Generation Of Osteoclasts Research Articles

OPG gene-modified adipose-derived stem cells improve bone formation around implants in osteoporotic rat maxillae

BackgroundOsteoporosis is a significant barrier to the use of dental implants in the elderly for the treatment of tooth defects. Adipose derived stem cells (ADSCs) have demonstrated extensive potential for tissue repair and regeneration. The present study aimed to investigate the effectiveness of ADSCs engineered to express high levels of osteoprotegerin (OPG) for the treatment of bone loss in implant dentistry caused by estrogen deficiency. MethodsA rat model of osteoporosis was established through double oophorectomy, and the rats were treated by gene modified cells Adv-OPG-ADSCs. The effects of the treatment on maxilla tissue changes were evaluated using HE staining and micro-CT. Additionally, ALP and TRAP staining were used to assess osteoblast and osteoclast alterations. Finally, the changes in related osteoblast and osteoclast indicators were measured by RT-qPCR, Western blot, and ELISA. ResultsThe successfully generated high-OPG-expressing ADSCs led to increase of cell viability, proliferation, and osteoblast differentiation. Treatment with Adv-OPG-ADSCs significantly ameliorated maxillary morphology, trabecular volume reduction, and bone mineral density decline in the model of estrogen-deficient maxillary implant dentistry. Furthermore, the treatment was beneficial to promoting the generation of osteoblasts and inhibiting the generation of osteoclast. Adv-OPG-ADSCs increased OPG, ALP, OCN, and Runx-2 expressions in the maxilla while suppressing RANKL expression, and also increased the concentration of COL I and PINP, as well as decreased the concentration of CTX-1. ConclusionAdv-OPG-ADSCs promote the formation of osteoblasts and inhibit the generation of osteoclasts, thereby inhibiting bone absorption, facilitating bone formation, and promoting the repair of maxillary bone after dental implantation in the presence of osteoporosis-related complications, especially in the setting of estrogen deficiency, providing scientific basis for the application of Adv-OPG-ADSCs in the treatment of implant related osteoporosis.

Evaluation of Biological Mechanisms of Quanduzhong Capsule for Treating Osteoporosis by Integrating Untargeted Metabolomics and Network Pharmacology

AbstractOsteoporosis (OP) is a metabolic disease characterized by bone formation and resorption disturbances. Quanduzhong Capsule (QDZC) is a common treatment for OP in China; however, the effective components and metabolites of the drug after oral administration remain largely unknown. This study aims to identify the active components, analyze the metabolite changes, and investigate the underlying mechanism against OP. In the study, ovariectomy-induced rat OP model was established, then treated with QDZC. Alendronate sodium tablets (ASTs) were used as a reference drug. The chemical constituents of QDZC were analyzed by UPLC-QTOF-MS (ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry) and network pharmacology. The metabolomics was used to analyze differences in serum metabolites of rats in different groups [Sham, Model, Model + QDZC, and Model + AST] at 4, 8, and 12 weeks. Body weight and bone mineral density (BMD) were assessed. Enzyme-linked immunosorbent assay was used to determine serum levels of Akt, p-Akt, ERK, and p-ERK. Our data suggested 86 different chemicals from QDZC, including nine core compounds. QDZC significantly regulated 25 biomarkers linked to arachidonic acid metabolism and unsaturated fatty acid biosynthesis, and promoted serum expression of Akt, p-Akt, ERK, and p-ERK. QDZC might act by activating PI3K-Akt and MAPK signaling pathways. In addition, QDZC may use arachidonic acid derivatives to inhibit osteoclast generation and bone resorption and enhance calcitriol formation to improve calcium absorption and increase bone mass.

Guizhi Shaoyao Zhimu granules attenuate bone destruction in mice with collagen-induced arthritis by promoting mitophagy of osteoclast precursors to inhibit osteoclastogenesis

