Vitamin A enhanced periosteal osteoclastogenesis is associated with increased number of tissue-derived macrophages/osteoclast progenitors.

A deleterious effect of elevated levels of vitamin A on bone health has been reported in clinical studies. Mechanistic studies in rodents have shown that numbers of periosteal osteoclasts are increased, while endocortical osteoclasts are simultaneously decreased by vitamin A treatment. The present study investigated the in vitro and in vivo effect of all-trans retinoic acid (ATRA), the active metabolite of vitamin A, on periosteal osteoclast progenitors. Mouse calvarial bone cells were cultured in media containing ATRA, with or without the osteoclastogenic cytokine receptor activator of nuclear factor kappa B-ligand (RANKL), on plastic dishes or bone discs. Whereas ATRA did not stimulate osteoclast formation alone, the compound robustly potentiated the formation of RANKL-induced bone resorbing osteoclasts. This effect was due to stimulation by ATRA (half-maximal stimulation ∼3 nM) on the numbers of macrophages/osteoclast progenitors in the bone cell cultures, as assessed by mRNA and protein expression of several macrophage and osteoclast progenitor cell markers, such as macrophage colony-stimulating factor receptor, receptor activator of nuclear factor kappa B, F4/80, and CD11b, as well as by flow cytometry (FACS) analysis of CD11b+/F480+/Gr1- cells. The stimulation of macrophage numbers in the periosteal cell cultures was not mediated by increased macrophage colony-stimulating factor or interleukin-34. In contrast, ATRA did not enhance macrophages in bone marrow cell cultures. Importantly, ATRA treatment upregulated the mRNA expression of several macrophage-related genes in the periosteum of tibia in adult mice. These observations demonstrate a novel mechanism by which vitamin A enhances osteoclast formation specifically on periosteal surfaces.

NOTCH2 promotes osteoclast maturation and metabolism and modulates the transcriptome profile during osteoclastogenesis.

Notch signaling plays a key regulatory role in bone remodeling and NOTCH2 enhances osteoclastogenesis, an effect that is mostly mediated by its target gene Hes1. In the present study, we explored mechanisms responsible for the enhanced osteoclastogenesis in bone marrow-derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice. Notch2tm1.1Ecan mice are osteopenic and have enhanced osteoclastogenesis. Bulk RNA-Seq and gene set enrichment analysis of Notch2tm1.1Ecan BMMs cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand revealed enrichment of genes associated with enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis. These pathways were not enhanced in the context of a Hes1 inactivation. Analysis of single cell RNA-Seq data of pooled control and Notch2tm1.1Ecan BMMs treated with M-CSF or M-CSF and receptor activator of NF-κB ligand for 3 days identified 11 well-defined cellular clusters. Pseudotime trajectory analysis indicated a trajectory of clusters expressing genes associated with osteoclast progenitors, osteoclast precursors, and mature cells. There were an increased number of cells expressing gene markers associated with the osteoclast and with an unknown, albeit related, cluster in Notch2tm1.1Ecan than in control BMMs as well as enhanced expression of genes associated with osteoclast progenitors and precursors in Notch2tm1.1Ecan cells. In conclusion, BMM cultures display cellular heterogeneity, and NOTCH2 enhances osteoclastogenesis, increases mitochondrial and metabolic activity of osteoclasts, and affects cell cluster allocation in BMMs.

MDSCs are important osteoclast precursors primed by B cells in rheumatoid arthritis.

Osteoclast-mediated bone erosion and deformation represent significant pathological features in rheumatoid arthritis (RA). Myeloid-derived suppressor cells (MDSCs) and B cells have emerged as key contributors to the progression of RA. Nevertheless, their involvement, especially the interaction in RA osteoclastogenesis remains elusive. In this study, our results revealed a marked expansion of MDSCs in RA patients, and importantly, their abundance was positively correlated with radiographic damage evaluated by the Sharp/van der Heijde score. Notably, MDSCs derived from both RA patients and arthritic mice exhibited a heightened propensity to differentiate into osteoclasts compared with those from healthy individuals. Intriguingly, we observed that B cells from RA patients could augment the osteoclastogenic potential of MDSCs, which was also observed in arthritic mice. The impact of B cells on MDSC-mediated osteoclastogenesis was found to be most pronounced in switched memory B cells, followed by CD21low B cells and naïve B cells. MDSCs from B-cell-deficient mice exhibited diminished capacity to differentiate into osteoclasts, accompanied by distinct gene expression profiles associated with osteoclastogenesis. Taken together, our findings suggested that MDSCs were important osteoclast precursors primed by B cells in RA, serving as novel therapeutic targets for the persistent disease.

