Osteoclastic Resorption Research Articles

Vasorin as an actor of bone turnover?

Bone diseases are increasing with aging populations and it is important to identify clues to develop innovative treatments. Vasn, which encodes vasorin (Vasn), a transmembrane protein involved in the pathophysiology of several organs, is expressed during the development in intramembranous and endochondral ossification zones. Here, we studied the impact of Vasn deletion on the osteoblast and osteoclast dialog through a cell Coculture model. In addition, we explored the bone phenotype of Vasn KO mice, either constitutive or tamoxifen-inducible, or with an osteoclast-specific deletion. First, we show that both osteoblasts and osteoclasts express Vasn. Second, we report that, in both KO mouse models but not in osteoclast-targeted KO mice, Vasn deficiency was associated with an osteopenic bone phenotype, due to an imbalance in favor of osteoclastic resorption. Finally, through the Coculture experiments, we identify a dysregulation of the Wnt/β-catenin pathway together with an increase in RANKL release by osteoblasts, which led to an enhanced osteoclast activity. This study unravels a direct role of Vasn in bone turnover, introducing a new biomarker or potential therapeutic target for bone pathologies.

The PDE4 Inhibitors Roflumilast and Rolipram Rescue ADO2 Osteoclast Resorption Dysfunction.

Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption caused by heterozygous missense mutations in the chloride channel 7 (CLCN7). Adenylate cyclase, which catalyzes the formation of cAMP, is critical for lysosomal acidification in osteoclasts. We found reduced cAMP levels in ADO2 osteoclasts compared to wild-type (WT) osteoclasts, leading us to examine whether regulating cAMP would improve ADO2 osteoclast activity. Although forskolin, a known activator of adenylate cyclase and cAMP levels, negatively affected osteoclast number, it led to an overall increase in ADO2 and WT osteoclast resorption activity in vitro. Next, we examined cAMP hydrolysis by the phosphodiesterase 4 (PDE4) proteins in ADO2 versus WT osteoclasts. QPCR analysis revealed higher expression of the three major PDE4 subtypes (4a, 4b, 4d) in ADO2 osteoclasts compared in WT, consistent with reduced cAMP levels in ADO2 osteoclasts. In addition, we found that the PDE4 antagonists, rolipram and roflumilast, stimulated ADO2 and WT osteoclast formation in a dose-dependent manner. Importantly, roflumilast and rolipram displayed a concentration-dependent increase in osteoclast resorption activity which was greater in ADO2 than WT osteoclasts. Moreover, treatment with roflumilast rescued cAMP levels in ADO2 OCLs. The key findings from our studies demonstrate that osteoclasts from ADO2 mice exhibit reduced cAMP levels and PDE4 inhibition rescues cAMP levels and ADO2 osteoclast activity dysfunction in vitro. The mechanism of action of PDE4 inhibitors and their ability to reduce the high bone mass of ADO2 mice in vivo are currently under investigation. Importantly, these studies advance the understanding of the mechanisms underlying the ADO2 osteoclast dysfunction which is critical for the development of therapeutic approaches to treat clinically affected ADO2 patients.

Murine IRF8 Mutation Offers New Insight into Osteoclast and Root Resorption

Interferon regulatory factor 8 (IRF8), a transcription factor expressed in immune cells, functions as a negative regulator of osteoclasts and helps maintain dental and skeletal homeostasis. Previously, we reported that a novel mutation in the IRF8 gene increases susceptibility to multiple idiopathic cervical root resorption (MICRR), a form of tooth root resorption mediated by increased osteoclast activity. The IRF8 G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. To investigate the molecular basis of MICRR and IRF8 function in osteoclastogenesis, we generated Irf8 knock-in (KI) mice using CRISPR/Cas9 technique modeling the human IRF8 G388S mutation. The heterozygous (Het) and homozygous (Homo) Irf8 KI mice showed no gross morphological defects, and the development of hematopoietic cells was unaffected and similar to wild-type (WT) mice. The Irf8 KI Het and Homo mice showed no difference in macrophage gene signatures important for antimicrobial defenses and inflammatory cytokine production. Consistent with the phenotype observed in MICRR patients, Irf8 KI Het and Homo mice demonstrated significantly increased osteoclast formation and resorption activity in vivo and in vitro when compared to WT mice. The oral ligature–inserted Het and Homo mice displayed significantly increased root resorption and osteoclast-mediated alveolar bone loss compared to WT mice. The increased osteoclastogenesis noted in KI mice is due to the inability of IRF8 G388S mutation to inhibit NFATc1-dependent transcriptional activation and downstream osteoclast specific transcripts, as well as its impact on autophagy-related pathways of osteoclast differentiation. This translational study delineates the IRF8 domain important for osteoclast function and provides novel insights into the IRF8 mutation associated with MICRR. IRF8 G388S mutation mainly affects osteoclastogenesis while sparing immune cell development and function. These insights extend beyond oral health and significantly advance our understanding of skeletal disorders mediated by increased osteoclast activity and IRF8’s role in osteoclastogenesis.