BackgroundGuizhi Shaoyao Zhimu decoction, a traditional Chinese medicine formula used empirically for the treatment of rheumatoid arthritis (RA), has been shown to alleviate bone destruction in rats with collagen-induced arthritis (CIA). PurposeThe aim of this study is to characterize the effects of Guizhi Shaoyao Zhimu granules (GSZGs) on bone destruction in RA and the underlying mechanism. Study designA CIA arthritis model using DBA/1 mice. The animals were divided into a normal group; CIA model group; low, medium, and high-dose GSZG groups (3, 6, and 9 g/kg/day); and a methotrexate group (1.14 mg/kg/w). In vitro, a cytokine induced osteoclastogenesis model was established. MethodsAfter 28 days of treatment, the paw volume was measured, bone destruction was examined by micro-CT, and the generation of osteoclasts in bone tissue was evaluated via tartrate-resistant acid phosphatase (TRAP) staining. Furthermore, the inhibitory effect and underlying mechanism of action of GSZG on RANKL-induced osteoclastogenesis were investigated in vitro. ResultsThe in vivo analyses demonstrated that the paw volume and degree of bone erosion of mice in the medium- and high-dose GSZG groups were significantly decreased compared to the CIA model group. In addition, GSZG treatment suppressed the excessive generation of osteoclasts in the bone tissue of CIA mice. In vitro, GSZG inhibited RANKL-induced osteoclastogenesis and osteoclast-mediated bone resorption. Specifically, it only inhibited the generation of osteoclast precursors (OCPs); it had no significant effect on the fusion of OCPs or maturation of osteoclasts. Finally, we showed that the inhibitory effect of GSZG on osteoclastogenesis was related to the promotion of PTEN-induced kinase protein 1 (PINK1)/Parkin pathway-mediated mitophagy of osteoclast precursors, which was verified using a PINK1 knockdown small interfering RNA in OCPs. ConclusionThese findings indicate that GSZG is a candidate for the treatment of bone destruction in RA and provide a more detailed elucidation of the mechanism of GSZG anti-RA bone erosion, i.e., inhibition of the ROS/NF-κB axis through the PINK1/Parkin-mediated mitochondrial autophagic pathway to inhibit osteoclast precursor production, compared to the published literature.

Application of biomaterials in treating early osteonecrosis of the femoral head: Research progress and future perspectives.

Osteonecrosis of the femoral head (ONFH), a progressive pathological process of femoral head ischemia and osteocyte necrosis, is a refractory orthopedic disease caused by multiple etiologies and there is no complete cure at present. With the extension of ONFH duration, osteocyte apoptosis and trabecular bone loss can decrease the load-bearing capacity of the femoral head, which leads to the collapse of the articular cartilage and subchondral bone. Therefore, an urgent clinical need exists to develop effective treatment strategies of early-stage ONFH for maintaining the hip joint function and preventing femoral head collapse. In recent years, extensive attention has been paid to the application of diverse biomaterials in treating early ONFH for sustaining the normal morphology and function of the autologous femoral head, and slowing disease progression. Herein, we review the research progress of bone grafts, metallic materials, bioceramics, bioglasses and polymer materials for early ONFH treatment, and discuss the biological mechanisms of bone repair and regeneration in the femoral-head necrotic area. We propose suggestions for future research directions, from a special perspective of improving the local microenvironment in femoral head by facilitating vessel-associated osteoclasts (VAOs) generation and coupling of bone-specific angiogenesis and osteogenesis, as well as inhibiting bone-associated osteoclasts (BAOs) and BAO-mediated bone resorption. This review can provide ideas for the research, development, and clinical application of biomaterials for treating early ONFH. STATEMENT OF SIGNIFICANCE: We believe that at least three aspects of this manuscript make it interesting to readers of the Acta Biomaterialia. First, we briefly summarize the incidence, pathogenesis, risk factors, classification criteria and treatment of early osteonecrosis of the femoral head (ONFH). Second, we review the research progress in biomaterials for early ONFH treatment and the biological mechanisms of bone repair and regeneration in femoral-head necrotic area. Third, we propose future research progress on improving the local microenvironment in femoral head by facilitating vessel-associated osteoclasts generation and coupling of bone-specific angiogenesis and osteogenesis, as well as inhibiting bone-associated osteoclasts and bone resorption. We hope this review can provide ideas for the research, development, and clinical application of biomaterials for treating early ONFH.

Messages from the Mineral: How Bone Cells Communicate with Other Tissues.

Bone is a highly dynamic tissue, and the constant actions of bone-forming and bone-resorbing cells are responsible for attaining peak bone mass, maintaining bone mass in the adults, and the subsequent bone loss with aging and menopause, as well as skeletal complications of diseases and drug side-effects. It is now accepted that the generation and activity of bone-forming osteoblasts and bone-resorbing osteoclasts is modulated by osteocytes, osteoblast-derived cells embedded in the bone matrix. The interaction among bone cells occurs through direct contact and via secreted molecules. In addition to the regulation of bone cell function, molecules released by these cells are also able to reach the circulation and have effects in other tissues and organs in healthy individuals. Moreover, bone cell products have also been associated with the establishment or progression of diseases, including cancer and muscle weakness. In this review, we will discuss the role of bone as an endocrine organ, and the effect of selected, osteoblast-, osteocyte-, and osteoclast-secreted molecules on other tissues.