Tristetraprolin regulates the skeletal phenotype and osteoclastogenic potential through monocytic myeloid-derived suppressor cells.

Tristetraprolin (TTP; also known as NUP475, GOS24, or TIS11), encoded by Zfp36, is an RNA-binding protein that regulates target gene expression by promoting mRNA decay and preventing translation. Although previous studies have indicated that TTP deficiency is associated with systemic inflammation and a catabolic-like skeletal phenotype, the mechanistic underpinnings remain unclear. Here, using both TTP-deficient (TTPKO) and myeloid-specific TTPKO (cTTPKO) mice, we reveal that global absence or loss of TTP in the myeloid compartment results in a reduced bone microarchitecture, whereas gain-of-function TTP knock-in (TTPKI) mice exhibit no significant loss of bone microarchitecture. Flow cytometry analysis revealed a significant immunosuppressive immune cell phenotype with increased monocytic myeloid-derived suppressor cells (M-MDSCs) in TTPKO and cTTPKO mice, whereas no significant changes were observed in TTPKI mice. Single-cell transcriptomic analyses of bone marrow myeloid progenitor cell populations indicated a dramatic increase in early MDSC marker genes for both cTTPKO and TTPKO bone marrow populations. Consistent with these phenotypic and transcriptomic data, in vitro osteoclastogenesis analysis of bone marrow M-MDSCs from cTTPKO and TTPKO displayed enhanced osteoclast differentiation and functional capacity. Focused transcriptomic analyses of differentiated M-MDSCs showed increased osteoclast-specific transcription factors and cell fusion gene expression. Finally, functional data showed that M-MDSCs from TTP loss-of-function mice were capable of osteoclastogenesis and bone resorption in a context-dependent manner. Collectively, these findings indicate that TTP plays a central role in regulating osteoclastogenesis through multiple mechanisms, including induction of M-MDSCs that appear to regulate skeletal phenotype.

Multinucleation resets human macrophages for specialized functions at the expense of their identity.

Macrophages undergo plasma membrane fusion and cell multinucleation to form multinucleated giant cells (MGCs) such as osteoclasts in bone, Langhans giant cells (LGCs) as part of granulomas or foreign-body giant cells (FBGCs) in reaction to exogenous material. How multinucleation per se contributes to functional specialization of mature mononuclear macrophages remains poorly understood in humans. Here, we integrate comparative transcriptomics with functional assays in purified mature mononuclear and multinucleated human osteoclasts, LGCs and FBGCs. Strikingly, in all three types of MGCs, multinucleation causes a pronounced downregulation of macrophage identity. We show enhanced lysosome-mediated intracellular iron homeostasis promoting MGC formation. The transition from mononuclear to multinuclear state is accompanied by cell specialization specific to each polykaryon. Enhanced phagocytic and mitochondrial function associate with FBGCs and osteoclasts, respectively. Moreover, human LGCs preferentially express B7-H3 (CD276) and can form granuloma-like clusters in vitro, suggesting that their multinucleation potentiates T cell activation. These findings demonstrate how cell-cell fusion and multinucleation reset human macrophage identity as part of an advanced maturation step that confers MGC-specific functionality.

Fin ray branching is defined by TRAP+ osteolytic tubules in zebrafish.

The shaping of bone structures relies on various cell types and signaling pathways. Here, we use the zebrafish bifurcating fin rays during regeneration to investigate bone patterning. We found that the regenerating fin rays form via two mineralization fronts that undergo an osteoblast-dependent fusion/stitching until the branchpoint, and that bifurcation is not simply the splitting of one unit into two. We identified tartrate-resistant acid phosphatase-positive osteolytic tubular structures at the branchpoints, hereafter named osteolytic tubules (OLTs). Chemical inhibition of their bone-resorbing activity strongly impairs ray bifurcation, indicating that OLTs counteract the stitching process. Furthermore, by testing different osteoactive compounds, we show that the position of the branchpoint depends on the balance between bone mineralization and resorption activities. Overall, these findings provide a unique perspective on fin ray formation and bifurcation, and reveal a key role for OLTs in defining the proximo-distal position of the branchpoint.

