Excessive Bone Resorption Research Articles

Autophagy Regulator Rufy 4 Promotes Osteoclastic Bone Resorption by Orchestrating Cytoskeletal Organization via Its RUN Domain

Rufy4, a protein belonging to the RUN and FYVE domain-containing protein family, participates in various cellular processes such as autophagy and intracellular trafficking. However, its role in osteoclast-mediated bone resorption remains uncertain. In this study, we investigated the expression and role of the Rufy4 gene in osteoclasts using small interfering RNA (siRNA) transfection and gene overexpression systems. Our findings revealed a significant increase in Rufy4 expression during osteoclast differentiation. Silencing Rufy4 enhanced osteoclast differentiation, intracellular cathepsin K levels, and formation of axial protrusive structures but suppressed bone resorption. Conversely, overexpressing wild-type Rufy4 in osteoclasts hindered differentiation while promoting podosome formation and bone resorption. Similarly, overexpression of a Rufy4 variant lacking the RUN domain mimics the effects of Rufy4 knockdown, significantly increasing intracellular cathepsin K levels, promoting osteoclastogenesis, and elongated axial protrusions formation, yet inhibiting bone resorption. These findings indicate that Rufy4 plays a critical role in osteoclast differentiation and bone resorption by regulating the cytoskeletal organization through its RUN domain. Our study provides new insights into the molecular mechanisms governing osteoclast activity and underscores Rufy4’s potential as a novel therapeutic target for bone disorders characterized by excessive bone resorption.

12460 Bilateral Parietal Thinning: A Rare Condition Of Unclear Etiology

Abstract Disclosure: T.L. Madsen-Barbosa: None. M. Cheikh: None. I. Marcano: None. Background: Bilateral parietal bone thinning (BPT) is an infrequent and often underdiagnosed condition characterized by the external thinning of the skull's parietal bone. The pathophysiology is unclear, but some studies suggest a link to osteoporosis. We present a case of osteoporosis diagnosed on evaluation of BPT. Case Description: A 77-year-old woman with a history of chronic kidney disease (CKD stage IV) and type 2 diabetes was referred to the Bone Health Clinic for evaluation of low bone density. She initially presented to Neurosurgery clinic for evaluation of head indentations. A dual energy x-ray absorptiometry (DEXA) scan showed values in osteopenia range (left total hip bone mineral density (BMD) 0.678 and T-score of -2.2, left femoral neck BMD 0.272 and T-score of -1.6, and left 33% radius BMD 0.577 and T-score of -2.0), leading to the referral. The patient reported painless indentations in the parietal bones that had been progressing for four years, with no history of head trauma, inflammatory disease, or prolonged steroid use. A head computed tomography (CT) scan showed symmetric thinning of the skull’s parietal bones, with an average thickness of 2.34 mm (compared to 5.1 mm four years earlier). Review of prior images also revealed a T11 compression fracture on spine X-ray from 2019. Laboratory tests showed elevated parathyroid hormone (PTH) 155 pg/mL (reference range 15-65 pg/mL), low vitamin D 25-OH 10.6 ng/mL (reference range 30-100 ng/mL), elevated alkaline phosphatase 142 units/L (reference range 46-116 units/L), and high creatinine level 2.7 mg/dL (reference range 0.5-0.8 mg/dL), other laboratory tests were normal. After discussion with the patient and Nephrology, Denosumab therapy along with Calcium and Vitamin D supplementation were started in November 2023. The patient continues the treatment with antiresorptive medication every 6 months and head CT scan to monitor the skull thinning changes. Discussion: BPT is rare, with only approximately 150 cases reported. Crucial aspects such as its etiology, pathophysiology, treatment, and prognosis remain unknown. It can result from various conditions like trauma, systemic mastocytosis, diabetes mellitus, hereditary diseases, or idiopathic causes and mainly affects women of advanced age. Osteoporosis is suggested as a possible cause based on histological findings on prior studies that propose BPT may result from osteoporosis-induced decreased bone formation rather than increased bone destruction. The reason for excessive bone resorption in the skull of osteoporotic patients remains unclear. Our case highlights progressive and symmetric BPT possibly related to systemic conditions like osteoporosis, with a focus on treatment options. Conclusion: This case sheds light on the clinical presentation, imaging findings, pathogenesis, and treatment options for the rare condition of bilateral parietal bone thinning. Presentation: 6/3/2024

