Bone Anabolism Research Articles

A magnetoelastic bone fixation device for controlled mechanical stimulation at femoral fractures in rodents

Studies have shown that mechanical stimulations can provide anabolic bone formation and counteract conditions such as osteoporosis in intact bones, as well as promote healing of bone fractures. However, many of these studies are based on external mechanical loading platforms or bulky wearable force loading systems, which are inconvenient and impractical in clinical settings especially for the treatment of bone injuries. To overcome this limitation, a bone fixation device was developed to provide localized mechanical strains across a bone fracture site. The fixation device, designed for a rodent’s femoral fracture (critical-size segmental defect), is consisted of a magnetoelastic (ME) actuator that changes its physical dimension when exposed to magnetic fields. The strain produced by the ME actuator is transferred to the bone fixation device, which acts directly on the bone-implant interface to produce a highly focused and localized mechanical loading. The actuation generated by the fixation device was evaluated, and results show the device was able to provide up to 150 με at 30 Hz. Mechanical characterization of the fixation device showed its off-axis compressive stiffness was higher than 160 N mm−1, which was comparable to existing fixation devices used for the same rodent’s femoral fracture model. Furthermore, the fixation device was implanted in rodent subjects for 8 weeks to validate its safety and biocompatibility in vivo. Results indicate the device is biocompatible, showing no adverse impact on bone regeneration.

Bilateral atypical femoral fractures during denosumab therapy in a patient with adult-onset hypophosphatasia.

SummaryHypophosphatasia (HPP) is a rare and under-recognised genetic defect in bone mineralisation. Patients presenting with fragility fractures may be mistakenly diagnosed as having osteoporosis and prescribed antiresorptive therapy, a treatment which may increase fracture risk. Adult-onset HPPhypophosphatasia was identified in a 40-year-old woman who presented with bilateral atypical femoral fractures after 4 years of denosumab therapy. A low serum alkaline phosphatase (ALP) and increased serum vitamin B6 level signalled the diagnosis, which was later confirmed by identification of two recessive mutations of the ALPL gene. The patient was treated with teriparatide given the unavailability of ALP enzyme-replacement therapy (asfotase alfa). Fracture healing occurred, but impaired mobility persisted. HPP predisposes to atypical femoral fracture (AFF) during antiresorptive therapy; hence, bisphosphonates and denosumab are contraindicated in this condition. Screening patients with fracture or ‘osteoporosis’ to identify a low ALP level is recommended.Learning pointsHypophosphatasia (HPP) is a rare and under-recognised cause of bone fragility produced by impaired matrix mineralisation that can be misdiagnosed as a fragility fracture due to age-related bone loss.Antiresorptive therapy is contraindicated in HPP.Low serum alkaline phosphatase (ALP) provides a clue to the diagnosis.Elevated serum vitamin B6 (an ALP substrate) is indicative of HPP, while identification of a mutation in the ALPL gene is confirmatory.Enzyme therapy with recombinant ALP (asfotase alfa) is currently prohibitively costly.Treatment with anabolic bone agents such as teriparatide has been reported, but whether normally mineralized bone is formed requires further study.

262. Does preoperative bone mineral density impact fusion success in anterior cervical spine surgery: a prospective cohort study

