Long-Term Effects of Reproduction and Lactation on the Rat Supraspinatus Tendon and Proximal Humerus.

During pregnancy and breastfeeding, women undergo hormonal fluctuations required for fetal development, parturition, and infant growth. These changes have secondary consequences on the maternal musculoskeletal system, increasing the risk for joint pain and osteoporosis. Though hormone levels return to prepregnancy levels postpartum, women may experience lasting musculoskeletal pain. Sex disparities exist in the prevalence of musculoskeletal disorders, but it remains unclear how reproductive history may impact sex differences. Specifically, the effects of both reproductive history and sex on the rotator cuff have not been studied. Pregnancy and lactation affect bone microstructure, suggesting possible impairments at the enthesis of rotator cuff tendons, where tears commonly occur. Therefore, our objective was to evaluate how reproductive history affects sex differences of the supraspinatus tendon and proximal humerus using male, virgin female, and female rats with a history of reproduction (referred to as reproductive females). We hypothesized tendon mechanical properties and humeral bone microstructure would be inferior in reproductive females compared to virgin females. Results showed sex differences independent of reproductive history, including greater tendon midsubstance modulus but lower subchondral bone mineral density (BMD) in females. When considering reproductive history, reproductive rats exhibited reduced tendon insertion site modulus and trabecular bone micro-architecture compared to virgin females with no differences from males. Overall, our study identified long-term changes in supraspinatus tendon mechanical and humeral trabecular bone properties that result following pregnancy and lactation, highlighting the importance of considering reproductive history in investigations of sex differences in the physiology and pathology of rotator cuff injuries.

Proteasome inhibitor bortezomib is a novel therapeutic agent for focal radiation-induced osteoporosis.

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.

Mechanical Regulation of the Maternal Skeleton during Reproduction and Lactation.

PURPOSE OF REVIEW: This review summarizes recently published data on the effects of pregnancy and lactation on bone structure, mechanical properties, and mechano-responsiveness in an effort to elucidate how the balance between the structural and metabolic functions of the skeleton is achieved during these physiological processes. RECENT FINDINGS: While pregnancy and lactation induce significant changes in bone density and structure to provide calcium for fetal/infant growth, the maternal physiology also comprises several innate compensatory mechanisms that allow for the maintenance of skeletal mechanical integrity. Both clinical and animal studies suggest that pregnancy and lactation lead to adaptations in cortical bone structure to allow for rapid calcium release from the trabecular compartment while maintaining whole bone stiffness and strength. Moreover, extents of lactation-induced bone loss and weaning-induced recovery are highly dependent on a given bone's load-bearing function, resulting in better protection of the mechanical integrity at critical load-bearing sites. The recent discovery of lactation-induced osteocytic perilacunar/canalicular remodeling (PLR) indicates a new means for osteocytes to modulate mineral homeostasis and tissue-level mechanical properties of the maternal skeleton. Furthermore, lactation-induced PLR may also play an important role in maintaining the maternal skeleton's load-bearing capacity by altering osteocyte's microenvironment and modulating the transmission of anabolic mechanical signals to osteocytes. Both clinical and animal studies show that parity and lactation have no adverse, or a positive effect on bone strength later in life. The skeletal effects during pregnancy and lactation reflect an optimized balance between the mechanical and metabolic functions of the skeleton.

CREB mediates brain serotonin regulation of bone mass through its expression in ventromedial hypothalamic neurons.

Serotonin is a bioamine regulating bone mass accrual differently depending on its site of synthesis. It decreases accrual when synthesized in the gut, and increases it when synthesized in the brain. The signal transduction events elicited by gut-derived serotonin once it binds to the Htr1b receptor present on osteoblasts have been identified and culminate in cAMP response element-binding protein (CREB) regulation of osteoblast proliferation. In contrast, we do not know how brain-derived serotonin favors bone mass accrual following its binding to the Htr2c receptor on neurons of the hypothalamic ventromedial nucleus (VMH). We show here--through gene expression analysis, serotonin treatment of wild-type and Htr2c(-/-) hypothalamic explants, and cell-specific gene deletion in the mouse--that, following its binding to the Htr2c receptor on VMH neurons, serotonin uses a calmodulin kinase (CaMK)-dependent signaling cascade involving CaMKKß and CaMKIV to decrease the sympathetic tone and increase bone mass accrual. We further show that the transcriptional mediator of these events is CREB, whose phosphorylation on Ser 133 is increased by CaMKIV following serotonin treatment of hypothalamic explants. A microarray experiment identified two genes necessary for optimum sympathetic activity whose expression is regulated by CREB. These results provide a molecular understanding of how serotonin signals in hypothalamic neurons to regulate bone mass accrual and identify CREB as a critical determinant of this function, although through different mechanisms depending on the cell type, neuron, or osteoblast in which it is expressed.