DOT1L decelerates the development of osteoporosis by inhibiting SRSF1 transcriptional activity via microRNA-181-mediated KAT2B inhibition.

OBJECTIVE: Our study explored the function of DOT1L in osteoporosis (OP) via the microRNA (miR)-181/KAT2B/SRSF1 axis. METHODS: Osteoclast (OC) number was evaluated via TRAP staining, and serum CTXI, PINP, and ALP contents were tested by ELISA. Following identification of bone marrow mesenchymal stem cells (BMSCs), OC differentiation was induced by M-CSF and RANKL, followed by the detection of OC differentiation and the expression of bone resorption-related genes, DOT1L, miR-181, KAT2B, and SRSF1. RESULTS: Overexpressed DOT1L or miR-181 stimulated calcified nodule formation and increased alkaline phosphatase activity and osteogenic marker gene expression. KAT2B knockdown enhanced the osteogenic differentiation of BMSCs by reducing SRSF1 acetylation. The enhancement of OC differentiation induced by overexpressed SRSF1 was inhibited by simultaneous DOT1L or miR-181 overexpression. DOT1L suppressed OP development in vivo via the miR-181/KAT2B/SRSF1 axis. CONCLUSION: DOT1L overexpression slowed down bone loss and promoted bone formation via the miR-181/KAT2B/SRSF1 axis, thereby alleviating OP development.

Mechanosensitivity and mechanotransductive properties of osteoclasts.

Mechanical loading is essential for maintaining bone health. Here, we aimed to investigate the role of ATP and ADP in the mechanotransduction of bone-resorptive osteoclasts. Single osteoclast in primary cultures from 10 to 12-wk-old mice was mechanically stimulated by a gentle touch with a micropipette. Changes in cytosolic free calcium [Ca2+]i were analyzed in Fura-2 loaded osteoclasts. The cell injury was assessed by analyzing the cellular Fura-2 loss and classified as severe or mild using k-means. Osteoclasts responded to mechanical stimuli with transient calcium elevation (primary responders) and transduced these signals to neighboring cells, which responded with delayed calcium elevations (secondary responders). Severely injured osteoclasts had higher calcium transients than mildly injured cells. Fluid shear stress similarly induced reversible cell injury in osteoclasts. Secondary responses were abolished by treatment with A-804598, a specific inhibitor of P2X7, but not suramin, a broad P2 receptor blocker. Osteoclasts responded to ATP and ADP with concentration-dependent changes in [Ca2+]i. We performed osteoclast micropipette stimulation in the presence of phosphoenolpyruvate and pyruvate kinase which converted all ADP in solution to ATP, or with hexokinase converting all ATP to ADP. Osteoclasts with mild membrane injury demonstrated similar calcium responses in ATP and ADP-rich environments. However, when the mechanotransductive signal to severe osteoclast injury was converted to ADP, the fraction of secondary responders and their [Ca2+]i amplitude was higher. This study suggests the importance of osteoclast mechanobiology and the role of ADP-mediated signaling in conditions of altered mechanical loading associated with bone loss.NEW & NOTEWORTHY Osteoclasts are rarely considered as cells that participate in mechanical signaling in bone. We show that osteoclasts are capable of sensing and transmitting mechanical signals to neighboring cells. Mechanical stimulation commonly induces minor repairable membrane injury in osteoclasts. ATP and especially ADP were found to play important roles in the mechanoresponsiveness of osteoclasts. This study highlights the importance of osteoclast mechanobiology especially in conditions of altered mechanical loading associated with bone loss, such as in microgravity.

Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice.