<h3>BACKGROUND CONTEXT</h3> Patients with osteoporosis have an imbalance in anabolic versus catabolic bone homeostasis. It has been theorized that osteoporosis may impact fusion rates in lumbar spine surgery. However, to our knowledge this has not been investigated in cervical spine surgery. As our population ages, we are encountering ever increasing numbers of patients with osteoporosis and osteopenia coming in for elective surgery. <h3>PURPOSE</h3> The purpose of this study is to identify risk factors for pseudarthrosis in patients undergoing anterior cervical spine surgery. <h3>STUDY DESIGN/SETTING</h3> This was a prospective, single-center study conducted at an academic medical center between 2002 and 2018. <h3>PATIENT SAMPLE</h3> Seventy-nine patients who underwent 1-4 level anterior cervical spine fusions between the years of 2002-2018, and had a bone mineral density examination performed preoperatively, completed the study with one year of clinical and radiographic follow-up. <h3>OUTCOME MEASURES</h3> Bone density as measured by T-score, Hounsfield units and fusion status were measured. <h3>METHODS</h3> We prospectively collected and analyzed data on patients undergoing anterior cervical spine fusions involving between 1-4 levels between the years of 2002-2018 in which a bone mineral density (BMD) examination was clinically indicated. Based on the World Health Organization (WHO) classification using DEXA scan for measurement of BMD, normal bone density was defined as T-score greater than or equal to -1 SD, osteopenia as having a T-score less than -1 and greater than -2.5 SD, and osteoporosis as having a T-score less than or equal to -2.5 SD. Patients were followed for a minimum of one year. CT scans performed postoperatively were used to evaluate fusion. Three Hounsfield unit measurements were obtained on the vertebrae above and below the fusion levels and averaged. A total of 79 patients completed the study with one-year of clinical and radiographic follow-up. BMD, number of levels fused, smoking status, age, sex and ACE inhibitors/ARBs were analyzed to assess their impact on fusion success. Fusion was established when there was continuous bone bridging between the vertebrae on CT scan. Multivariate analysis was performed to identify potential risk factors for pseudarthrosis utilizing JMP 10.0.0 statistical software. (SAS Institute Inc, Carey, North Carolina). <h3>RESULTS</h3> Fourteen patients (18%) developed pseudarthrosis at one-year postoperatively. The mean age at surgery was 58.8 ±10.5 (30 to 84 years). The mean BMD was -0.45 ±1.4 (-3 to 4). Osteopenia and osteoporosis were identified as risk factors for pseudarthorsis at a minimum of 1 year follow up. Number of levels fused, smoking status, Hounsfield units, ACE/ARB medications, age or gender were not statistically significant risk factors correlated with development of pseudarthrosis although the study may have been underpowered to detect these differences. <h3>CONCLUSIONS</h3> This study suggests that preoperative osteopenia or osteoporosis may be correlated with a higher incidence of pseudarthrosis in anterior cervical spine surgery, even more so than smoking or number of levels fused. While the number of patients enrolled in this study is small, the results are convincing enough to warrant screening and treatment of osteoporosis as well as the optimization of preoperative bone stock in all potential candidates for cervical spine fusion. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Gli1+ progenitors mediate bone anabolic function of teriparatide via Hh and Igf signaling.

SUMMARYTeriparatide is the most widely prescribed bone anabolic drug in the world, but its cellular targets remain incompletely defined. The Gli1+ metaphyseal mesenchymal progenitors (MMPs) are a main source for osteoblasts in postnatal growing mice, but their potential response to teriparatide is unknown. Here, by lineage tracing, we show that teriparatide stimulates both proliferation and osteoblast differentiation of MMPs. Single-cell RNA sequencing reveals heterogeneity among MMPs, including an unexpected chondrocyte-like osteoprogenitor (COP). COP expresses the highest level of Hedgehog (Hh) target genes and the insulin-like growth factor 1 receptor (Igf1r) among all cell clusters. COP also expresses Pth1r and further upregulates Igf1r upon teriparatide treatment. Inhibition of Hh signaling or deletion of Igf1r from MMPs diminishes the proliferative and osteogenic effects of teriparatide. The study therefore identifies COP as a teriparatide target wherein Hh and insulin-like growth factor (Igf) signaling are critical for the osteoanabolic response in growing mice.

Targeted Remodeling of the Tumor Microenvironment Via Mechanical Stimulation Defines a Novel Therapeutic Strategy in Myeloma Bone Disease