A novel method to fabricate monetite granules for bone graft applications.

Monetite granules were reported to be able to balance osteoclastic resorption and new bone formation. However, to date, the dehydration of preset brushite has been the well-known method for preparing monetite granules. In the present study, for the first time, monetite granules could be prepared from the phase transformation of calcium sulfate dihydrate (CSD) granules through immersion in NaH2PO4 solution under hydrothermal conditions. CSD granules could be fully transformed into monetite granules at a reaction temperature of 125°C for 24 h. The obtained monetite granules were eight times more soluble in acetate buffer than in Tris-HCl buffer. Furthermore, monetite granules were two times more soluble in acetate buffer but comparable in Tris-HCl buffer compared to xenograft HA. The initial cytotoxicity test indicated that the novel monetite granules were nontoxic. In short, novel monetite granules were successfully prepared, exhibited better solubility in osteoclastic simulation than xenograft HA and were nontoxic.

Bone resorption by osteoclasts involves fine tuning of RHOA activity by its microtubule-associated exchange factor GEF-H1.

Bone health is controlled by the balance between bone formation by osteoblasts and degradation by osteoclasts. A disequilibrium in favor of bone resorption leads to osteolytic diseases characterized by decreased bone density. Osteoclastic resorption is dependent on the assembly of an adhesion structure: the actin ring, also called podosome belt or sealing zone, which is composed of a unique patterning of podosomes stabilized by microtubules. A better understanding of the molecular mechanisms regulating the crosstalk between actin cytoskeleton and microtubules network is key to find new treatments to inhibit bone resorption. Evidence points to the importance of the fine tuning of the activity of the small GTPase RHOA for the formation and maintenance of the actin ring, but the underlying mechanism is not known. We report here that actin ring disorganization upon microtubule depolymerization is mediated by the activation of the RHOA-ROCK signaling pathway. We next show the involvement of GEF-H1, one of RHOA guanine exchange factor highly expressed in osteoclasts, which has the particularity of being negatively regulated by sequestration on microtubules. Using a CRISPR/Cas9-mediated GEF-H1 knock-down osteoclast model, we demonstrate that RHOA activation upon microtubule depolymerization is mediated by GEF-H1 release. Interestingly, although lower levels of GEF-H1 did not impact sealing zone formation in the presence of an intact microtubule network, sealing zone was smaller leading to impaired resorption. Altogether, these results suggest that a fine tuning of GEF-H1 through its association with microtubules, and consequently of RHOA activity, is essential for osteoclast sealing zone stability and resorption function.

Osteoclast-specific Plastin 3 knockout in mice fail to develop osteoporosis despite dramatic increased osteoclast resorption activity.

PLS3 loss-of-function mutations in humans and mice cause X-linked primary osteoporosis. However, it remains largely unknown how PLS3 mutations cause osteoporosis and which function PLS3 plays in bone homeostasis. A recent study showed that ubiquitous Pls3 KO in mice results in osteoporosis. Mainly osteoclasts were impacted in their function However, it has not been proven if osteoclasts are the major cell type affected and responsible for osteoporosis development in ubiquitous Pls3 KO mice. Here, we generated osteoclast-specific Pls3 KO mice. Additionally, we developed a novel polyclonal PLS3 antibody that showed specific PLS3 loss in immunofluorescence staining of osteoclasts in contrast to previously available antibodies against PLS3, which failed to show PLS3 specificity in mouse cells. Moreover, we demonstrate that osteoclast-specific Pls3 KO causes dramatic increase in resorptive activity of osteoclasts in vitro. Despite these findings, osteoclast-specific Pls3 KO in vivo failed to cause any osteoporotic phenotype in mice as proven by micro-CT and three-point bending test. This demonstrates that the pathomechanism of PLS3-associated osteoporosis is highly complex and cannot be reproduced in a system singularly focused on one cell type. Thus, the loss of PLS3 in alternative bone cell types might contributes to the osteoporosis phenotype in ubiquitous Pls3 KO mice.