Hem1 (hematopoietic protein 1), a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins, is essential for lymphopoiesis and innate immunity as well as for the transition of hematopoiesis from the fetal liver to the bone marrow. However, the role of Hem1 in bone cell differentiation and bone remodeling is unknown. Here, we show that deletion of Hem1 resulted in a markedly increase in bone mass because of defective bone resorption in mice of both sexes. Hem1-deficient osteoclast progenitors were able to differentiate into osteoclasts, but the osteoclasts exhibited impaired osteoclast fusion and decreased bone-resorption activity, potentially because of decreased mitogen-activated protein kinase and tyrosine kinase c-Abl activity. Transplantation of bone marrow hematopoietic stem and progenitor cells from wildtype into Hem1 knockout mice increased bone resorption and normalized bone mass. These findings indicate that Hem1 plays a pivotal role in the maintenance of normal bone mass.

Interleukin-35 protein inhibits osteoclastogenesis and attenuates collagen-induced arthritis in mice.

Rheumatoid arthritis (RA) is a chronic autoimmune disease. Its pathological features include synovial inflammation, bone erosion, and joint structural damage. Our previous studies have shown that interleukin (IL)-35 is involved in the pathogenesis of bone loss in RA patients. In this study, we are further evaluating the efficacy of IL-35 on collagen-induced arthritis (CIA) in the mouse model. Male DBA/1J mice (n = 10) were initially immunized, 2 µg/mouse IL-35 was injected intraperitoneally every week for 3 weeks after the establishment of the CIA model. Clinical arthritis, histopathological analysis, and three-dimensional micro-computed tomography (3D micro-CT) were determined after the mice were anesthetized on the 42th day. In vitro, RANKL/M-CSF induced mouse preosteoclasts (RAW264.7 cells line) was subjected to antiarthritis mechanism study in the presence of IL-35. The results of clinical arthritis, histopathological analysis, and 3D micro-CT, the expression of RANK/RANKL/OPG axis, inflammatory cytokines, and osteoclastogenesis-related makers demonstrated decreasing severity of synovitis and bone destruction in the ankle joints after IL-35 treatment. Furthermore, IL-35 attenuated inflammatory cytokine production and the expression of osteoclastogenesis-related makers in a mouse preosteoclasts cell line RAW264.7. The osteoclastogenesis-related makers were significantly reduced in IL-35 treated RAW264.7 cells line after blockage with the JAK/STAT1 signaling pathway. These results demonstrated that IL-35 protein could inhibits osteoclastogenesis and attenuates CIA in mice. We concluded that IL-35 can exhibit anti-osteoclastogenesis effects by reducing the expression of inflammatory cytokines and osteoclastogenesis-related makers, thus alleviating bone destruction in the ankle joint and could be a potential therapeutic target for RA.

Inhibition of caspase-8 cascade restrains the osteoclastogenic fate of bone marrow cells.

Osteoclasts are multinucleated cells of hematopoietic origin, with a pivotal role in bone development and remodeling. Failure in osteoclast differentiation and activation leads to various bone disorders; thus, attention has focused on a search of molecules involved in osteoclast regulatory pathways. Caspase-8 appears to be an interesting candidate for further exploration, due to its potential function in bone development and homeostasis. Mouse bone marrow cells were differentiated into osteoclasts by RANKL stimulation. Increased activation of caspase-8 and its downstream executioner caspases (caspase-3 and caspase-6) was found during osteoclastogenesis. Subsequent inhibition of caspase-8, caspase-3, or caspase-6, respectively, during osteoclast differentiation showed distinct changes in the formation of TRAP-positive multinucleated cells and reduced expression of osteoclast markers including Acp5, Ctsk, Dcstamp, and Mmp9. Analysis of bone matrix resorption confirmed significantly reduced osteoclast function after caspase inhibition. The results clearly showed the role of caspases in the proper development of osteoclasts and contributed new knowledge about non-apoptotic function of caspases.

mmu-miR-185 regulates osteoclasts differentiation and migration by targeting Btk.