Multiple myeloma (MM) patients develop devastating osteolytic bone disease causing non-healing bone fractures and pain. MM cells in the bone marrow microenvironment stimulate osteoclast activity and inhibit osteoblast and osteocyte function leading to massive bone resorption and therefore destruction. Bones have the capacity to locally adapt to changing loads and for self-repair and remodeling as dramatically seen in athletes during training. In this study we determined if mechanical loading can counterbalance osteolytic bone disease in the presence of established MM. We hypothesized that mechanical stimulation increases bone formation and decreases bone resorption by activating known bone (re)modeling pathways, which are deleteriously altered by MM cells in bone. We performed in vivo cyclic axial compressive tibial loading (Fig. 1) to MOPC315.BM BALB/c injected mice (n=19) with established MM bone disease (Fig. 2, bioluminescence imaging). The bone (re)modeling response to mechanical loading (5d/wk, over 3 wks) was investigated by longitudinal in vivo microCT imaging at days 14, 18, 23, 28 and 33 (Fig. 3, 4). Loading was administered to the left tibia starting 14 days after i.t. injection of left limbs of MOPC315.BM (n=10) and PBS (n=7) mice (left limb: εmax=2000µε determined by strain gauging, right limb was nonloaded). 15 mice served as nonloaded control mice (Fig. 3). We also analyzed a group of loaded, but not injected mice (n=10). MicroCT data (Fig. 3), such as cortical thickness (Ct.Th), cortical bone area (Ct.Ar) of loaded left limbs demonstrate that metaphyseal cortical bone formation was significantly greater and cortical porosity (Ct.Po) was lower compared to nonloaded control limbs. Our data provide for the first time an indication that mechanical loading counteracts MM induced bone loss over time. To further study mechanisms of mechanotransduction we used a small scale cell culture bioreactor system to analyze mechanical stimulation of human mesenchymal stem cells (hMSCs) and the translation of mechanical forces into biochemical signals. The bioreactor system allows for analysis of molecular mechanisms of mechanotransduction using a transcription factor activator protein (AP1) luciferase reporter gene construct. Cyclic stretching of telomerase immortalized hMSCs (hMSC-TERT cells) which were stably transfected by an AP1 construct resulted in stimulation of AP1 activity as measured by luciferase gene expression. We analyzed how MM cells interfere with mechanotransduction when co-cultured with hMSC-TERT cells. Our data show that in presence of MOPC315.BM cells and 2 (MM1S, INA-6) out of 4 human MM cell lines hMSCs can be mechanically stimulated and have osteogenic activity. In contrast the human MM cell lines Amo-1 and U266 inhibit the hMSC-TERT response to mechanical stimulation under these conditions. Biochemical and molecular genetic studies are underway to determine how MM cells in bone alter known bone (re)modeling pathways and which yet unanticipated factors regulate the anabolic response to mechanical loading in the MOPC315.BM model. Our study provides a first fundamental understanding of the mechano-biological mechanisms of anabolic bone adaption during MM bone disease in response to mechanical stimuli. We conclude that mechanical loading in the form of exercise has the potential to be a potent novel anabolic strategy for primary and secondary prevention of MM bone disease. Moreover dissection of the underlying molecular pathways will identify new targets for innovative antimyeloma strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Energy Metabolism of Osteocytes.

In this review, we provide a recent update on bioenergetic pathways in osteocytes and identify potential future areas of research interest. Studies have identified a role for regulation of bone formation and bone resorption through osteocyte mechanosensing and osteocyte secreted factors. Nevertheless, there is a paucity of studies on the bioenergetics and energy metabolism of osteocytes, which are required for the regulation of bone remodeling. Osteocytes are cells of the osteoblast lineage embedded in bone. The osteocyte lacunocanalicular network within the skeletal matrix is exposed to a unique hypoxic environment. Therefore, the bioenergetic requirements of these cells could differ from other bone cells due to its location in the ossified matrix and its role in bone regulation transduced by mechanical signals. Recent findings highlighted in this review provide some evidence that metabolism of these cells is dependent on their location due to the substrates present in the microenvironment and metabolic cues from stress pathways. Both glycolysis (glucose metabolism) and oxidative phosphorylation (mitochondrial dynamics, ROS generation) affect osteocyte function and viability. In this review, we provide evidence that is currently available about information regarding bioenergetics pathways in osteocytes. We discuss published studies showing a role for hypoxia-driven glucose metabolism in regulating osteocyte bioenergetics. We also provide information on various substrates that osteocytes could utilize to fuel energetic needs, namely pyruvate, amino acids, and fatty acids. This is based on some preliminary experimental evidence that is available in literature. The role of parathyroid hormone PTH and parathryoid hormone-related peptide PTHrP in bone anabolism and resorption, along with regulation of metabolic pathways in the cells of the skeletal niche, needs to be explored further. Mitochondrial metabolism has a role in osteocyte bioenergetics through substrate utilization, location of the osteocyte in the bone cortex, and mitochondrial biogenesis. While there are limitations in studying metabolic flux in traditional cell lines, there are now novel cell lines and sophisticated tools available to study osteocyte bioenergetics to help harness its potential in vivo in the future.