IN VITRO MODELS MIMICKING THE BONE REMODELLING CYCLE

Metabolic bone diseases, such as osteoporosis and osteopetrosis, result from an imbalanced bone remodeling process. In vitro bone models are often used to investigate either bone formation or resorption independently, while in vivo, these processes are coupled. Combining these processes in a co-culture is challenging as it requires finding the right medium components to stimulate each cell type involved without interfering with the other cell type's differentiation. Furthermore, differentiation stimulating factors often comprise growth factors in supraphysiological concentrations, which can overshadow the cell-mediated crosstalk and coupling.To address these challenges, we aimed to recreate the physiological bone remodeling process, which follows a specific sequence of events starting with cell activation and bone resorption by osteoclasts, reversal, followed by bone formation by osteoblasts. We used a mineralized silk fibroin scaffold as a bone-mimetic template, inspired by bone's extracellular matrix composition and organization. Our model supported osteoclastic resorption and osteoblastic mineralization in the specific sequence that represents physiological bone remodeling.We also demonstrated how culture variables, such as different cell ratios, base media, and the use of osteogenic/osteoclast supplements, and the application of mechanical load, can be adjusted to represent either a high bone turnover system or a self-regulating system. The latter system did not require the addition of osteoclastic and osteogenic differentiation factors for remodeling, therefore avoiding growth factor use.Our in vitro model for bone remodeling has the potential to reduce animal experiments and advance in vitro drug development for bone remodeling pathologies like osteoporosis. By recreating the physiological bone remodeling cycle, we can investigate cell-cell and cell-matrix interactions, which are essential for understanding bone physiology and pathology. Furthermore, by tuning the culture variables, we can investigate bone remodeling under various conditions, potentially providing insights into the mechanisms underlying different bone disorders.

Functional and analytical recapitulation of osteoclast biology on demineralized bone paper

Osteoclasts are the primary target for osteoporosis drug development. Recent animal studies revealed the crucial roles of osteoblasts in regulating osteoclastogenesis and the longer lifespans of osteoclasts than previously thought with fission and recycling. However, existing culture platforms are limited to replicating these newly identified cellular processes. We report a demineralized bone paper (DBP)-based osteoblast culture and osteoclast assay platform that replicates osteoclast fusion, fission, resorption, and apoptosis with high fidelity and analytical power. An osteoid-inspired DBP supports rapid and structural mineral deposition by osteoblasts. Coculture osteoblasts and bone marrow monocytes under biochemical stimulation recapitulate osteoclast differentiation and function. The DBP-based bone model allows longitudinal quantitative fluorescent monitoring of osteoclast responses to bisphosphonate drug, substantiating significantly reducing their number and lifespan. Finally, we demonstrate the feasibility of humanizing the bone model. The DBP-based osteo assay platforms are expected to advance bone remodeling-targeting drug development with improved prediction of clinical outcomes.

Exosome-targeted delivery of METTL14 regulates NFATc1 m6A methylation levels to correct osteoclast-induced bone resorption.