Immune modulation as a potential treatment strategy to salvage the bone metabolism after periprosthetic joint infection

Background: In arthroplasty, periprosthetic joint infection (PJI) is one of the most disastrous postoperative complications with high long-term failure rates despite successful revision surgery. PJI causes a pro-inflammatory environment in the surrounding bone stock that subsequently results in compromised bone metabolism. This increases the risk for unstructured bone-resorption potentially leading to aseptic loosening. We hypothesize that PD-1-positive monocytes and monocyte-derived osteoclasts play a significant role in this pathology. In this study, we assessed the presence of PD-1-positive monocytes and their osteoclastogenic differentiation potential, as well as resulting osteoclast activity in PJI. We also investigated anti-PD-1-antibody as a potential salvage therapy. Material and Methods: Peripheral blood specimens were collected from 15 patients undergoing knee arthroplasty (ctrl) or prosthesis explantation due to PJI. Peripheral blood mononuclear cells were isolated and stained for CD33, CD11b, and CD14. Osteoclastic differentiation was induced ± PD-L1 and ± anti-PD-1-antibody. Osteoclast number and function were determined by pit assay, TRAP staining and qPCR. Results: Monocyte cell count (0.86±0.35 v 0.21±0.03/nl, p=.02) and PD-1 expression on monocytes (92.50±6.83 v 35.34±15.43%, p<.01) waselevated in patients with PJI. After differentiation, in PJI, total osteoclast surface area was larger (47.40±3.19% v 26.75±3.49%, p<.01) and increased significantly further (+64.04±1.72%, p<.01) after PD-L1 stimulation. Addition of PD-1 inhibitor reduced osteoclast surface area in PJI (36.51±3.95 v 64.04±1.72%, p<.01). Expression of osteoclast differentiation marker Nfatc1 and Ctsk were higher in the PJI group ( Nfatc1: 1.09±0.20 v 0.45±0.06, p<.01; Ctsk: 2.27±1.00 v 0.73±0.23, p=.02), but did not differ in Mmp9 and Acp5 expression. In PJI, the expression of all four genes were upregulated after PD-L1 stimulation, while PD-1 inhibitor treatment reversed this effect. PD-L1 stimulation led to increased osteoclast bone resorption compared to without stimulation (7.65±3.15 v 2.49±0.67%, p=.05), while addition of PD-1 inhibitor showed no influence in the ctrl group. In PJI, osteoclast surface resorption was increased after PD-L1 stimulation (20.69±6.54 v 8.06±1.54, p=.01). Conversely, osteoclast function was reduced after treatment with PD-1 inhibitor (4.23±0.55 v 8.06±1.54, p=.03). Conclusion: Elevated numbers of monocytic osteoclast-progenitor cells and increased osteoclastic differentiation and function may contribute to decreased bone volume and impact prosthesis osseointegration in patients with PJI. Our results show PD-L1 can stimulate osteoclast generation and function and that this can be reversed by PD-1 inhibitor. This effect is particularly prominent in PJI. Targeting PD-1 can be a potential therapy to inhibit monocyte-mediated osteoclastogenesis while retaining anti-microbial activity of macrophages to limit the impact of PJI on the bone stock. Dr. Arne Kienzle is participant in the BIH-Charité Junior Clinician Scientist Program funded by the Charité - Universitätsmedizin Berlin and the Berlin Institute of Health. The authors wish to acknowledge the support of the non-profit German Arthritis Society (Deutsche Arthrose-Hilfe e.V.) and its president Helmut H. Huberti, MD by grant P482. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Regulatory function and mechanism of autophagy on osteoclast