Histomorphometry evaluation of bone anabolism promoted by prostaglandin E1 and its relation to hypercalcemia.

At present, parathyroid hormone is the only existing anabolic bone therapy but produces hypercalcemia. Prostaglandin E1 (PGE1) has been suggested as a bone anabolic agent that allows bone modeling formation without producing hypercalcemia. This study aimed to corroborate these PGE1 properties. For 22 days, rabbits (n = 30) were divided into three groups (n = 10 each group) and received intravenous solutions: vehicle (control group), palate disjunction + vehicle (sham group), and palate disjunction + 50 μg of PGE1 (PGE1 group). On days 1, 3, and 22, palatine suture X-rays weretaken. On day 22, bone formation markers were analyzed, and the rabbits were sacrificed. Bone palate undecalcified samples were processed. Histomorphometry software was used to analyze bone parameters, and the mineralization front was stained with toluidine blue. Scalloped lines reflect remodeling-based bone formation (RBF), and smooth lines reflect modeling-based formation (MBF). X-rays showed more significant palatal disjunction in the PGE1 group; this group exhibited significant calcitriol serum increments. Hypercalciuria was observed in the PGE1 group, and resorption markers (N-telopeptides) remained stable. Sutural bones in the PGE1 group exhibited significant anabolism in structural parameters. RBF was 20%, and MBF was 6% in the sham group; in the PGE1 group, RBF was 8.6%, and MBF was 17%. In the PGE1 group, mineralization was significantly accelerated, but resorption remained stable. This model suggests that PGE1 favors palate disjunction, calcitriol synthesis, and shortens the mineralization. Therefore, PGE1 is an important bone anabolic molecule predominantly of modeling-based form and no hypercalcemia.

Multiomic analysis of stretched osteocytes reveals processes and signalling linked to bone regeneration and cancer

Exercise is a non-pharmacological intervention that can enhance bone regeneration and improve the management of bone conditions like osteoporosis or metastatic bone cancer. Therefore, it is gaining increasing importance in an emerging area of regenerative medicine—regenerative rehabilitation (RR). Osteocytes are mechanosensitive and secretory bone cells that orchestrate bone anabolism and hence postulated to be an attractive target of regenerative exercise interventions. However, the human osteocyte signalling pathways and processes evoked upon exercise remain to be fully identified. Making use of a computer-controlled bioreactor that mimics exercise and the latest omics approaches, RNA sequencing (RNA-seq) and tandem liquid chromatography-mass spectrometry (LC-MS), we mapped the transcriptome and secretome of mechanically stretched human osteocytic cells. We discovered that a single bout of cyclic stretch activated network processes and signalling pathways likely to modulate bone regeneration and cancer. Furthermore, a comparison between the transcriptome and secretome of stretched human and mouse osteocytic cells revealed dissimilar results, despite both species sharing evolutionarily conserved signalling pathways. These findings suggest that osteocytes can be targeted by exercise-driven RR protocols aiming to modulate bone regeneration or metastatic bone cancer.

Lactoferrin Is a Potential Activator of the Vitamin D Receptor in Its Regulation of Osteogenic Activities in C57BL/6J Mice and MC3T3-E1 Cells.