Osteoporosis has a profound influence on public health. First-line bisphosphonates often cause osteonecrosis of the jaw meanwhile inhibiting osteoclasts. Therefore, it is important to develop effective treatments. The results of this study showed that the increased level of NFATc1 m6A methylation caused by zoledronic acid (ZOL), with 4249A as the functional site, is highly correlated with the decreased bone resorption of osteoclasts. Upstream, METTL14 regulates osteoclast bone absorption through the methylation functional site of NFATc1. Downstream, YTHDF1 and YTHDF2 show antagonistic effects on the post-transcriptional regulation of NFATc1 after the m6A methylation level is elevated by METTL14. In this study, meRIP-Seq, luciferase reporter assays, meRIP and other methods were used to elucidate the NFATc1 regulatory mechanism of osteoclasts from the perspective of RNA methylation. In addition, EphA2 overexpression on exosomes is an effective biological method for targeted delivery of METTL14 into osteoclasts. Importantly, this study shows that METTL14 released by exosomes can increase the m6A methylation level of NFATc1 to inhibit osteoclasts, help postmenopausal osteoporosis patients preserve bone mass, and avoid triggering osteonecrosis of the jaw, thus becoming a new bioactive molecule for the treatment of osteoporosis.

Comparative evaluation of osseointegration of new percutaneous implants made of Ti Grade 4 ultrafine‑grained alloy

Introduction It has been shown that titanium implants with a structured surface provide an increased rate of osseointegration what makes their application quite promising.The purpose of this work was to conduct a comparative evaluation of the efficiency of osseointegration of new percutaneous implants for prosthetics made of ultrafine-grained Ti Grade 4 alloy.Materials and methods The study was carried out on 12 male rabbits of the Soviet Chinchilla breed. Six rabbits of the control group had implants made of Ti6Al4V powder using selective laser sintering technology that were osseointegrated into the tibia, 6 rabbits of the experimental group had implants made of Ti Grade 4 by equal channel angular pressing. The formation of the "bone-implant" block was examined 26 weeks after the implantation.Results Histologically, after 26 weeks of the experiment, porous changes, enlargement of the Haversian canals, and pronounced osteoclastic resorption were not detected in the animals of the experimental group throughout the stump in the compact plate. Around the implant, a bony case repeating the bone shape was formed, represented by lamellar bone tissue. Using X-ray electron probe microanalysis, it was found that in the substrate formed on the surface of the implant in rabbits of the experimental group, there was significantly more calcium in all areas over the implant relative to the animals of the control group. In the control group, relative to the experimental group, an increased level of C-reactive protein in blood serum was retained longer. Complications and significant clinical and laboratory abnormalities were not found in both groups during the entire experiment.Discussion Our data are consistent with the results of other experimental studies, which unambiguously noted that titanium implants with a structured surface show increased osseointegration characteristics in comparative studies relative to implants without modification of the structure of the material of the threaded surface. The absence of complications and undesirable reactions of the animal organism also indicates the acceptable safety of the tested products.Conclusion Osseointegration of a percutaneous implant that has a mixed nanocrystalline and ultrafine-grained structure was more effective than the reference implant. This makes the use of such implant promising for solving clinical problems in prosthetics.

Bioactive composite hydrogel with effects of robust promoting osteogenesis and immunomodulation for osteoporotic bone regeneration

Osteoporotic bone defect is an intractable challenge in clinical practice, involving impaired bone repair ability and abnormal immune response. Despite the development of various tissue-engineered scaffolds for osteoporotic bone repair, the pathogenesis of osteoporosis has been lacking consideration, resulting in poor efficacy. Here, we integrated short-chain chitosan (CS) and nanoparticulate hydroxyapatite (nHAp) into a covalent tetra-armed poly (ethylene glycol) (tetra-PEG) network to create a composite hydrogel (PEG/nHAp/CS, PHC). The constructed PHC hydrogel exhibited ability of enhancing osteogenesis with high mechanical strength, excellent surface properties, and good biocompatibility. PHC hydrogel also had immunomodulatory effects of promoting M2 macrophage polarization and suppressing M1 macrophage polarization via antagonizing TLR4/NF-κB signaling. PHC hydrogel further increased osteogenic capacity and inhibited osteoclast formation and resorption indirectly. Parathyroid hormone (PTH) could be effective loaded and sustainable released in the PHC hydrogel (PTH@PHC), in which the osteogenic ability was further enhanced via activation of cAMP/PKA/CREB signaling. By using an osteoporotic calvaria bone defect rat model, we demonstrated that the PTH@PHC hydrogel was able to improve bone regeneration and bone defects healing. Findings of this study indicated that the bioactive composite hydrogel has powerful osteogenic-promoting potency and immunomodulation, which provides a promising therapeutic strategy for future clinical repair of osteoporotic bone defect.