BACKGROUND: Bones undergo a constant remodeling, a process involving osteoclast-mediated bone resorption and osteoblast-mediated bone formation, crucial for maintaining healthy bone mass. We previously observed that miR-185 depletion may promote bone formation by regulating Bgn expression and the BMP/Smad signaling pathway. However, the effects of miR-185-5p on the osteoclasts and bone remodeling have not been elucidated, warranting further exploration. METHODS: Tartrate-resistant acid phosphatase staining was utilized to assess the differentiation ability of bone marrow mononuclear macrophages (BMMs) from mmu-miR-185 gene knockout (KO) mice and wild-type (WT) mice. A reverse transcriptase-quantitative PCR was conducted to compare differences in miR-185-5p and osteoclast marker molecules, including Trap, Dcstamp, Ctsk and Nfatc1, between the KO group and WT group BMMs. Western blot analysis was employed to observe the expression of osteoclast marker molecules. A cell-counting kit-8 was used to analyze cell proliferation ability. Transwell experiments were conducted to detect cell migration. Dual-luciferase reporter assays were employed to confirm whether Btk is a downstream target gene of miR-185-5p. RESULTS: miR-185 depletion promoted osteoclast differentiation in bone marrow-derived monocytes/macrophages. Overexpression of miR-185-5p in RAW264.7 cells inhibited differentiation and migration of osteoclasts. Furthermore, Btk was identified as a downstream target gene of miR-185-5p, suggesting that miR-185-5p may inhibit osteoclast differentiation and migration by targeting Btk. CONCLUSIONS: miR-185 regulates osteoclasts differentiation, with overexpression of miR-185-5p inhibiting osteoclast differentiation and migration in vitro. Additionally, miR-185-5p may modulate osteoclastic differentiation and migration by regulating Btk expression.

Impdh2 deficiency suppresses osteoclastogenesis through mitochondrial oxidative phosphorylation and alleviates ovariectomy-induced osteoporosis.

Abnormalities in osteoclastic generation or activity disrupt bone homeostasis and are highly involved in many pathologic bone-related diseases, including rheumatoid arthritis, osteopetrosis, and osteoporosis. Control of osteoclast-mediated bone resorption is crucial for treating these bone diseases. However, the mechanisms of control of osteoclastogenesis are incompletely understood. In this study, we identified that inosine 5'-monophosphate dehydrogenase type II (Impdh2) positively regulates bone resorption. By histomorphometric analysis, Impdh2 deletion in mouse myeloid lineage cells (Impdh2LysM-/- mice) showed a high bone mass due to the reduced osteoclast number. qPCR and western blotting results demonstrated that the expression of osteoclast marker genes, including Nfatc1, Ctsk, Calcr, Acp5, Dcstamp, and Atp6v0d2, was significantly decreased in the Impdh2LysM-/- mice. Furthermore, the Impdh inhibitor MPA treatment inhibited osteoclast differentiation and induced Impdh2-cytoophidia formation. The ability of osteoclast differentiation was recovered after MPA deprivation. Interestingly, genome-wide analysis revealed that the osteoclastic mitochondrial biogenesis and functions, such as oxidative phosphorylation, were impaired in the Impdh2LysM-/- mice. Moreover, the deletion of Impdh2 alleviated ovariectomy-induced bone loss. In conclusion, our findings revealed a previously unrecognized function of Impdh2, suggesting that Impdh2-mediated mechanisms represent therapeutic targets for osteolytic diseases.

Unique cartilage matrix-associated protein inhibits osteoclast differentiation by alleviating RANKL-induced reactive oxygen species.

Unique cartilage matrix-associated protein (UCMA) is a γ-carboxyglutamic acid-rich secretory protein primarily expressed in adult cartilage. UCMA promotes osteoblast differentiation and reduces high glucose-induced reactive oxygen species (ROS) production in osteoblasts; however, its role in osteoclasts remains unclear. Since Ucma is not expressed in osteoclasts, treatment with recombinant UCMA protein (rUCMA) was employed to investigate the effect of UCMA on osteoclasts. The rUCMA-treated osteoclasts exhibited significantly reduced osteoclast differentiation, resorption activity, and osteoclast-specific gene expression. Moreover, rUCMA treatment reduced RANKL-induced ROS production and increased the expression of antioxidant genes in osteoclasts. This study demonstrates that UCMA effectively inhibits RANKL-stimulated osteoclast differentiation and oxidative stress.

A novel method to efficiently differentiate human osteoclasts from blood-derived monocytes.