Lactoferrin (LF) has been shown to promote bone anabolism, and the vitamin D receptor (VDR) mediates the effects of vitamin D on bone. We hypothesized that LF improves bone health by increasing VDR expression. We sought to determine the role of VDR activation in LF-induced osteogenic activity in vivo and in vitro and the underlying molecular mechanisms. Sixty male C57BL/6J mice (aged 4 wk) were randomly assigned into 6 groups and fed vitamin D-deficient (VDD; 0 IU/kg) or vitamin D-normal diet (VDN; 1000 IU cholecalciferol/kg) and administered placebo or LF (100 or 1000 mg/kg body weight) by gavage for 24 wk. Trabecular bone structure was analyzed using micro-CT, and VDR expression was assessed by immunohistochemistry. In vitro, MC3T3-E1 cells were treated with 100 μg LF/mL to evaluate its effect on VDR expression. Finally, the direct recruitment of LF to the Vdr promoter was confirmed by chromatin immunoprecipitation assay. In addition, cells were transfected with pGL3-basic Vdr vector for monitoring Vdr promoter activation using luciferase assays. LF supplementation at 100 and 1000 mg/kg revealed an ∼6.5% (P<0.05) increase in bone mineral density in mice on VDD diet and exhibited an enhanced expression of VDR in bone compared with control. This increased expression of VDR was also observed in the bone of mice on the VDN diet, but the effect was more pronounced in VDD diet. In vitro, compared with the control group, Vdr mRNA expression was 18 times greater (P<0.05) and peaked at 2 h posttreatment of LF. By cotransfection of the pGL3-basic Vdr vector, LF induced luciferase activity by 30% (P<0.05) in MC3T3-E1 cells. In vivo and in vitro, LF, a potential activator of VDR, promotes osteogenesis. This suggests that dairy products, which are rich in LF, may serve as a functional food to improve bone health.

Osteocyte mechanosensing following short-term and long-term treatment with sclerostin antibody.

Sclerostin antibody romosozumab (EVENITY™, romosozumab-aqqg) has a dual mechanism of action on bone, increasing bone formation and decreasing bone resorption, leading to increases in bone mass and strength, and a decreased risk of fracture, and has been approved for osteoporosis treatment in patients with high risk of fragility fractures. The bone formation aspect of the response to sclerostin antibody treatment has thus far been best described as having two phases: an immediate and robust phase of anabolic bone formation, followed by a long-term response characterized by attenuated bone accrual. We herein test the hypothesis that following the immediate pharmacologic anabolic response, the changes in bone morphology result in altered (lesser) mechanical stimulation of the resident osteocytes, initiating a negative feedback signal quantifiable by a reduced osteocyte signaling response to load. This potential desensitization of the osteocytic network is probed via a novel ex vivo assessment of intracellular calcium (Ca2+) oscillations in osteocytes below the anteromedial surface of murine tibiae subjected to load after short-term (2weeks) or long-term (8weeks) treatment with sclerostin antibody or vehicle control. We found that for both equivalent load levels and equivalent strain levels, osteocyte Ca2+ dynamics are maintained between tibiae from the control mice and the mice that received long-term sclerostin antibody treatment. Furthermore, under matched strain environments, we found that short-term sclerostin antibody treatment results in a reduction of both the number of responsive cells and the speed of their responses, which we attribute largely to the probability that the observed cells in the short-term group are relatively immature osteocytes embedded during initial pharmacologic anabolism. Within this study, we demonstrate that osteocytes embedded following long-term sclerostin antibody treatment exhibit localized Ca2+ signaling akin to those of mature osteocytes from the vehicle group, and thus, systemic attenuation of responses such as circulating P1NP and bone formation rates likely occur as a result of processes downstream of osteocyte Ca2+ signaling.

SLPI is a critical mediator that controls PTH-induced bone formation

Osteoclastic bone resorption and osteoblastic bone formation/replenishment are closely coupled in bone metabolism. Anabolic parathyroid hormone (PTH), which is commonly used for treating osteoporosis, shifts the balance from osteoclastic to osteoblastic, although it is unclear how these cells are coordinately regulated by PTH. Here, we identify a serine protease inhibitor, secretory leukocyte protease inhibitor (SLPI), as a critical mediator that is involved in the PTH-mediated shift to the osteoblastic phase. Slpi is highly upregulated in osteoblasts by PTH, while genetic ablation of Slpi severely impairs PTH-induced bone formation. Slpi induction in osteoblasts enhances its differentiation, and increases osteoblast–osteoclast contact, thereby suppressing osteoclastic function. Intravital bone imaging reveals that the PTH-mediated association between osteoblasts and osteoclasts is disrupted in the absence of SLPI. Collectively, these results demonstrate that SLPI regulates the communication between osteoblasts and osteoclasts to promote PTH-induced bone anabolism.