Clinical Evaluation of Bone Strength and Fracture Risk.

PURPOSE OF REVIEW: This paper seeks to evaluate and compare recent advances in the clinical assessment of the changes in bone mechanical properties that take place as a result of osteoporosis and other metabolic bone diseases and their treatments. RECENT FINDINGS: In addition to the standard of DXA-based areal bone mineral density (aBMD), a variety of methods, including imaging-based structural measurements, finite element analysis (FEA)-based techniques, and alternate methods including ultrasound, bone biopsy, reference point indentation, and statistical shape and density modeling, have been developed which allow for reliable prediction of bone strength and fracture risk. These methods have also shown promise in the evaluation of treatment-induced changes in bone mechanical properties. Continued technological advances allowing for increasingly high-resolution imaging with low radiation dose, together with the expanding adoption of DXA-based predictions of bone structure and mechanics, as well as the increasing awareness of the importance of bone material properties in determining whole-bone mechanics, lead us to anticipate substantial future advances in this field.

PTH1-34 blocks radiation-induced osteoblast apoptosis by enhancing DNA repair through canonical Wnt pathway.

Focal radiotherapy for cancer patients has detrimental effects on bones within the radiation field and the primary clinical signs of bone damage include the loss of functional osteoblasts. We reported previously that daily injection of parathyroid hormone (PTH, 1-34) alleviates radiation-induced osteopenia in a preclinical radiotherapy model by improving osteoblast survival. To elucidate the molecular mechanisms, we irradiated osteoblastic UMR 106-01 cells and calvarial organ culture and demonstrated an anti-apoptosis effect of PTH1-34 on these cultures. Inhibitor assay indicated that PTH exerts its radioprotective action mainly through protein kinase A/ß-catenin pathway. γ-H2AX foci staining and comet assay revealed that PTH efficiently promotes the repair of DNA double strand breaks (DSBs) in irradiated osteoblasts via activating the ß-catenin pathway. Interestingly, Wnt3a alone also blocked cell death and accelerated DNA repair in primary osteoprogenitors, osteoblastic and osteocytic cells after radiation through the canonical signaling. Further investigations revealed that both Wnt3a and PTH increase the amount of Ku70, a core protein for initiating the assembly of DSB repair machinery, in osteoblasts after radiation. Moreover, down-regulation of Ku70 by siRNA abrogated the prosurvival effect of PTH and Wnt3a on irradiated osteoblasts. In summary, our results identify a novel role of PTH and canonical Wnt signaling in regulating DSB repair machinery and apoptosis in osteoblasts and shed light on using PTH1-34 or Wnt agonist as possible therapy for radiation-induced osteoporosis.

Enhanced individual trabecular repair and its mechanical implications in parathyroid hormone and alendronate treated rat tibial bone.

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.

Kidney transplantation with early corticosteroid withdrawal: paradoxical effects at the central and peripheral skeleton.

The use of early corticosteroid withdrawal (ECSW) protocols after kidney transplantation has become common, but the effects on fracture risk and bone quality are unclear. We enrolled 47 first-time adult transplant recipients managed with ECSW into a 1-year study to evaluate changes in bone mass, microarchitecture, biomechanical competence, and remodeling with dual energy x-ray absorptiometry (DXA), high-resolution peripheral quantitative computed tomography (HRpQCT), parathyroid hormone (PTH) levels, and bone turnover markers obtained at baseline and 3, 6, and 12 months post-transplantation. Compared with baseline, 12-month areal bone mineral density by DXA did not change significantly at the spine and hip, but it declined significantly at the 1/3 and ultradistal radii (2.2% and 2.9%, respectively; both P<0.001). HRpQCT of the distal radius revealed declines in cortical area, density, and thickness (3.9%, 2.1%, and 3.1%, respectively; all P<0.001), trabecular density (4.4%; P<0.001), and stiffness and failure load (3.1% and 3.5%, respectively; both P<0.05). Findings were similar at the tibia. Increasing severity of hyperparathyroidism was associated with increased cortical losses. However, loss of trabecular bone and bone strength were most severe at the lowest and highest PTH levels. In summary, ECSW was associated with preservation of bone mineral density at the central skeleton; however, it was also associated with progressive declines in cortical and trabecular bone density at the peripheral skeleton. Cortical decreases related directly to PTH levels, whereas the relationship between PTH and trabecular bone decreases was bimodal. Studies are needed to determine whether pharmacologic agents that suppress PTH will prevent cortical and trabecular losses and post-transplant fractures.