Yunnan Baiyao Inhibits Periodontitis by Suppressing the Autophagic Flux

BackgroundYunnan Baiyao (YNBY), a traditional Chinese medicine, is renowned for its anti-inflammatory properties. Recent studies have suggested that YNBY plays a significant role in inhibiting osteoclast differentiation and autophagy, which are essential processes in inflammation and bone resorption associated with periodontitis. However, the precise relationship between autophagy and the mechanism by which YNBY inhibits osteoclastogenesis remains unexplored.The primary objective of this study was to investigate the inhibitory effects of YNBY on the process of osteoclastogenesis and its potential in preventing inflammatory bone loss. MethodsThe animals were subjected to sacrifice at intervals of 2, 4, and 6 weeks postintervention whilst under deep anaesthesia, and specimens were subsequently collected. The specimens were subjected to hematoxylin and eosin (HE) staining, in addition to tartrate-resistant acid phosphatase (TRAP) staining and subsequently imaged employing a digital scanner. The confirmation of osteoclast (OC) differentiation and autophagic flux was achieved through various techniques, including western blotting, transmission electron microscopy (TEM), TRAP staining, pit formation assay, and immunofluorescence. ResultsThe microcomputed tomography images provided evidence of the effective inhibition of alveolar bone absorption at 2, 4, and 6 weeks following YNBY treatment. Additionally, the histomorphometric evaluations of tissue segments stained with HE and TRAP, which involved measuring the distance between the alveolar bone crest (ABC) and cementoenamel junction (CEJ) and quantifying TRAP-positive OCs, yielded comparable results to those obtained through computed tomography analysis. YNBY treatment resulted in a decrease in the CEJ–ABC distance and inhibition of OC differentiation. Furthermore, in vitro studies showed that the autophagy modulators rapamycin (RAP) and 3-methyladenine (3-MA) significantly affected OC differentiation and function. YNBY attenuated the impact of RAP on the differentiation of OCs, autophagy-related factor activation, and bone resorption. ConclusionsWe hypothesise that YNBY suppresses the differentiation of OC and bone resorption by blocking autophagy. This study reveals that targeting autophagy might be a new alternative treatment methodology for periodontitis treatment.

Integration of osteoclastogenesis through addition of PBMCs in human osteochondral explants cultured ex vivo

The preservation of tissue specific cells in their native 3D extracellular matrix in bone explants provides a unique platform to study remodeling. Thus far, studies involving bone explant cultures showed a clear focus on achieving bone formation and neglected osteoclast activity and resorption. To simulate the homeostatic bone environment ex vivo, both key elements of bone remodeling need to be represented. This study aimed to assess and include osteoclastogenesis in human osteochondral explants through medium supplementation with RANKL and M-CSF and addition of peripheral blood mononuclear cells (PBMCs), providing osteoclast precursors.Osteochondral explants were freshly harvested from human femoral heads obtained from hip surgeries and cultured for 20 days in a two-compartment culture system. Osteochondral explants preserved viability and cellular abundance over the culture period, but histology demonstrated that resident osteoclasts were no longer present after 4 days of culture. Quantitative extracellular tartrate resistant acid phosphatase (TRAP) analysis confirmed depletion of osteoclast activity on day 4 even when stimulated with RANKL and M-CSF. Upon addition of PBMCs, a significant upregulation of TRAP activity was measured from day 10 onwards. Evaluation of bone loss trough μCT registration and measurement of extracellular cathepsin K activity revealed indications of enhanced resorption upon addition of PBMCs. Based on the results we suggest that an external source of osteoclast precursors, such as PBMCs, needs to be added in long-term bone explant cultures to maintain osteoclastic activity, and bone remodeling.