Osteoclast (OC) is multinucleated, bone-resorbing cells originated from monocyte/macrophage lineage of cells, excessive production and abnormal activation of which could lead to many bone metabolic diseases, such as osteoporosis, osteoarthritis, etc. Autophagy, as a highly conserved catabolic process in eukaryotic cells, which plays an important role in maintaining cell homeostasis, stress damage repair, proliferation and differentiation. Recent studies have found that autophagy was also involved in the regulation of osteoclast generation and bone resorption. On the one hand, autophagy could be induced and activated by various factors in osteocalsts, such as nutrient deficiency, hypoxia, receptor activator of nuclear factor(NF)-κB ligand(RANKL), inflammatory factors, wear particles, microgravity environment, etc, different inducible factors, such as RANKL, inflammatory factors, wear particles, could interact with each other and work together. On the other hand, activated autophagy is involved in regulating various stages of osteoclast differentiation and maturation, autophagy could promote proliferation of osteoclasts, inhibiting apoptosis, and promoting differentiation, migration and bone resorption of osteoclast. The classical autophagy signaling pathway mediated by mammalian target of rapamycin complex 1(mTORC1) is currently a focus of research, and it could be regulated by upstream signalings such as phosphatidylinositol 3 kinase(PI-3K)/protein kinase B (PKB), AMP-activated protein kinase(AMPK). However, the paper found that mTORC1-mediated autophagy may play a bidirectional role in regulating differentiation and function of osteoclasts, and its underlying mechanism needs to be further ciarified. Integrin αvβ3 and Rab protein families are important targets for autophagy to play a role in osteoclast migration and bone resorption, respectively. In view of important role of osteoclast in the occurrence of various bone diseases, it is of great significance to elucidate the role of autophagy on osteoclast and its mechanism for the treatment of various bone diseases. The autophagy pathway could be used as a new therapeutic target for the treatment of clinical bone diseases such as osteoporosis.

Oscillating Fluid Flow Activated Osteocyte Lysate-Based Hydrogel for Regulating Osteoblast/Osteoclast Homeostasis to Enhance Bone Repair.

As major regulators on bone formation/resorption in response to mechanical stimuli, osteocytes have shown great promise for restoring bone injury. However, due to the unmanageable and unabiding cell functions in unloading or diseased environments, the efficacy of osteogenic induction by osteocytes has been enormously limited. Herein, a facile method of oscillating fluid flow (OFF) loading for cell culture is reported, which enables osteocytes to initiate only osteogenesis and not the osteolysis process. After OFF loading, multiple and sufficient soluble mediators are produced in osteocytes, and the collected osteocyte lysates invariably induce robust osteoblastic differentiation and proliferation while restraining osteoclast generation and activity under unloading or pathological conditions. Mechanistic studies confirm that elevated glycolysis and activation of the ERK1/2 and Wnt/β-catenin pathways are the major contributors to the initiation of osteoinduction functions induced by osteocytes. Moreover, an osteocyte lysate-based hydrogel is designed to establish a stockpile of "active osteocytes" to sustainably deliver bioactive proteins, resulting in accelerated healing through regulation of endogenous osteoblast/osteoclast homeostasis.

Isoimperatorin attenuates bone loss by inhibiting the binding of RANKL to RANK

Osteoporosis, an immune disease characterized by bone mass loss and microstructure destruction, is often seen in postmenopausal women. Isoimperatorin (ISO), a bioactive, natural furanocoumarin isolated from many traditional Chinese herbal medicines, has therapeutic effects against various diseases; however, its effect on bone homeostasis remains unclear. In this study, we investigated the effect of ISO on the differentiation and activation of osteoclast and its molecular mechanism in vitro, and evaluated the effect of ISO on bone metabolism by ovariectomized (OVX) rat model. In vitro experiments showed that ISO affected RANKL-induced MAPK, NFAT, NFATc1 trafficking and expression, osteoclast F-actin banding, osteoclast-characteristic gene expression, ROS inhibitory activity, and calcium oscillations, NF-κB signaling pathway. In vivo experiments showed that oral administration of ISO effectively reduced bone loss caused by ovariectomy and retained bone mass.Collectively, ISO inhibits RANK/RANKL binding, thereby reducing the activity of NFATc1, calcium, and ROS and inhibiting osteoclast generation. In addition, ISO protects bone mass by slowing osteoclast production and downregulating NFATc1 gene and protein expression in the bone tissue microenvironment and inhibits OVX-induced bone loss in vivo.

Specific targeting of inflammatory osteoclastogenesis by the probiotic yeast S. boulardii CNCM I-745 reduces bone loss in osteoporosis.

Bone destruction is a hallmark of chronic inflammation, and bone-resorbing osteoclasts arising under such a condition differ from steady-state ones. However, osteoclast diversity remains poorly explored. Here, we combined transcriptomic profiling, differentiation assays and in vivo analysis in mouse to decipher specific traits for inflammatory and steady-state osteoclasts. We identified and validated the pattern-recognition receptors (PRR) Tlr2, Dectin-1, and Mincle, all involved in yeast recognition as major regulators of inflammatory osteoclasts. We showed that administration of the yeast probiotic Saccharomyces boulardii CNCM I-745 (Sb) in vivo reduced bone loss in ovariectomized but not sham mice by reducing inflammatory osteoclastogenesis. This beneficial impact of Sb is mediated by the regulation of the inflammatory environment required for the generation of inflammatory osteoclasts. We also showed that Sb derivatives as well as agonists of Tlr2, Dectin-1, and Mincle specifically inhibited directly the differentiation of inflammatory but not steady-state osteoclasts in vitro. These findings demonstrate a preferential use of the PRR-associated costimulatory differentiation pathway by inflammatory osteoclasts, thus enabling their specific inhibition, which opens new therapeutic perspectives for inflammatory bone loss.

