes in surface morphology, mass, and pore volume.

a rat model of supraspinatus overuse has suggested mechanisms governing tendon degeneration; however, delineating which changes are pathologic or simply physiologic adaptations to increased loading remains a question. The objective of this study was to develop and characterize a rat exercise model that induces systemic and local shoulder adaptations without mechanical injury to the supraspinatus tendon.exercise rats completed a treadmill training protocol for 12 weeks. Body, fat pad, and heart weights were determined. Supraspinatus tendon collagen content, cross-sectional area, and mechanical properties were measured. Supraspinatus muscle cross-sectional area, weight, and the expression of mitochondrial oxidative phosphorylation (OXPHOS) proteins were measured. Humeri were analyzed with μCT and mechanically tested.exercise decreased fat pad mass. Supraspinatus muscle hypertrophied and had increased OXPHOS proteins. Humerus trabecular bone had increased anisotropic orientation, and cortical bone showed increased bone and tissue mineral density. Importantly, the supraspinatus tendon did not have diminished mechanical properties, indicating that this protocol was not injurious to the tendon.this study establishes the first rat exercise protocol that induces adaptations in the shoulder. Future research can use this as a comparison model to study how the supraspinatus tendon adapts to loading and undergoes degeneration with overuse.

Combined parathyroid hormone (PTH) and bisphosphonate (alendronate—ALN) therapy has recently been shown to increase bone volume fraction and plate-like trabecular structure beyond either monotherapy. To identify the mechanism through which plate-like structure was enhanced, we used in vivo microcomputed tomography (μCT) of the proximal tibia metaphysis and individual trabecular dynamics (ITD) analysis to quantify connectivity repair (incidences of rod connection and plate perforation filling) and deterioration (incidences of rod disconnection and plate perforation). Three-month-old female, intact rats were scanned before and after a 12 day treatment period of vehicle (Veh, n = 5), ALN (n = 6), PTH (n = 6), and combined (PTH+ALN, n = 6) therapy. Additionally, we used computational simulation and finite element (FE) analysis to delineate the contributions of connectivity repair or trabecular thickening to trabecular bone stiffness. Our results showed that the combined therapy group had greater connectivity repair (5.8 ± 0.5% connected rods and 2.0 ± 0.3% filled plates) beyond that of the Veh group, resulting in the greatest net gain in connectivity. For all treatment groups, increases in bone volume due to thickening (5–31%) were far greater than those due to connectivity repair (2–3%). Newly formed bone contributing only to trabecular thickening caused a 10%, 41%, and 69% increase in stiffness in the ALN, PTH, and PTH+ALN groups, respectively. Moreover, newly formed bone that led to connectivity repair resulted in an additional improvement in stiffness, with the highest in PTH+ALN (by an additional 12%), which was significantly greater than either PTH (5.6%) or ALN (4.5%). An efficiency ratio was calculated as the mean percent increase in stiffness divided by mean percent increase in BV for either thickening or connectivity repair in each treatment. For all treatments, the efficiency ratio of connectivity repair (ALN: 2.9; PTH: 3.4; PTH+ALN: 4.4) was higher than that due to thickening (ALN: 2.0; PTH: 1.7; PTH+ALN: 2.2), suggesting connectivity repair required less new bone formation to induce larger gains in stiffness. We conclude that through rod connection and plate perforation filling PTH+ALN combination therapy improved bone stiffness in a more efficient and effective manner than either monotherapy.

Cancer radiotherapy is often complicated by a spectrum of changes in the neighboring bone from mild osteopenia to osteoradionecrosis. We previously reported that parathyroid hormone (PTH, 1–34), an anabolic agent for osteoporosis, reversed bone structural deterioration caused by multiple microcomputed tomography (microCT) scans in adolescent rats. To simulate clinical radiotherapy for cancer patients and to search for remedies, we focally irradiated the tibial metaphyseal region of adult rats with a newly available small animal radiation research platform (SARRP) and treated these rats with intermittent injections of PTH1–34. Using a unique 3D image registration method that we recently developed, we traced the local changes of the same trabecular bone before and after treatments, and observed that, while radiation caused a loss of small trabecular elements leading to significant decreases in bone mass and strength, PTH1–34 preserved all trabecular elements in irradiated bone with remarkable increases in bone mass and strength. Histomorphometry demonstrated that SARRP radiation severely reduced osteoblast number and activity, which were impressively reversed by PTH treatment. In contrast, suppressing bone resorption by alendronate failed to rescue radiation-induced bone loss and to block the rescue effect of PTH1–34. Furthermore, histological analyses revealed that PTH1–34 protected osteoblasts and osteocytes from radiation-induced apoptosis and attenuated radiation-induced bone marrow adiposity. Taken together, our data strongly support a robust radioprotective effect of PTH on trabecular bone integrity through preserving bone formation and shed light on further investigations of an anabolic therapy for radiation-induced bone damage.