Conditional knockout of PDK1 in osteoclasts suppressed osteoclastogenesis and ameliorated prostate cancer-induced osteolysis in murine model

BackgroundThe development and maintenance of normal bone tissue is maintained by balanced communication between osteoblasts and osteoclasts. The invasion of cancer cells disrupts this balance, leading to osteolysis. As the only bone resorbing cells in vivo, osteoclasts play important roles in cancer-induced osteolysis. However, the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in osteoclast resorption remains unclear.MethodsIn our study, we used a receptor activator of nuclear factor-kappa B (RANK) promoter‐driven Cre‐LoxP system to conditionally delete the PDK1 gene in osteoclasts in mice. We observed the effect of osteoclast‐specific knockout of PDK1 on prostate cancer-induced osteolysis. Bone marrow-derived macrophage cells (BMMs) were extracted and induced to differentiate osteoclasts in vitro to explore the role of PDK1 in osteoclasts.ResultsIn this study, we found that PDK1 conditional knockout (cKO) mice exhibited smaller body sizes when compared to the wild-type (WT) mice. Moreover, deletion of PDK1 in osteoclasts ameliorated osteolysis and rPDK1educed bone resorption markers in the murine model of prostate cancer-induced osteolysis. In vivo, we discovered that osteoclast‐specific knockout of suppressed RANKL-induced osteoclastogenesis, bone resorption function, and osteoclast-specific gene expression (Ctsk, TRAP, MMP-9, NFATc1). Western blot analyses of RANKL-induced signaling pathways showed that conditional knockout of PDK1 in osteoclasts inhibited the early nuclear factor κB (NF-κB) activation, which consequently suppressed the downstream induction of NFATc1.ConclusionThese findings demonstrated that PDK1 performs an important role in osteoclastogenesis and prostate cancer-induced osteolysis by modulating the PDK1/AKT/NF-κB signaling pathway.

SAT260 Asymptomatic Vitamin A Toxicity Causing Hypercalcemia

Abstract Disclosure: N. Seshan: None. G. Goswami: None. Background: Vitamin A toxicity is an overlooked cause of hypercalcemia. Most cases of hypercalcemia are secondary to primary hyperparathyroidism or malignancy. High levels of Vitamin A are thought to have a direct effect on the bone by stimulating osteoclast resorption or inhibiting osteoblastic formation, therefore increasing the fracture risk. Clinical Case: 56 year old female with history of alcoholic cirrhosis complicated by hepatic encephalopathy, acute kidney injury and now status post liver transplant five months prior that was admitted for asymptomatic hypercalcemia. Initially, hypercalcemia was thought to be secondary to cyclosporine, so medication was discontinued. Hypercalcemia resolved after hydration however she was again readmitted for hypercalcemia found on routine labs. She did not show any other signs of excess vitamin A. Lab work showed calcium 12.5 mg/dL (8.6 - 10.2mg/dL), albumin 4g/dL (3.4 - 4.8g/dL), normal 24 hour urine calcium excretion, eGFR 33mL/min, parathyroid hormone 16.9 pg/mL (8.7-77pg/mL), normal 1,25-OH and 25-OH vitamin D, PTHrP 25pmol/L, AM Cortisol 9mcg/dL, TSH 1.7 mIU/L (0.35 - 4.7mIU/L), serum protein electrophoresis unremarkable. Her vitamin A level was elevated to 113 ug/dL (30-75mcg/dL) and was diagnosed with hypercalcemia due to elevated vitamin A levels. Treatment involved hydration and discontinuation of her multivitamin containing vitamin A. Current limited literature suggests discontinuing vitamin A supplements will normalize vitamin A levels over time. Her vitamin A levels improved over several months. Incidentally, due to complaints of back aches from prior injury, imaging showed acute compression fracture deformities of two thoracic vertebrae. Bone density scan showed normal bone density. Bone turnover markers were elevated indicative of increased bone resorption. Clinical Lesson/Conclusion: Hypervitaminosis A is a rare etiology of hypercalcemia. Vitamin A toxicity may be due to excess ingestion of preformed vitamin A through supplements and animal sources. Though not well understood, the proposed mechanism of vitamin A actions on the bone involves stimulation of bone resorption through increase in osteoclastic activity and inhibition of bone formation through osteoblastic suppression, leading to high serum calcium and increasing the risk of fractures. We present a unique case in a patient who developed hypercalcemia secondary to hypervitaminosis A. Presentation: Saturday, June 17, 2023

Influence of the sintering atmosphere on the physico-chemical properties and the osteoclastic resorption of β-tricalcium phosphate cylinders.