BACKGROUND: Osteoclasts are the tissue-specific macrophage population of the bone and unique in their bone-resorbing activity. Hence, they are fundamental for bone physiology in health and disease. However, efficient protocols for the isolation and study of primary human osteoclasts are scarce. In this study, we aimed to establish a protocol, which enables the efficient differentiation of functional human osteoclasts from monocytes. RESULTS: Human monocytes were isolated through a double-density gradient from donor blood. Compared to standard differentiation schemes in polystyrene cell culture dishes, the yield of multinuclear osteoclasts was significantly increased upon initial differentiation of monocytes to macrophages in fluorinated ethylene propylene (FEP) Teflon bags. This initial differentiation phase was then followed by the development of terminal osteoclasts by addition of Receptor Activator of NF-κB Ligand (RANKL). High concentrations of RANKL and Macrophage colony-stimulating factor (M-CSF) as well as an intermediate cell density further supported efficient cell differentiation. The generated cells were highly positive for CD45, CD14 as well as the osteoclast markers CD51/ITGAV and Cathepsin K/CTSK, thus identifying them as osteoclasts. The bone resorption of the osteoclasts was significantly increased when the cells were differentiated from macrophages derived from Teflon bags compared to macrophages derived from conventional cell culture plates. CONCLUSION: Our study has established a novel protocol for the isolation of primary human osteoclasts that improves osteoclastogenesis in comparison to the conventionally used cultivation approach.

The abortive SARS-CoV-2 infection of osteoclast precursors promotes their differentiation into osteoclasts.

The Coronavirus Disease 2019 (COVID-19) pandemic has resulted in the loss of millions of lives, although a majority of those infected have managed to survive. Consequently, a set of outcomes, identified as long COVID, is now emerging. While the primary target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the respiratory system, the impact of COVID-19 extends to various body parts, including the bone. This study aims to investigate the effects of acute SARS-CoV-2 infection on osteoclastogenesis, utilizing both ancestral and Omicron viral strains. Monocyte-derived macrophages, which serve as precursors to osteoclasts, were exposed to both viral variants. However, the infection proved abortive, even though ACE2 receptor expression increased postinfection, with no significant impact on cellular viability and redox balance. Both SARS-CoV-2 strains heightened osteoclast formation in a dose-dependent manner, as well as CD51/61 expression and bone resorptive ability. Notably, SARS-CoV-2 induced early pro-inflammatory M1 macrophage polarization, shifting toward an M2-like profile. Osteoclastogenesis-related genes (RANK, NFATc1, DC-STAMP, MMP9) were upregulated, and surprisingly, SARS-CoV-2 variants promoted RANKL-independent osteoclast formation. This thorough investigation illuminates the intricate interplay between SARS-CoV-2 and osteoclast precursors, suggesting potential implications for bone homeostasis and opening new avenues for therapeutic exploration in COVID-19.

The novel cytotoxic polybisphosphonate osteodex decreases bone resorption by enhancing cell death of mature osteoclasts without affecting osteoclastogenesis of RANKL-stimulated mouse bone marrow macrophages.

It has previously been demonstrated that the polybisphosphonate osteodex (ODX) inhibits bone resorption in organ-cultured mouse calvarial bone. In this study, we further investigate the effects by ODX on osteoclast differentiation, formation, and function in several different bone organ and cell cultures. Zoledronic acid (ZOL) was used for comparison. In retinoid-stimulated mouse calvarial organ cultures, ODX and ZOL significantly reduced the numbers of periosteal osteoclasts without affecting Tnfsf11 or Tnfrsf11b mRNA expression. ODX and ZOL also drastically reduced the numbers of osteoclasts in cell cultures isolated from the calvarial bone and in vitamin D3-stimulated mouse crude bone marrow cell cultures. These data suggest that ODX can inhibit osteoclast formation by inhibiting the differentiation of osteoclast progenitor cells or by directly targeting mature osteoclasts. We therefore assessed if osteoclast formation in purified bone marrow macrophage cultures stimulated by RANKL was inhibited by ODX and ZOL and found that the initial formation of mature osteoclasts was not affected, but that the bisphosphonates enhanced cell death of mature osteoclasts. In agreement with these findings, ODX and ZOL did not affect the mRNA expression of the osteoclastic genes Acp5 and Ctsk and the osteoclastogenic transcription factor Nfatc1. When bone marrow macrophages were incubated on bone slices, ODX and ZOL inhibited RANKL-stimulated bone resorption. In conclusion, ODX does not inhibit osteoclast formation but inhibits osteoclastic bone resorption by decreasing osteoclast numbers through enhanced cell death of mature osteoclasts.