Bone marrow Adipoq-lineage progenitors are a major cellular source of M-CSF that dominates bone marrow macrophage development, osteoclastogenesis, and bone mass.

M-CSF is a critical growth factor for myeloid lineage cells, including monocytes, macrophages, and osteoclasts. Tissue-resident macrophages in most organs rely on local M-CSF. However, it is unclear what specific cells in the bone marrow produce M-CSF to maintain myeloid homeostasis. Here, we found that Adipoq-lineage progenitors but not mature adipocytes in bone marrow or in peripheral adipose tissue, are a major cellular source of M-CSF, with these Adipoq-lineage progenitors producing M-CSF at levels much higher than those produced by osteoblast lineage cells. The Adipoq-lineage progenitors with high CSF1 expression also exist in human bone marrow. Deficiency of M-CSF in bone marrow Adipoq-lineage progenitors drastically reduces the generation of bone marrow macrophages and osteoclasts, leading to severe osteopetrosis in mice. Furthermore, the osteoporosis in ovariectomized mice can be significantly alleviated by the absence of M-CSF in bone marrow Adipoq-lineage progenitors. Our findings identify bone marrow Adipoq-lineage progenitors as a major cellular source of M-CSF in bone marrow and reveal their crucial contribution to bone marrow macrophage development, osteoclastogenesis, bone homeostasis, and pathological bone loss.

Inhibitory Effect of Jinwujiangu Prescription on Peripheral Blood Osteoclasts in Patients with Rheumatoid Arthritis and the Relevant Molecular Mechanism.

Rheumatoid arthritis (RA) is a chronic progressive autoimmune disease characterized with high recurrence, high disability, poor prognosis, and long treatment cycles. Versus western medicine, traditional Chinese medicine has the traits of definite efficacy, low toxicity, and side effects in the treatment of RA. Moreover, traditional Chinese medicine also has the advantages of multiple targets, multiple links, and multiple approaches. This study was committed to exploring the effect of Jinwujiangu prescription on peripheral blood osteoclasts in those patients with RA and relevant molecular mechanisms. We first identified 159 common targets by online pharmacology, and there were correlations among these targets; besides, the main signaling pathways involved were inclusive TNF signaling pathway, rheumatoid arthritis, IL-17 signaling pathway, NF-kappa B signaling pathway, Toll-like receptor signaling pathway, etc. Through experimental verification, we found that PBMC cells extracted from human peripheral blood could be successfully induced into osteoclasts, and Jinwujiangu prescription inhibited the generation of osteoclasts from PBMCs of RA patients. CCK-8 and flow cytometry showed that osteoclast viability was significantly decreased and osteoclast apoptosis was significantly increased in the HIF-1α interference group; low-, medium-, and high-dose Jinwujiangu prescription groups; sinapine group; and hydroxychloroquine control group. Moreover, Jinwujiangu prescription and sinapine could inhibit the production of cytokines in peripheral blood osteoclasts and inhibit autophagy in RA patients. The expression level of mTOR was significantly increased in both Jinwu middle- and high-dose groups. In conclusion, this study demonstrated that sinapine, the active target in Jinwujiangu prescription, can act as a HIF-1α inhibitor; activate the mTOR pathway; downregulate the level of autophagy rate, ATG5, beclin-1, and LC3 expression; and inhibit the occurrence of autophagy. The trial registration number of the study is KYW2021010.