One of the most widely used materials for bone graft substitution is β-Tricalcium phosphate (β-TCP; β-Ca3(PO4)2). β-TCP is typically produced by sintering in air or vacuum. During this process, evaporation of phosphorus (P) species occurs, leading to the formation of a calcium-rich alkaline layer. It was recently shown that the evaporation of P species could be prevented by co-sintering β-TCP with dicalcium phosphate (DCPA; CaHPO4; mineral name: monetite). The aim of this study was to see how a change of sintering atmosphere could affect the physico-chemical and biological properties of β-TCP. For this purpose, three experimental groups were considered: β-TCP cylinders sintered in air and subsequently polished to remove the surface layer (control group); the same polished cylinders after subsequent annealing at 500°C in air to generate a calcium-rich alkaline layer (annealed group); and finally, β-TCP cylinders sintered in a monetite-rich atmosphere and subsequently polished (monetite group). XPS analysis confirmed that cylinders from the annealed group had a significantly higher Ca/P molar ratio at their surface than that of the control group while this ratio was significantly lower for the cylinders from the monetite group. Sintering β-TCP in the monetite-rich atmosphere significantly reduced the grain size and increased the density. Changes of surface composition affected the activity of osteoclasts seeded onto the surfaces, since annealed β-TCP cylinders were significantly less resorbed than β-TCP cylinders sintered in the monetite-rich atmosphere. This suggests that an increase of the surface Ca/P molar ratio leads to a decrease of osteoclastic resorption. STATEMENT OF SIGNIFICANCE: Minimal changes of surface and bulk (< 1%) composition have major effects on the ability of osteoclasts to resorb β-tricalcium phosphate (β-TCP), one of the most widely used ceramics for bone substitution. The results presented in this study are thus important for the calcium phosphate community because (i) β-TCP may have up to 5% impurities according to ISO and ASTM standards and still be considered to be "pure β-TCP", (ii) β-TCP surface properties are generally not considered during biocompatibility assessment and (iii) a rationale can be proposed to explain the various inconsistencies reported in the literature on the biological properties of β-TCP.

Comparative effects of various running exercise modalities on femoral bone quality in rats.

It is now well established that physical exercise is an effective preventive method to reduce and treat certain chronic diseases, particularly musculoskeletal disorders. At the bone level, running exercise is well known for its positive effects on various parameters of bone quality. There is, however, no consensus regarding the effects of different running exercise modalities on bone quality. The objective of this study was to compare the effects of three treadmill running modalities: intermittent, moderate continuous, and a combination of both-on bone quality parameters in rats. Thirty-nine, 5-week-old, male Wistar rats were randomly divided in 4 groups: sedentary control (SED; n = 10), intermittent running exercise (IE; n = 10), continuous running exercise (CE; n = 10) and combined running exercise (COME; n = 9). Rats in running groups were exercised 45min/day, 5days/week, for 8 consecutive weeks. Femoral micro-architectural parameters were assessed by micro-CT; femoral osteocyte apoptosis, osteoclast resorption and bone histomorphometry were assessed by histology. Femoral trabecular thickness in the combined running group was increased (p < 0.0001) compared to respective results in the other running groups (0.13mm vs 0.11mm). The cortical thickness, osteocyte lacunae occupancy rate in the whole femur, numbers of apoptotic osteocytes and osteoclastic resorption surfaces were not significantly different between groups. Statistical differences were occasionally noted depending on the femoral anatomical region. These results suggest that the femur should not be considered as the better bone to study the effects of running protocols.

Garlic extract enhances bioceramic bone scaffolds through upregulating ALP & BGLAP expression in hMSC-monocyte co-culture