Injectable double-crosslinked bone cement with enhanced bone adhesion and improved osteoporotic pathophysiological microenvironment for osteoregeneration in osteoporosis

The osteoporotic bone defect caused by excessive activity of osteoclasts has posed a challenge for public healthcare. However, most existing bioinert bone cement fails to effectively regulate the pathological bone microenvironment and reconstruct bone homeostasis in the presence of osteoclast overactivity and osteoblast suppression. Herein, inspired by natural bone tissue, an in-situ modulation system for osteoporotic bone regeneration is developed by fabricating an injectable double-crosslinked PEGylated poly(glycerol sebacate) (PEGS)/calcium phosphate cement (CPC) loaded with sodium alendronate (ALN) (PEGS/CPC@ALN) adhesive bone cement. By incorporating ALN, the organic-inorganic interconnection within PEGS/CPC@ALN results in a 100 % increase in compression modulus and energy dissipation efficiency. Additionally, PEGS/CPC@ALN effectively adheres to the bone by bonding with amine and calcium ions present on the bone surface. Moreover, this in-situ regulation system comprehensively mitigates excessive bone resorption through the buffering effect of CPC to improve the acidic microenvironment of osteoporotic bone and the release of ALN to inhibit hyperactive osteoclasts, and facilitates stem cell proliferation and differentiation into osteoblasts through calcium ion release. Overall, the PEGS/CPC@ALN effectively regulates the pathological microenvironment of osteoporosis while promoting bone regeneration through synergistic effects of drugs and materials, thereby improving bone homeostasis and enabling minimally invasive treatment for osteoporotic defects.

Pharmacological impacts of tanshinone on osteogenesis and osteoclastogenesis: a review.

Tanshinone, a lipophilic component of Salvia miltiorrhiza, is used to treat diseases like atherosclerosis, hypertension, Alzheimer's disease, and diabetes mellitus through its pharmacological activities like anti-inflammatory, anti-oxidant, and anti-tumor. Excessive inflammation is the primary cause of bone diseases such as osteoporosis and rheumatoid arthritis, affecting more than millions of people across the globe. Recently, tanshinone has shown potential benefits against bone diseases by modulating signaling pathways accountable for the proliferation and differentiation of bone cells. In vitro and in vivo studies reported that tanshinone promotes osteoblast formation and mineralization and suppresses excessive bone resorption during disease conditions. In this review, we have summarized the beneficial effects of tanshinone and other extracts of Salvia miltiorrhiza for bone health and their potential molecular targets in signaling.

S100P binds to RAGE and activates ERK/NF-κB signaling to promote osteoclast differentiation and activity

S100 calcium-binding protein P (S100P) is a secretory protein that is expressed in various healthy tissues and tumors. Megakaryocyte-secreted S100P promotes osteoclast differentiation and function; however, its receptor and cellular signaling in osteoclasts remain unclear. Receptor for advanced glycation end products (RAGE), which is the receptor for S100P on cancer cells, was expressed in osteoclast precursors, and S100P-RAGE binding was confirmed through co-immunoprecipitation. Additionally, the phosphorylation of ERK and NF-κB was increased in S100P-stimulated osteoclast precursors but was inhibited by addition of the RAGE antagonistic peptide (RAP). S100P-induced osteoclast differentiation and excessive bone resorption activity were also reduced by the addition of RAP. This study demonstrates that S100P, upon binding with RAGE, activates the ERK and NF-κB signaling pathways in osteoclasts, leading to increased cell differentiation and bone resorption activity.

Membrane-free stem cell components suppress osteoclast differentiation: Implications for oral regenerative treatment