Accuracy of individual trabecula segmentation based plate and rod finite element models in idealized trabecular bone microstructure.

Currently, specimen-specific micro finite element (µFE) analysis based micro computed tomography (µCT) images have become a major computational tool for the assessment of the mechanical properties of human trabecular bone. Despite the fine characterization of the three-dimensional (3D) trabecular microstructure based on high-resolution µCT images, conventional µFE models with each voxel converted to an element are not efficient in predicting the nonlinear failure behavior of bone due to a prohibitive computational cost. Recently, a highly efficient individual trabecula segmentation (ITS)-based plate and rod (PR) modeling technique has been developed by substituting individual plates and rods with shell and beam elements, respectively. In this technical brief, the accuracy of novel PR µFE models was examined in idealized microstructure models over a broad range of trabecular thicknesses. The Young's modulus and yield strength predicted by simplified PR models strongly correlated with those of voxel models at various voxel sizes. The conversion from voxel models to PR models resulted in an ∼762-fold reduction in the largest model size and significantly accelerated the nonlinear FE analysis. The excellent predictive power of the PR µFE models, demonstrated in an idealized trabecular microstructure, provided a quantitative mechanical basis for this promising tool for an accurate and efficient assessment of trabecular bone mechanics and fracture risk.

Engineering anatomically shaped human bone grafts.

The ability to engineer anatomically correct pieces of viable and functional human bone would have tremendous potential for bone reconstructions after congenital defects, cancer resections, and trauma. We report that clinically sized, anatomically shaped, viable human bone grafts can be engineered by using human mesenchymal stem cells (hMSCs) and a "biomimetic" scaffold-bioreactor system. We selected the temporomandibular joint (TMJ) condylar bone as our tissue model, because of its clinical importance and the challenges associated with its complex shape. Anatomically shaped scaffolds were generated from fully decellularized trabecular bone by using digitized clinical images, seeded with hMSCs, and cultured with interstitial flow of culture medium. A bioreactor with a chamber in the exact shape of a human TMJ was designed for controllable perfusion throughout the engineered construct. By 5 weeks of cultivation, tissue growth was evidenced by the formation of confluent layers of lamellar bone (by scanning electron microscopy), markedly increased volume of mineralized matrix (by quantitative microcomputer tomography), and the formation of osteoids (histologically). Within bone grafts of this size and complexity cells were fully viable at a physiologic density, likely an important factor of graft function. Moreover, the density and architecture of bone matrix correlated with the intensity and pattern of the interstitial flow, as determined in experimental and modeling studies. This approach has potential to overcome a critical hurdle-in vitro cultivation of viable bone grafts of complex geometries-to provide patient-specific bone grafts for craniofacial and orthopedic reconstructions.

Structural role of osteocyte lacunae on mechanical properties of bone matrix: A cohesive finite element study.

Despite the extensive studies on biological function of osteocytes, there are limited studies that evaluated the structural role of osteocyte lacunae on local mechanical properties of the bone matrix. As a result, the goal of this study was to elucidate the independent contribution of osteocyte lacunae structure on mechanical properties and fracture behavior of the bone matrix uncoupled from its biological effects and bone tissue composition variation. This study combined cohesive finite element modeling with experimental data from a lactation rat model to evaluate the influence of osteocyte lacunar area porosity, density, size, axis ratio, and orientation on the elastic modulus, ultimate strength, and ultimate strain of the bone matrix as well as on local crack formation and propagation. It also performed a parametric study to isolate the influence of a single osteocyte lacunae structural property on the mechanical properties of the bone matrix. The experimental measurements demonstrated statistically significant differences in lacunar size between ovariectomized rats with lactation history and virgin groups (both ovariectomized and intact) and in axis ratio between rats with lactation history and virgins. There were no differences in mechanical properties between virgin and lactation groups as determined by the finite element simulations. However, there were statistically significant linear relationships between the physiological range of osteocyte lacunar area porosity, density, size, and orientation and the elastic modulus and ultimate strength of the bone matrix in virgin and lactation rats. The parametric study also revealed similar but stronger relationships between elastic modulus and ultimate strength and lacunar density, size, and orientation. The simulations also demonstrated that the osteocyte lacunae guided the crack propagation through local stress concentrations. In summary, this study enhanced the limited knowledge on the structural role of osteocyte lacunae on local mechanical properties of the bone matrix. These data are important in gaining a better understanding of the mechanical implications of the local modifications due to osteocytes in the bone matrix.