Bone homeostasis is predicated by osteoblast and osteoclast cell cycles where gene expressions are responsible for their differentiation from human mesenchymal stem cells (hMSC) and monocytes, respectively. The pro-osteogenic potential of an hMSC-monocyte co-culture can be measured through complementary DNA (mRNA synthesis) within the nucleus, known as quantitative polymerase chain reaction (qPCR). Through this technique, the effects of garlic extract (allicin) release from calcium phosphate bone scaffolds on gene expression of bone forming and bone remodeling cells was explored. Results show this complex biomaterial system enhances hMSC differentiation through the upregulation of bone-forming proteins. Osteoblastic gene markers alkaline phosphatase (ALP) and osteocalcin (BGLAP), are respectively upregulated by 3-fold and 1.6-fold by day 14. These mature osteoblasts then upregulate the receptor activator of nuclear factor-kB ligand (RANKL) which recruits osteoclast cells, as captured by a nearly 2-fold higher osteoclast expression of tartrate-resistance acid-phosphatase (ACP5). This also activates antagonist osteoprotegerin (OPG) expression in osteoblasts, decreasing osteoclast resorption potential and ACP5 expression by day 21. The pro-osteogenic environment with garlic extract release is further quantified by a 4× increase in phosphatase activity and visibly captured in immunofluorescent tagged confocal images. Also corroborated by enhanced collagen formation in a preliminary in vivo rat distal femur model, this work collectively reveals how garlic extract can enhance bioceramic scaffolds for bone tissue regenerative applications.

Abr, a Rho-regulating protein, modulates osteoclastogenesis by enhancing lamellipodia formation by interacting with poly(ADP-ribose) glycohydrolase.

Osteoclasts are multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage lineage. During osteoclast differentiation, Rho GTPases are involved in various processes, including cell migration, adhesion, and polarity. However, the role of Rho-regulatory molecules in the regulation of osteoclast differentiation remains unclear. In this study, among these genes, we focused on active breakpoint cluster region-related (Abr) protein that is a multifunctional regulator of Rho GTPases. We examined using knockdown and overexpression experiments in RANKL-stimulated RAW-D macrophages whether Abr regulates osteoclast differentiation and cell morphology. We observed an increase in Abr expression during osteoclast differentiation and identified expression of a variant of the Abr gene in osteoclasts. Knockdown of Abr suppressed osteoclast differentiation and resorption. Abr knockdown markedly inhibited the expression of osteoclast markers, such as Nfatc1, c-fos, Src, and Ctsk in osteoclasts. Conversely, overexpression of Abr enhanced the formation of multinucleated osteoclasts, bone resorption activity, and osteoclast marker gene expression. Moreover, Abr overexpression accelerated lamellipodia formation and induced the formation of well-developed actin in osteoclasts. Importantly, the Abr protein interacted with poly(ADP-ribose) glycohydrolase (PARG) and Rho GTPases, including RhoA, Rac1/2/3, and Cdc42 in osteoclasts. Taken together, these results indicate that Abr modulates osteoclastogenesis by enhancing lamellipodia formation via its interaction with PARG.

Cilengitide inhibits osteoclast adhesion through blocking the αvβ3-mediated FAK/Src signaling pathway

The remodeling of actin cytoskeleton of osteoclasts on the bone matrix is essential for osteoclastic resorption activity. A specific regulator of the osteoclast cytoskeleton, integrin αvβ3, is known to provide a key role in the degradation of mineralized bone matrixes. Cilengitide is a potent inhibitor of integrins and is capable of affecting αvβ3 receptors, and has anti-tumor and anti-angiogenic and apoptosis-inducing effects. However, its function on osteoclasts is not fully understood. Here, the cilengitide role on nuclear factor κB ligand-receptor activator (RANKL)-induced osteoclasts was explored. Cells were cultured with varying concentrations of cilengitide (0,0.002,0.2 and 20 μM) for 7 days, followed by detected via Cell Counting Kit-8, staining for tartrate resistant acid phosphatase (TRAP), F-actin ring formation, bone resorption assays, adhesion assays, immunoblotting assays, and real-time fluorescent quantitative PCR. Results demonstrated that cilengitide effectively restrained the functionality and formation of osteoclasts in a concentration-dependent manner, without causing any cytotoxic effects. Mechanistically, cilengitide inhibited osteoclast-relevant genes expression; meanwhile, cilengitide downregulated the expression of key signaling molecules associated with the osteoclast cytoskeleton, including focal adhesion kinase (FAK), integrin αvβ3 and c-Src. Therefore, this results have confirmed that cilengitide regulates osteoclast activity by blocking the integrin αvβ3 signal pathway resulting in diminished adhesion and bone resorption of osteoclasts.