Osteoclast generation from RAW 264.7 and PBMC cells. The set up in our lab

Osteoclast generation from RAW 264.7 and PBMC cells. The set up in our lab

MiR-218-5p-Modified Bone Marrow Mesenchymal Stem Cells Mediate the Healing Effect of EphrinB2-EphB4 Signals on Alveolar Bone Defect

Abnormally expressed miR-218-5p involves in alveolar bone defect. We intend to investigate whether miR-218-5p-modified bone marrow mesenchymal stem cells (BMSCs) mediates the healing effects of EphrinB2-EphB4 signals on the alveolar bone defect. Fifty germ-free rats (6-month-old) were utilized in this study. The grouping was set up as follows: blank group, model group, miR-218-5p group, EphrinB2-EphB4 antagonist group, and positive control group (10 rats in each group). HE staining was employed to quantify bone resorption lacunae number. And the following indicators were monitored: miR-218-5p expression, differentiation status of osteoblasts, concentrations of TNF-α/IL-10/ IL-8, and EphrinB2 and EphB4 expression. As shown in HE staining, massive infiltration of inflammatory cells was denoted at the alveolar bone defective sites in rats from model group. However, infiltration of inflammatory cells in lesions was moderate in rats from EphrinB2-EphB4 antagonist group and positive control group, which was accompanied by formation of small bone islands. Furthermore, lesser infiltration of inflammatory cells was denoted at the alveolar bone defective sites in rats from the miR-218-5p group, which also exhibited a larger number of newly formed bone trabeculae growing toward the center of lesions. On the 3rd day of culture, absorption lacunae were rare in the model group, while remaining undetectable in other groups. On the 7th day of culture, bone resorption lacunae number in samples from model group was significantly higher in comparison with that in other groups. Meanwhile, it was reduced significantly in miR-218-5p group. However, it was increased in EphrinB2-EphB4 antagonist group and positive control group (P <0.05). An elevation of the intracellular miR-218-5p level was denoted in the modified BMSCs in comparison with those unmodified BMSCs (P < 0.05). In comparison with blank group, other groups exhibited significantly elevated ALP levels, among which model group showed highest level. However, decline of ALP levels was denoted in positive control group, EphrinB2-EphB4 antagonist group and miR-218-5p group, with lowest ALP level in miR-218-5p group (P <0.05). Except blank group, rats in other groups exhibited a significant elevation of TNF-α, IL-10 and IL-8 in the serum, among which those in the model group displayed the most remarkable increase of these cytokines. Rats in miR-218-5p group, EphrinB2-EphB4 antagonist group and positive control group exhibited significantly reduced levels of IL-8, IL-10 and TNF-α in the serum, with miR-218-5p group showing lowest levels (P < 0.05). In comparison with the blank group, other groups showed significantly enhanced protein expression of EphrinB2 and EphB4, among which the model group displayed the most remarkable enrichment of these proteins. In comparison with the model group, samples from the miR-218-5p group, EphrinB2-EphB4 antagonist group and positive control group exhibited significantly weakened expression of EphrinB2 and EphB4, among which the miR-218-5p group displayed the most remarkable decrease of these proteins (P <0.05). miR-218-5p-modified BMSCs can modulate the EphrinB2-EphB4 signal transduction pathway to produce two-way transmission, which included their inhibition of the osteoclast generation and their enhancement of the osteoclast differentiation. In this way, they aided in alleviating inflammatory response in alveolar bone defective lesions, thereby accelerating the healing process of alveolar bone defect. The function of miR-218-5p-modified BMSCs is mainly achieved in the healing process of the alveolar bone defect.

The effects of seeding density and osteoclastic supplement concentration on osteoclastic differentiation and resorption

The bone resorbing osteoclasts are a complex type of cell essential for in vivo bone remodeling. There is no consensus on medium composition and seeding density for in vitro osteoclastogenesis, despite the importance thereof on osteoclastic differentiation and activity. The aim of this study was to investigate the relative effect of monocyte or peripheral blood mononuclear cell (PBMC) seeding density, osteoclastic supplement concentration and priming on the in vitro generation of functional osteoclasts, and to explore and evaluate the usefulness of commonly used markers for osteoclast cultures. Morphology and osteoclast formation were analyzed with fluorescence imaging for tartrate resistant acid phosphatase (TRAP) and integrin β3 (Iβ3). TRAP release was analyzed from supernatant samples, and resorption was analyzed from culture on Corning® Osteo Assay plates. In this study, we have shown that common non-standardized culturing conditions of monocyte or PBMCs had a significant effect on the in vitro generation of functional osteoclasts. We showed how increased osteoclastic supplement concentrations supported osteoclastic differentiation and resorption but not TRAP release, while priming resulted in increased TRAP release as well. Increased monocyte seeding densities resulted in more and large TRAP positive bi-nuclear cells, but not directly in more multinucleated osteoclasts, resorption or TRAP release. Increasing PBMC seeding densities resulted in more and larger osteoclasts and more resorption, although resorption was disproportionally low compared to the monocyte seeding density experiment. Exploration of commonly used markers for osteoclast cultures demonstrated that Iβ3 staining was an excellent and specific osteoclast marker in addition to TRAP staining, while supernatant TRAP measurements could not accurately predict osteoclastic resorptive activity. With improved understanding of the effect of seeding density and osteoclastic supplement concentration on osteoclasts, experiments yielding higher numbers of functional osteoclasts can ultimately improve our knowledge of osteoclasts, osteoclastogenesis, bone remodeling and bone diseases.