Background/purposeMembrane-free stem cell components (MFSCCs) have been developed by removing cell membranes with antigens to overcome the limitations associated with cell-based therapies and isolate effective peptides. MFSCCs have been reported to have effects on oral infection sites. Chronic inflammatory diseases cause excessive bone resorption. This study investigated the effects of MFSCCs on osteoclast differentiation in the context of the high prevalence of inflammatory bone resorption. Materials and methodsBone marrow macrophages (BMMs) were treated with macrophage colony-stimulating factor and receptor activator of nuclear factor kappa-B ligand. Osteoclast differentiation was assessed based on the MFSCC concentrations. Tartrate-resistant acid phosphatase (TRAP)-stained mature osteoclasts and multinucleated cells derived from BMMs were analyzed using light microscopy. The messenger RNA (mRNA) expression levels of genes related to osteoclast differentiation were measured using real-time polymerase chain reaction (RT-PCR). The relative expression levels of the key transcription factors c-fos and nuclear factor of activated T cells (NFATc1) were determined using quantitative RT-PCR and western blotting. ResultsAfter treatment with MFSCCs, the cell viability was similar, depending on the level of BMMs. As the MFSCC concentration increased, the number of TRAP-positive cells decreased. The mRNA and protein expression of cathepsin K, TRAP, dendritic cell-specific transmembrane protein, c-fos, and NFATc1 decreased as the MFSCC concentration increased. ConclusionOur findings demonstrate that MFSCCs suppress osteoclast differentiation by downregulating transcription factors, particularly, c-fos and NFATc1. Therefore, MFSCCs may serve as a conservative treatment option for chronic inflammatory bone resorption diseases of the oral cavity by suppressing excessive bone resorption.

A maternal brain hormone that builds bone

In lactating mothers, the high calcium (Ca2+) demand for milk production triggers significant bone loss1. Although oestrogen normally counteracts excessive bone resorption by promoting bone formation, this sex steroid drops precipitously during this postpartum period. Here we report that brain-derived cellular communication network factor 3 (CCN3) secreted from KISS1 neurons of the arcuate nucleus (ARCKISS1) fills this void and functions as a potent osteoanabolic factor to build bone in lactating females. We began by showing that our previously reported female-specific, dense bone phenotype2 originates from a humoral factor that promotes bone mass and acts on skeletal stem cells to increase their frequency and osteochondrogenic potential. This circulatory factor was then identified as CCN3, a brain-derived hormone from ARCKISS1 neurons that is able to stimulate mouse and human skeletal stem cell activity, increase bone remodelling and accelerate fracture repair in young and old mice of both sexes. The role of CCN3 in normal female physiology was revealed after detecting a burst of CCN3 expression in ARCKISS1 neurons coincident with lactation. After reducing CCN3 in ARCKISS1 neurons, lactating mothers lost bone and failed to sustain their progeny when challenged with a low-calcium diet. Our findings establish CCN3 as a potentially new therapeutic osteoanabolic hormone for both sexes and define a new maternal brain hormone for ensuring species survival in mammals.

Multiscale effects of the calcimimetic drug, etelcalcetide on bone health of rats with secondary hyperparathyroidism induced by chronic kidney disease

Chronic kidney disease-induced secondary hyperparathyroidism (CKD-SHPT) heightens fracture risk through impaired mineral homeostasis and elevated levels of uremic toxins (UTs), which in turn enhance bone remodeling. Etelcalcetide (Etel), a calcium-sensing receptor (CaSR) agonist, suppresses parathyroid hormone (PTH) in hyperparathyroidism to reduce excessive bone resorption, leading to increased bone mass. However, Etel's effect on bone quality, chemical composition, and strength is not well understood. To address these gaps, we established a CKD-SHPT rat model and administered Etel at a human-equivalent dose concurrently with disease induction. The effects on bone and mineral homeostasis were compared with a CKD-SHPT (vehicle-treated group) and a control group (rats without SHPT). Compared with vehicle-treated CKD-SHPT rats, Etel treatment improved renal function, reduced circulating UT levels, improved mineral homeostasis parameters, decreased PTH levels, and prevented mineralization defects. The upregulation of mineralization-promoting genes by Etel in CKD-SHPT rats might explain its ability to prevent mineralization defects. Etel preserved both trabecular and cortical bones with attendant suppression of osteoclast function, besides increasing mineralization. Etel maintained the number of viable osteocytes to the control level, which could also contribute to its beneficial effects on bone. CKD-SHPT rats displayed increased carbonate substitution of matrix and mineral, decreased crystallinity, mineral-to-matrix ratio, and collagen maturity, and these changes were mitigated by Etel. Further, Etel treatment prevented CKD-SHPT-induced deterioration in bone strength and mechanical behavior. Based on these findings, we conclude that in CKD-SHPT rats, Etel has multiscale beneficial effects on bone that involve remodeling suppression, mineralization gene upregulation, and preservation of osteocytes.