Background a rat model of supraspinatus overuse has suggested mechanisms governing tendon degeneration; however, delineating which changes are pathologic or simply physiologic adaptations to increased loading remains a question. The objective of this study was to develop and characterize a rat exercise model that induces systemic and local shoulder adaptations without mechanical injury to the supraspinatus tendon. Methods exercise rats completed a treadmill training protocol for 12 weeks. Body, fat pad, and heart weights were determined. Supraspinatus tendon collagen content, cross-sectional area, and mechanical properties were measured. Supraspinatus muscle cross-sectional area, weight, and the expression of mitochondrial oxidative phosphorylation (OXPHOS) proteins were measured. Humeri were analyzed with μCT and mechanically tested. Results exercise decreased fat pad mass. Supraspinatus muscle hypertrophied and had increased OXPHOS proteins. Humerus trabecular bone had increased anisotropic orientation, and cortical bone showed increased bone and tissue mineral density. Importantly, the supraspinatus tendon did not have diminished mechanical properties, indicating that this protocol was not injurious to the tendon. Conclusion this study establishes the first rat exercise protocol that induces adaptations in the shoulder. Future research can use this as a comparison model to study how the supraspinatus tendon adapts to loading and undergoes degeneration with overuse.

Aging shifts bone remodeling toward a negative balance between bone formation and resorption, causing bone loss and increased fracture risk. Anti-resorptive agents are commonly used to inhibit bone resorption and stabilize bone mass. While they are effective to prevent further bone loss, there is also a great need for anabolic agents which can reverse bone deterioration and regain lost skeletal integrity. Intermittent parathyroid hormone (PTH) treatment is the only FDA-approved anabolic treatment for osteoporosis, which greatly stimulates bone formation. Combined therapy of anti-resorptive drugs, such as alendronate (ALN), and PTH have been proposed and are expected to further stimulate bone formation. However, studies show conflicting results regarding the effectiveness of combined treatments: some have reported the addition of ALN to impair PTH function [1, 2], while others suggest an improvement over PTH monotherapy [3, 4]. The first objective of this study is to document the immediate changes of individual trabecular structures due to PTH and combined therapy within 12 days using in vivo micro computed tomography (μCT). As PTH is typically prescribed for 1 to 3 years to osteoporotic patients, a treatment of 12 days for rats (approximately equivalent to one year of human life) may be more clinically relevant than long-term treatment studies on rats. The secondary purpose of this study was to gain insight into the mechanism of combined versus PTH treatments through a bone dynamic imaging strategy to track events over an individual remodeling site. We hypothesized that PTH and combined treatments would immediately enhance bone formation on the trabecular surface.Copyright © 2013 by ASME

Spinal cord injury (SCI)-induced bone loss represents the most severe osteoporosis with no effective treatment. Past animal studies have focused primarily on long bones at the acute stage using adolescent rodents. To mimic chronic SCI in human patients, we performed a comprehensive analysis of long-term structural and mechanical changes in axial and appendicular bones in adult rats after SCI. In this experiment, 4-month-old Fischer 344 male rats received a clinically relevant T13 contusion injury. Sixteen weeks later, sublesional femurs, tibiae, and L4 vertebrae, supralesional humeri, and blood were collected from these rats and additional non-surgery rats for micro-computed tomography (µCT), micro-finite element, histology, and serum biochemical analyses. At trabecular sites, extreme losses of bone structure and mechanical competence were detected in the metaphysis of sublesional long bones after SCI, while the subchondral part of the same bones showed much milder damage. Marked reductions in bone mass and strength were also observed in sublesional L4 vertebrae but not in supralesional humeri. At cortical sites, SCI induced structural and strength damage in both sub- and supralesional long bones. These changes were accompanied by diminished osteoblast number and activity and increased osteoclast number and activity. Taken together, our study revealed site-specific effects of SCI on bone and demonstrated sustained inhibition of bone formation and elevation of bone resorption at the chronic stage of SCI.