Syndecan-3 enhances anabolic bone formation through WNT signaling.

Osteoporosis is the most common age‐related metabolic bone disorder, which is characterized by low bone mass and deterioration in bone architecture, with a propensity to fragility fractures. The best treatment for osteoporosis relies on stimulation of osteoblasts to form new bone and restore bone structure, however, anabolic therapeutics are few and their use is time restricted. Here, we report that Syndecan‐3 increases new bone formation through enhancement of WNT signaling in osteoblasts. Young adult Sdc3−/− mice have low bone volume, reduced bone formation, increased bone marrow adipose tissue, increased bone fragility, and a blunted anabolic bone formation response to mechanical loading. This premature osteoporosis‐like phenotype of Sdc3−/− mice is due to delayed osteoblast maturation and impaired osteoblast function, with contributing increased osteoclast‐mediated bone resorption. Indeed, overexpressing Sdc3 in osteoblasts using the Col1a1 promoter rescues the low bone volume phenotype of the Sdc3−/− mice, and also increases bone volume in WT mice. Mechanistically, SDC3 enhances canonical WNT signaling in osteoblasts through stabilization of Frizzled 1, making SDC3 an attractive target for novel bone anabolic drug development.

Loss of KDM4B exacerbates bone-fat imbalance and mesenchymal stromal cell exhaustion in skeletal aging.

Skeletal aging is a complex process, characterized by a decrease in bone formation, an increase in marrow fat, and stem cell exhaustion. Loss of H3K9me3, a heterochromatin mark, has been proposed to be associated with aging. Here, we report that loss of KDM4B in mesenchymal stromal cells (MSCs) exacerbated skeletal aging and osteoporosis by reducing bone formation and increasing marrow adiposity via increasing H3K9me3. KDM4B epigenetically coordinated β-catenin/Smad1-mediated transcription by removing repressive H3K9me3. Importantly, KDM4B ablation impaired MSC self-renewal and promoted MSC exhaustion by inducing senescence-associated heterochromatin foci formation, providing a mechanistic explanation for stem cell exhaustion with aging. Moreover, while KDM4B was required for parathyroid hormone-mediated bone anabolism, KDM4B depletion accelerated bone loss and marrow adiposity induced by a high-fat diet. Our results suggest that the epigenetic rejuvenation and reversing bone-fat imbalance might be new strategies for preventing and treating skeletal aging and osteoporosis by activating KDM4B in MSCs.

Fluid-structure interaction (FSI) modeling of bone marrow through trabecular bone structure under compression.

The present study has sought to investigate the fluid characteristic and mechanical properties of trabecular bone using fluid-structure interaction (FSI) approach under different trabecular bone orientations. This method imposed on trabecular bone structure at both longitudinal and transverse orientations to identify effects on shear stress, permeability, stiffness and stress regarded to the trabeculae. Sixteen FSI models were performed on different range trabecular cubes of 27mm3 with eight models developed for each longitudinal and transverse direction. Results show that there was a moderate correlation between permeability and porosity, and surface area in the longitudinal and transverse orientations. For the longitudinal orientation, the permeability values varied between 3.66 × 10-8 and 1.9 × 10-7 and the sheer stress values varied between 0.05 and 1.8Pa, whilst for the transverse orientation, the permeability values varied between 5.95 × 10-10 and 1.78 × 10-8 and the shear stress values varied between 0.04 and 3.1Pa. Here, transverse orientation limits the fluid flow from passing through the trabeculae due to high shear stress disturbance generated within the trabecular bone region. Compared to physiological loading direction (longitudinal orientation), permeability is higher within the range known to trigger a response in bone cells. Additionally, shear stresses also increase with bone surface area. This study suggests the shear stress within bone marrow in real trabecular architecture could provide the mechanical signal to marrow cells that leads to bone anabolism and can depend on trabecular orientation.