Metabolic Bone Disease due to Renal Tubular Acidosis as a Primary Manifestation in a Patient with Sjogren’s Syndrome: A Case Report

Background: Renal tubular acidosis (RTA) is a group of diseases where metabolic acidosis develops due to inability of renal tubules to maintain acid base balance despite normal glomerular filtration rate. Uncorrected acidosis in RTA can cause excess osteoclastic bone resorption and result in severe metabolic bone disease like rickets, osteomalacia and pathological fracture. Sjogren’s syndrome can cause distal RTA (type 1). Here, we report a case of a 35 years old female who presented with metabolic bone disease secondary to RTA resulting from Sjogren’s syndrome.

Gallium and silver-doped titanium surfaces provide enhanced osteogenesis, reduce bone resorption and prevent bacterial infection in co-culture

Bacterial infection remains a significant problem associated with orthopaedic surgeries leading to surgical site infection (SSI). This unmet medical need can become an even greater complication when surgery is due to malignant bone tumor. In the present study, we evaluated in vitro titanium (Ti) implants subjected to gallium (Ga) and silver (Ag)-doped thermochemical treatment as strategy to prevent SSI and improve osteointegration in bone defects caused by diseases such as osteoporosis, bone tumor, or bone metastasis. Firstly, as Ga has been reported to be an osteoinductive and anti-resorptive agent, its performance in the mixture was proved by studying human mesenchymal stem cells (hMSC) and pre-osteoclasts (RAW264.7) behaviour. Then, the antibacterial potential provided by Ag was assessed by resembling “The Race for the Surface” between hMSC and Pseudomonas aeruginosa in two co-culture methods. Moreover, the presence of quorum sensing molecules in the co-culture was evaluated. The results highlighted the suitability of the mixture to induce osteodifferentiation and reduce osteoclastogenesis in vitro. Furthermore, the GaAg surface promoted strong survival rate and retained osteoinduction potential of hMSCs even after bacterial inoculation. Therefore, GaAg-modified titanium may be an ideal candidate to repair bone defects caused by excessive bone resorption, in addition to preventing SSI. Statement of significanceThis article provides important insights into titanium for fractures caused by osteoporosis or bone metastases with high incidence in surgical site infection (SSI) because in this situation bacterial infection can become a major disaster. In order to solve this unmet medical need, we propose a titanium implant modified with gallium and silver to improve osteointegration, reduce bone resorption and avoid bacterial infection. For that aim, we study osteoblast and osteoclast behavior with the main novelty focused on the antibacterial evaluation. In this work, we recreate “the race for the surface” in long-term experiments and study bacterial virulence factors (quorum sensing). Therefore, we believe that our article could be of great interest, providing a great impact on future orthopedic applications.

Development and experimental validation of an energy metabolism-related gene signature for diagnosing of osteoporosis

Osteoporosis is usually caused by excessive bone resorption and energy metabolism plays a critical role in the development of osteoporosis. However, little is known about the role of energy metabolism-related genes in osteoporosis. This study aimed to explore the important energy metabolism-related genes involved in the development of osteoporosis and develop a diagnosis signature for osteoporosis. The GSE56814, GSE62402, and GSE7158 datasets were downloaded from the NCBI Gene Expression Omnibus. The intersection of differentially expressed genes between high and low levels of body mineral density (BMD) and genes related to energy metabolism were screened as differentially expressed energy metabolism genes (DE-EMGs). Subsequently, a DE-EMG-based diagnostic model was constructed and differential expression of genes in the model was validated by RT-qPCR. Furthermore, a receiver operating characteristic curve and nomogram model were constructed to evaluate the predictive ability of the diagnostic model. Finally, the immune cell types in the merged samples and networks associated with the selected optimal DE-EMGs were constructed. A total of 72 overlapped genes were selected as DE-EMGs, and a five DE-EMG based diagnostic model consisting B4GALT4, ADH4, ACAD11, B4GALT2, and PPP1R3C was established. The areas under the curve of the five genes in the merged training dataset and B4GALT2 in the validation dataset were 0.784 and 0.790, respectively. Moreover, good prognostic prediction ability was observed using the nomogram model (C index = 0.9201; P = 5.507e−14). Significant differences were observed in five immune cell types between the high- and low-BMD groups. These included central memory, effector memory, and activated CD8 T cells, as well as regulatory T cells and activated B cells. A network related to DE-EMGs was constructed, including hsa-miR-23b-3p, DANCR, 17 small-molecule drugs, and two Kyoto Encyclopedia of Genes and Genomes pathways, including metabolic pathways and pyruvate metabolism. Our findings highlighted the important roles of DE-EMGs in the development of osteoporosis. Furthermore, the DANCR/hsa-miR-23b-3p/B4GALT4 axis might provide novel molecular insights into the process of osteoporosis development.