PARK2 Induces Osteoclastogenesis through Activation of the NF-κB Pathway.

Osteoclast generation from monocyte/macrophage lineage precursor cells needs to be tightly regulated to maintain bone homeostasis and is frequently over-activated in inflammatory conditions. PARK2, a protein associated with Parkinson's disease, plays an important role in mitophagy via its ubiquitin ligase function. In this study, we investigated whether PARK2 is involved in osteoclastogenesis. PARK2 expression was found to be increased during the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. PARK2 gene silencing with siRNA significantly reduced osteoclastogenesis induced by RANKL, LPS (lipopolysaccharide), TNFα (tumor necrosis factor α), and IL-1β (interleukin-1β). On the other hand, overexpression of PARK2 promoted osteoclastogenesis. This regulation of osteoclastogenesis by PARK2 was mediated by IKK (inhibitory κB kinase) and NF-κB activation while MAPK (mitogen-activated protein kinases) activation was not involved. Additionally, administration of PARK2 siRNA significantly reduced osteoclastogenesis and bone loss in an in vivo model of inflammatory bone erosion. Taken together, this study establishes a novel role for PARK2 as a positive regulator in osteoclast differentiation and inflammatory bone destruction.

Autophagy attenuates osteoarthritis in mice by inhibiting chondrocyte pyroptosis and improving subchondral bone remodeling.

Osteoarthritis (OA) is an age-related degenerative disease characterized by cartilage degeneration and abnormal bone remodeling in the subchondral bone. Autophagy maintains cellular homeostasis by self-phagocytosis. However, the underlying mechanisms of autophagy on the pathological progression of OA are still unknown. This study assessed the effects of autophagy on cartilage and subchondral bone in a mouse OA model. A mouse OA model was induced using destabilization of the medial meniscus (DMM) surgery. Assessment was performed by histomorphology, microcomputed tomography (micro-CT), immunohistochemical, immunofluorescent, and tartrate-resistant acid phosphatase (TRAP) staining. Our data revealed that autophagy can significantly delay the pathological progression of OA by increasing the thickness of hyaline cartilage and decreasing the thickness of calcified cartilage, increasing the subchondral bone volume fraction and bone mineralization density, and decreasing trabecular separation in the early stages of OA (2 weeks), whereas the opposite is true in the late stages of OA (8 weeks). Mechanistically, activation of autophagy in cartilage increased the expression of type II collagen (Col II), decreased the expression of matrix metalloproteinase 13 (MMP 13) and decreased the pyroptosis mediated by NOD-like receptor protein 3 (NLRP3) inflammasome by decreasing the expression of NLRP3, caspase-1, gasdermin D (GSDMD), and IL-1β. In the subchondral bone, activation of autophagy decreased the generation of mature osteoclasts at the early stages of OA (2 weeks) mainly by reducing the receptor activator for nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) ratio, while it decreased osteoblastogenesis by reducing Runt-related transcription factor 2 (Runx2) expression significantly in the late stages of OA (8 weeks). In conclusion, autophagy may delay the pathological progression of OA in mice by inhibiting chondrocyte pyroptosis and improving subchondral bone remodeling.

Abstract 6374: BMP6 overcomes multiple myeloma-induced bone disease by inducing the expression of small leucine-rich proteoglycans