Instrumented nanoindentation in musculoskeletal research.

Musculoskeletal tissues, such as bone, cartilage, and muscle, are natural composite materials that are constructed with a hierarchical structure ranging from the cell to tissue level. The component differences and structural complexity, together, require comprehensive multiscale mechanical characterization. In this review, we focus on nanoindentation testing, which is used for nanometer to sub-micrometer length scale mechanical characterization. In the following context, we will summarize studies of nanoindentation in musculoskeletal research, examine the critical factors that affect nanoindentation testing results, and briefly summarize other commonly used techniques that can be conjoined with nanoindentation for synchronized imaging and colocalized characterization.

Distinct Responses of Modeling- and Remodeling-Based Bone Formation to the Discontinuation of Intermittent Parathyroid Hormone Treatment in Ovariectomized Rats.

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).

Short Cyclic Regimen With Parathyroid Hormone (PTH) Results in Prolonged Anabolic Effect Relative to Continuous Treatment Followed by Discontinuation in Ovariectomized Rats.

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).

Activating EGFR Signaling Attenuates Osteoarthritis Development Following Loading Injury in Mice.

Posttraumatic osteoarthritis (PTOA) results in joint pain, loss of joint function, and impaired quality of daily life in patients with limited treatment options. We previously demonstrated that epidermal growth factor receptor (EGFR) signaling is essential for maintaining chondroprogenitors during articular cartilage development and homeostasis. Here, we used a nonsurgical, loading-induced PTOA mouse model to investigate the protective action of EGFR signaling. A single bout of cyclic tibial loading at a peak force of 6 N injured cartilage at the posterior aspect of lateral femoral condyle. Similar loading at a peak force of 9 N ruptured the anterior cruciate ligament, causing additional cartilage damage at the medial compartment and ectopic cartilage formation in meniscus and synovium. Constitutively overexpression of an EGFR ligand, heparin binding EGF-like growth factor (HBEGF), in chondrocytes significantly reduced cartilage injury length, synovitis, and pain after 6 N loading and mitigated medial side cartilage damage and ectopic cartilage formation after 9 N loading. Mechanistically, overactivation of EGFR signaling protected chondrocytes from loading-induced apoptosis and loss of proliferative ability and lubricant synthesis. Overexpressing HBEGF in adult cartilage starting right before 6 N loading had similar beneficial effects. In contrast, inactivating EGFR in adult cartilage led to accelerated PTOA progression with elevated cartilage Mankin score and synovitis score and increased ectopic cartilage formation. As a therapeutic approach, we constructed a nanoparticle conjugated with the EGFR ligand TGFα. Intra-articular injections of this nanoconstruct once every 3 weeks for 12 weeks partially mitigated PTOA symptoms in cartilage and synovium after 6 N loading. Our findings demonstrate the anabolic actions of EGFR signaling in maintaining articular cartilage during PTOA development and shed light on developing a novel nanomedicine for PTOA. © 2022 American Society for Bone and Mineral Research (ASBMR).

Dissociation of the neuronal regulation of bone mass and energy metabolism by leptin in vivo.

The leptin regulation of bone remodeling, which has been documented through studies of loss-of-function mutations of this hormone or of its receptor in mice and humans, still raised several unanswered questions. For instance, it has been assumed but not formally demonstrated that this regulation occurs through neuronal means. Likewise, it has not been possible until now to dissociate the influence leptin exerts on appetite and energy expenditure from this function. We show here through mouse genetic studies that a deletion of the leptin receptor in neurons results in an increase in bone formation and bone resorption, resulting in a high bone mass as seen in leptin-deficient mice. In contrast, the same deletion in osteoblasts only does not influence bone remodeling. Furthermore, through the use of l/l mice, a model of gain of function in leptin signaling harboring a Y985L substitution in the leptin receptor, we show that leptin signaling inhibits bone mass accrual by up-regulating sympathetic activity independently of any change in appetite or energy expenditure. This work establishes that in vivo leptin regulates bone mass accrual by acting through neuronal means and provides a direct demonstration that this function of leptin can occur independently of its regulation of energy metabolism.

Peak trabecular bone microstructure predicts rate of estrogen-deficiency-induced bone loss in rats.

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.

Activation, development, and attenuation of modeling- and remodeling-based bone formation in adult rats.

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.

Maternal bone adaptation to mechanical loading during pregnancy, lactation, and post-weaning recovery.

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.