Bitter Taste Receptor as a Therapeutic Target in Orthopaedic Disorders.

Non-gustatory, extraoral bitter taste receptors (T2Rs) are G-protein coupled receptors that are expressed throughout the body and have various functional responses when stimulated by bitter agonists. Presently, T2Rs have been found to be expressed in osteoclasts and osteocytes where osteoclasts were capable of detecting bacterial quorum-sensing molecules through the T2R38 isoform. In the innate immune system, stimulating T2Rs induces anti-inflammatory and anti-pathogenic effects through the phospholipase C/inositol triphosphate pathway, which leads to intracellular calcium release from the endoplasmic reticulum. The immune cells with functional responses to T2R activation also play a role in bone inflammation and orthopaedic disorders. Furthermore, increasing intracellular calcium levels in bone cells through T2R activation can potentially influence bone formation and resorption. With recent studies finding T2R expression in bone cells, we examine the potential of targeting this receptor to treat bone inflammation and to promote bone anabolism.

Molecular hydrogen enhances osteogenesis in Danio rerio embryos.

Molecular hydrogen (H2 ) has been reported to have important biological effects on bone tissue in several in vitro and in vivo models. Danio rerio (zebrafish) embryo is a good model to study osteogenesis because of its transparency, size and rapid development. In zebrafish embryo, hydrogen-rich water (HRW) does not affect vitality or growth rate up to 15%. In addition, 7% HRW treatment enhances zebrafish embryo osteogenesis, increasing the vertebral mineralization rate. In conclusion, we demonstrated the beneficial effects of hydrogen on anabolic bone functions in vivo.

Osteolytic effects of tumoral estrogen signaling in an estrogen receptor-positive breast cancer bone metastasis model.

Aim:Estrogen receptor α-positive (ER+) subtypes of breast cancer have the greatest predilection for forming osteolytic bone metastases (BMETs). Because tumor-derived factors mediate osteolysis, a possible role for tumoral ERα signaling in driving ER+ BMET osteolysis was queried using an estrogen (E2)-dependent ER+ breast cancer BMET model.Methods:Female athymic Foxn1nu mice were inoculated with human ER+ MCF-7 breast cancer cells via the left cardiac ventricle post-E2 pellet placement, and age- and dose-dependent E2 effects on osteolytic ER+ BMET progression, as well as direct bone effects of E2, were determined.Results:Osteolytic BMETs, which did not form in the absence of E2 supplementation, occurred with the same frequency in young (5-week-old) vs. skeletally mature (16-week-old) E2 (0.72 mg)-treated mice, but were larger in young mice where anabolic bone effects of E2 were greater. However, in mice of a single age and across a range of E2 doses, anabolic E2 bone effects were constant, while osteolytic ER+ BMET lesion incidence and size increased in an E2-dose-dependent fashion. Osteoclasts in ER+ tumor-bearing (but not tumor-naive) mice increased in an E2-dose dependent fashion at the bone-tumor interface, while histologic tumor size and proliferation did not vary with E2 dose. E2-inducible tumoral secretion of the osteolytic factor parathyroid hormone-related protein (PTHrP) was dose-dependent and mediated by ERα, with significantly greater levels of secretion from ER+ BMET-derived tumor cells.Conclusion:These results suggest that tumoral ERα signaling may contribute to ER+ BMET-associated osteolysis, potentially explaining the greater predilection for ER+ tumors to form clinically-evident osteolytic BMETs.