Two Weeks of Continuous Opioid Treatment in an Adenine-Induced Mouse Model of Chronic Kidney Disease Exacerbates the Bone Inflammatory State and Increases Osteoclasts.

Patients with chronic kidney disease (CKD) report high pain levels, but reduced renal clearance eliminates many analgesic options; therefore, 30-50% of CKD patients have chronic opioid prescriptions. Opioid use in CKD is associated with higher fracture rates. Opioids may directly alter bone turnover directly through effects on bone cells and indirectly via increasing inflammation. We hypothesized that continuous opioid exposure would exacerbate the high bone turnover state of CKD and be associated with elevated measures of inflammation. Male C57Bl/6J mice after 8 weeks of adenine-induced CKD (AD) and non-AD controls (CON) had 14-day osmotic pumps (0.25-µL/hr release) containing either saline or 50-mg/mL oxycodone (OXY) surgically implanted in the subscapular region. After 2 weeks, all AD mice had elevated blood urea nitrogen, parathyroid hormone, and serum markers of bone turnover compared to controls with no effect of OXY. Immunohistochemical staining of the distal femur showed increased numbers of osteocytes positive for the mu opioid and for toll-like receptor 4 (TLR4) due to OXY. Osteocyte protein expression of tumor necrosis factor-α (TNF-α) and RANKL were higher due to both AD and OXY so that AD + OXY mice had the highest values. Trabecular osteoclast-covered surfaces were also significantly higher due to both AD and OXY, resulting in AD + OXY mice having 4.5-fold higher osteoclast-covered surfaces than untreated CON. These data demonstrate that opioids are associated with a pro-inflammatory state in osteocytes which increases the pro-resorptive state of CKD.

Etiological Mechanisms and Genetic/Biological Modulation Related to PTH1R in Primary Failure of Tooth Eruption.

Primary failure of eruption (PFE) is a rare disorder that is characterized by the inability of a molar tooth/teeth to erupt to the occlusal plane or to normally react to orthodontic force. This condition is related to hereditary factors and has been extensively researched over many years. However, the etiological mechanisms of pathogenesis are still not fully understood. Evidence from studies on PFE cases has shown that PFE patients may carry parathyroid hormone 1 receptor (PTH1R) gene mutations, and genetic detection can be used to diagnose PFE at an early stage. PTH1R variants can lead to altered protein structure, impaired protein function, and abnormal biological activities of the cells, which may ultimately impact the behavior of teeth, as observed in PFE. Dental follicle cells play a critical role in tooth eruption and root development and are regulated by parathyroid hormone-related peptide (PTHrP)-PTH1R signaling in their differentiation and other activities. PTHrP-PTH1R signaling also regulates the activity of osteoblasts, osteoclasts and odontoclasts during tooth development and eruption. When interference occurs in the PTHrP-PTH1R signaling pathway, the normal function of dental follicles and bone remodeling are impaired. This review provides an overview of PTH1R variants and their correlation with PFE, and highlights that a disruption of PTHrP-PTH1R signaling impairs the normal process of tooth development and eruption, thus providing insight into the underlying mechanisms related to PTH1R and its role in driving PFE.

Part II: A new perspective for modeling the bone remodeling process: Biology, mechanics, and pathologies.

In this paper, we explore the effects of biological (pathological) and mechanical damage on bone tissue within a benchmark model. Using the Finite Element Methodology, we analyze and numerically test the model's components, capabilities, and performance under physiologically and pathologically relevant conditions. Our findings demonstrate the model's effectiveness in simulating bone remodeling processes and self-repair mechanisms for micro-damage induced by biological internal conditions and mechanical external ones within bone tissue. This article is the second part of a series, where the first part presented the mathematical model and the biological and physical significance of the terms used in a simplified benchmark model. It explored the bone remodeling model's application, implementation, and results under physiological conditions.