Abstract Multiple myeloma (MM), the second most common hematologic malignancy in adults, is characterized by clonal proliferation of malignant plasma cells and tumor-induced complications, including MM-induced bone disease (MBD). Up to 90% of MM patients develop MBD during the course of their disease, which is a major cause of morbidity and mortality. MM therapy focuses primarily on agents that seek to eradicate the tumor cells and others which inhibit bone resorption. In contrast, no drugs are currently available to promote bone formation in MM patients and reverse MBD. Towards addressing this unmet therapeutic need, we applied a modified version of our “humanized” bone marrow (BM)-like scaffold (huBMsc)-based xenograft model, where MM cells and bone marrow mesenchymal stromal cells (BMMSCs) are co-implanted. In this system, we investigated the impact of MM cells on BMMSCs to identify markers that can be targeted with the intent to reverse MBD. After confirming that MM cells indeed inhibit bone formation in vivo, we performed transcriptional analysis of ex vivo expanded “unexposed” control BMMSCs vs. “MM-exposed” BMMSCs and found 332 differentially expressed genes. Next, we compared these data with transcriptional profiles of BMMSCs after in vitro differentiation towards osteoblasts, as well as publicly available data of transcriptional profiles of BMMSCs obtained from MM, ALL, and AML patients. Combined, this analysis identified 8, MM-specific, differentially expressed genes, including the small leucine-rich proteoglycans (SLRPs), ASPN, OGN and OMD, that are 1) suppressed in the presence of MM (xenograft model and patient-derived BMMSCs); 2) induced during in vitro osteogenic differentiation; and 3) not dysregulated in BMMSCs in other hematological malignancies. As reported previously, we observed that bone morphogenetic protein (BMP)6 can upregulate the expression of these SLRPs in BMMSCs, while inducing osteogenic differentiation. Moreover, incubation of MM cells with BMP6 in either mono- or co-cultures with stromal cell lines, resulted in reduced MM cell viability. In addition, pretreatment of BMMSCs with BMP6 completely abolished their support of MM proliferation. Interestingly, BMP6, in contrast to BMP2 and BMP4, did not induce, but inhibited receptor activator of nuclear kappa-B ligand (RANKL)-dependent osteoclast generation. Collectively, our analyses identify BMP6 as a master regulator that promotes osteogenic differentiation, inhibits RANKL-dependent osteoclastogenesis and can directly induce MM cell death. Therefore, by reshaping the BM microenvironment to become less tumor-supportive, BMP6 represents an attractive candidate for MM treatment. Citation Format: Jiaxian Wang, Thomas Baardemans, Ricardo de Matos Simoes, Olga Dashevsky, Huipin Yuan, Joost D. de Bruijn, Anton C. Martens, Constantine S. Mitsiades, Richard W. Groen. BMP6 overcomes multiple myeloma-induced bone disease by inducing the expression of small leucine-rich proteoglycans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6374.

Osteoblasts and osteoclasts generation from iPSCs to study CFTR role in cystic fibrosis-related bone disease

Osteoblasts and osteoclasts generation from iPSCs to study CFTR role in cystic fibrosis-related bone disease

Genetic Deletion of Menin in Mouse Mesenchymal Stem Cells: An Experimental and Computational Analysis.

ABSTRACTLoss‐of‐function mutations in the MEN1 tumor‐suppressor gene cause the multiple endocrine neoplasia type 1 syndrome. Menin, the MEN1 gene product, is expressed in many tissues, including bone, where its function remains elusive. We conditionally inactivated menin in mesenchymal stem cells (MSCs) using paired‐related homeobox 1 (Prx1)‐Cre and compared resultant skeletal phenotypes of Prx1‐Cre;Men1 f/f menin‐knockout mice (KO) and wild‐type controls using in vivo and in vitro experimental approaches and mechanics simulation. Dual‐energy X‐ray absorptiometry demonstrated significantly reduced bone mineral density, and 3‐dimensional micro‐CT imaging revealed a decrease in trabecular bone volume, altered trabecular structure, and an increase in trabecular separation in KO mice at 6 and 9 months of age. Numbers of osteoblasts were unaltered, and dynamic histomorphometry demonstrated unaltered bone formation; however, osteoclast number and activity and receptor activator of NF‐κB ligand/osteoprotegerin (RANKL/OPG) mRNA profiles were increased, supporting increased osteoclastogenesis and bone resorption. In vitro, proliferative capabilities of bone marrow stem cells and differentiation of osteoblasts and mineralization were unaltered; however, osteoclast generation was increased. Gross femur geometrical alterations observed included significant reductions in length and in mid‐metaphyseal cross‐sectional area. Atomic force microscopy demonstrated significant decreases in elasticity of both cortical and trabecular bone at the nanoscale, whereas three‐point bending tests demonstrated a 30% reduction in bone stiffness; finite element analysis showed morphological changes of the femur microgeometry and a significantly diminished femur flexural rigidity. The biomechanical results demonstrated the detrimental outcome of the accelerated osteoclastic bone resorption. Our studies have a twofold implication; first, MEN1 deletion from MSCs can negatively regulate bone mass and bone biomechanics, and second, the experimental and computational biomechanical analyses employed in the present study should be applicable for improved phenotypic characterization of murine bone. Furthermore, our findings of critical menin function in bone may underpin the more severe skeletal phenotype found in hyperparathyroidism associated with loss‐of‐function of the MEN1 gene. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.