FoxO1 as the critical target of puerarin to inhibit osteoclastogenesis and bone resorption.

Elevated reactive oxygen species levels promote excessive osteoclastogenesis and bone resorption. Puerarin, a natural antioxidant, can prevent bone loss through its antioxidant effects; however, the underlying molecular mechanism remains unclear. This study aimed to explore the effects of puerarin on osteoclast differentiation and bone resorption by regulating the PI3K/AKT/FoxO1 signaling pathway. An ovariectomized (OVX) rat model of osteoporosis and H2O2-induced oxidative cell model of RAW 264.7 cells were established. The following indices were measured including bone μ-CT scanning and the protein expression levels of FoxO1, p-FoxO1, and catalase were detected using western blotting. Puerarin strongly alleviated oxidative stress-induced bone loss in OVX rats in vivo owing to its antioxidant effects. Puerarin improved the oxidative stress status of cells and inhibited osteoclast formation in vitro. Moreover, the protein expression of FoxO1 and its downstream target, catalase, was upregulated by puerarin. Puerarin improved the OPG/RANKL ratio, upregulated the protein expression and transcriptional activity of FoxO1, and suppressed the differentiation of RAW264.7 cells into osteoclasts. FoxO1 is a pivotal target of puerarin to confer anti-osteoporosis effects.

Abaloparatide promotes bone repair of vertebral defects in ovariectomized rats by increasing bone formation

Osteoporotic vertebral fracture (OVF) is the most common type of osteoporotic fracture and is associated with immobility and mortality. Bone anabolic agents, such as abaloparatide (ABL), are usually administered to patients with OVF to prevent subsequent fractures. Although several studies have shown that bone anabolic agents promote healing of long bone fractures, there is little evidence of their healing effect on vertebral bone fractures. In the present study, we investigated the effect of ABL on vertebral bone defects using ovariectomized (OVX) rats with vertebral body drill-hole defects, an animal model of OVF. Eight-week-old female Sprague-Dawley rats were subjected to OVX, followed by the 32–36 days of bone depletion period, once-daily subcutaneous ABL was administered to OVX rats at a dose of 30 μg/kg for a maximum of 6 weeks from the day of the vertebral defect surgery. We found that ABL significantly increased bone mineral content and improved trabecular structural parameters at the vertebral defect site. Moreover, ABL significantly increased bone strength of the defected vertebrae. Bone histochemical analysis revealed formation of thick trabecular bone networks at the defect site after ABL administration, consistent with an improvement in trabecular structural parameters by ABL. ABL increased ALPase- and PHOSPHO1-positive osteoblastic cells and ALPase/PCNA double-positive cells, indicating enhanced preosteoblast proliferation as well as bone formation at the defect site. On the other hand, ABL did not affect the number of cathepsin K-positive osteoclasts per bone surface, suggesting that ABL did not promote excessive bone resorption. Our findings suggest that ABL is useful not only for preventing secondary vertebral fractures but also for promoting bone healing in OVF.

Periostin Promotes the Proliferation, Differentiation and Mineralization of Osteoblasts from Ovariectomized Rats.

Perimenopausal period causes a significant amount of bone loss, which results in primary osteoporosis (OP). The Periostin (Postn) may play important roles in the pathogenesis of OP after ovariectomized (OVX) rats. To identify the roles of Postn in the bone marrow mesenchymal stem cell derived osteoblasts (BMSC-OB) in OVX rats, we investigated the expression of Wnt/β-catenin signaling pathways in BMSC-OB and the effects of Postn on bone formation by development of BMSC-OB cultures. Twenty-four female Sprague-Dawley rats at 6 months were randomized into 3 groups: sham-operated (SHAM) group, OVX group and OVX+Postn group. The rats were killed after 3 months, and their bilateral femora and tibiae were collected for BMSC-OB culture, Micro-CT Analysis, Bone Histomorphometric Measurement, Transmission Electron Microscopy and Immunohistochemistry Staining. The dose/time-dependent effects of Postn on the proliferation, differentiation and mineralization of BMSC-OB and the expression of osteoblastic markers were measured in in vitro experiments. We found increased Postn increased bone mass, promoted bone formation of trabeculae, Wnt signaling and the osteogenic activity in osteoblasts in sublesional femur. Postn have the function to enhance cell proliferation, differentiation and mineralization at a proper concentration and incubation time. Interestingly, in BMSC-OB from OVX rats treated with the different dose of Postn, the osteoblastic markers expression and Wnt/β-catenin signaling pathways were significantly promoted. The direct effect of Postn may lead to inhibit excessive bone resorption and increase bone formation through the Wnt/β-catenin signaling pathways after OVX. Postn may play a very important role in the pathogenesis of OP after OVX.