Probiotics Stimulate Bone Formation in Obese Mice via Histone Methylations

Rationale: Manipulation of the gut microbiome can prevent pathologic bone loss. However, the effects of probiotics on mitochondrial epigenetic remodeling and skeletal homeostasis in the high-fat diet (HFD)-linked obesity remains to be explored. Here, we examined the impact of probiotics supplementation on mitochondrial biogenesis and bone homeostasis through the histone methylation mechanism in HFD fed obese mice.Methods: 16S rRNA gene sequencing was performed to study the microbiota composition in the gut and microbial dysbiosis in obese mouse model. High resolution (microPET/CT) imaging was performed to demonstrate the obese associated colonic inflammation. Obese-associated upregulation of target miRNA in osteoblast was investigated using a microRNA qPCR array. Osteoblastic mitochondrial mass was evaluated using confocal imaging. Overexpression of mitochondrial transcription factor (Tfam) was used to investigate the glycolysis and mitochondrial bioenergetic metabolism using Tfam-transgenic (Tg) mice fed on HFD. The bone formation and mechanical strength was evaluated by microCT analysis and three-point bending analysis.Results: High-resolution imaging (µ-CT) and mechanical testing revealed that probiotics induced a significant increase of trabecular bone volume and bone mechanical strength respectively in obese mice. Probiotics or Indole-3-propionic acid (IPA) treatment directly to obese mice, prevents gut inflammation, and improved osteoblast mineralization. Mechanistically, probiotics treatment increases mitochondrial transcription factor A (Tfam) expression in osteoblasts by promoting Kdm6b/Jmjd3 histone demethylase, which inhibits H3K27me3 epigenetic methylation at the Tfam promoter. Furthermore, Tfam-transgenic (Tg) mice, fed with HFD, did not experience obesity-linked reduction of glucose uptake, mitochondrial biogenesis and mineralization in osteoblasts.Conclusions: These results suggest that the probiotics mediated changes in the gut microbiome and its derived metabolite, IPA are potentially be a novel agent for regulating bone anabolism via the gut-bone axis.

An MSC bone-homing compound, Rab001, increases bone mass and reduces the incidence of osteonecrosis in a glucocorticoid-induced osteonecrosis mouse model.

Currently, there are no effective medications to either prevent or slow the progression of atraumatic osteonecrosis (ON).The objective of this study is to determine the effects of bone-targeted delivery of mesenchymal stem cells on the prevalence of ON in a glucocorticoid (GC)-induced mouse model. Eight-week-old male BALB/c mice were randomized into groups that received placebo (PL), prednisolone (GC), or concurrent treatments with GC+mesenchymal stromal cells (MSCs), Rab001 or GC+Rab001 + MSCs. Human parathyroid hormone (hPTH) was used as a positive control for bone anabolism. Mice were killed after 30days, and quantitative measurements of bone mass, bone strength, prevalent ON at the distal femoral epiphysis (DFE) were performed. Angiogenesis was accessed by RNA-Seq, the circulating angiogenic markers, as well as by immunohistochemical staining. We have showed that a novel agent, Rab001 that can noncovalently bind to mesenchymal stem cells (MSC) and direct them to the bone, prevents the incidence of glucocorticoid-induced osteonecrosis in the mouse. In contrast, PTH, a bone anabolic treatment, preserves bone mass but sustains higher ON incidence than Rab001+/- MSC-treated mice. The results of these experiments reveal that glucocorticoids increase the prevalence of ON, and agents that prevent loss of bone vascularity appear to prevent the development of ON. This intervention might be useful in patients with early stages of atraumatic ON.

Enhancement of osteoblast differentiation using poly(ethylene sodium phosphate)

Biodegradable polyphosphoesters (PPEs) are of increasing interest due to their promising biomedical applications. Polyphosphodiesters (PPDEs), formed by polyphosphotriester (PPTE) dealkylation, have bone-targeting properties in vivo and therefore are promising polymer drug candidates for bone disease treatment. However, their effects on osteoblasts are still unclear. This study prepared two polymer structures, poly(methyl ethylene phosphate) (PMP) and poly(ethylene sodium phosphate) (PEP•Na), as PPTE and PPDE models, respectively, and investigated their effects on mouse osteoblastic cell (MC3T3-E1) differentiation. Results showed that PMP is inert toward osteoblast differentiation. In contrast, PEP•Na enhanced alkaline phosphatase (ALP) synthesis, matrix mineralization, and osteoblast-related gene expression. PEP•Na also enhanced Wnt signaling pathway–dependent Alp expression, in addition to its own internalization into the cytosol during 3 days of differentiation culture. These results showed that dealkylated PPEs are a polymer drug candidate for osteoporosis treatment, not only because of their bone-targeting properties but also because of the controllable effects of bone anabolism via osteoblast differentiation enhancement.