Fluid-solid coupling numerical simulation of entire rat caudal vertebrae under dynamic loading

Trabeculae bone undergoes directional growth along the applied force under physiological loading. The growth of bone structure relies on the coordinated interplay among osteocytes, osteoblasts, and osteoclasts. Under normal circumstances, bone remodeling maintains a state of equilibrium. Excessive bone formation can lead to osteosclerosis, while excessive bone resorption can result in osteoporosis and osteonecrosis. The investigation of the structural characteristics of trabeculae and the mechanotransduction between bone cells plays a vital role in the treatment of bone-related diseases. In this study, a fluid-solid coupling model of the entire vertebral bone was established based on micro-CT images obtained from rat tail vertebrae subjected to tensile loading experiments. The flow characteristics of bone marrow and the mechanical response of osteocytes in different regions under physiological loading were investigated. The results revealed a U-shaped distribution of wall fluid shear stress (FSS) along the longitudinal axis in trabecular bone, with higher FSS regions exhibiting greater mechanical stimulation on osteocytes. These findings elucidate a positive correlation between the mechanical microenvironment among osteocytes, osteoblasts, and osteoclasts, providing potential strategies for the prevention and treatment of bone diseases.

A Case of Osteofibrous Dysplasia at Birth: 2-Year Follow-Up

Osteofibrous dysplasia (OFD) is characterized by excessive resorption of long bone, especially tibia, with subsequent fibrous repair of the defect. OFD is an uncommon benign tumor of young children – neonatal presentation is restricted to single digits in literature. We describe a rare case of OFD in a newborn, which presented as unilateral painless lower limb deformity at birth. Radiological findings were suggestive and biopsy was diagnostic of OFD. Postbiopsy, the patient underwent expectant management given the generally slow progression of this condition, the possibility of postbiopsy regression, and the known risk of recurrence after surgical intervention. By 13 months, the lesion had undergone complete radiological resolution with spontaneous reduction of deformity. There has been no recurrence of disease at the same or another site till now (24 months age). There is no limb length discrepancy and motor milestones are appropriate for age; nevertheless, follow-up till puberty has been advised.

Evaluation of Different Subperiosteal Implant Thicknesses on Mechanical Strength and Stress on Bone by Finite Element Analysis.

Implants have always been within the interest of both clinicians and material scientists due to their places in reconstructive and prosthetics surgery. Excessive bone loss or resorption in some patients makes it difficult to design and manufacture the implants that bear the necessary loads to carry the final prosthetics. With this study; we tried to determine the minimum material thickness of the subperiosteal implants that can withstand the physiological forces. We have created a digital average bone structure based on actual patient data and designed different subperiosteal implants with 1, 1.5, and 2mm material thicknesses (M1, M2, M3) for this digital model. The designed implant models are subjected to 250 Newtons (N) of force, and the implant and bone are tested for the stress they are exposed to, the pressure they transmit to, and their mechanical strength with Finite Element Analysis with the physical parameters boot for the implant material and human bone. Results show us that under specific design parameters and thicknesses, the 1mm thickness design failed due to exceeding the yield stress limit of 415MPa with a 495,44MPa value. The thinnest implant showed plastic deformation and transmitted excessive forces, which may cause bone resorption due to residual stress. We determined that thinner subperiosteal implants down to 1.5mm that have the necessary material parameters for function and tissue support can be designed and manufactured with current technologies.

Improvement of osteoblast adhesion, viability, and mineralization by restoring the cell cytoskeleton after bisphosphonate discontinuation in vitro.

Bisphosphonates are prescribed to treat excessive bone resorption in patients with osteoporosis. However, its use is associated with potential adverse effects such as medication-related osteonecrosis of the jaw, prompting the introduction of the drug holiday concept in patients prior to dentoalveolar surgery. Furthermore, bisphosphonate discontinuation has been studied in vivo, in humans, and in animal models. However, it is not known whether this approach could affect bone cells in vitro. Therefore, the objective of this study was to investigate the potential effects of bisphosphonate discontinuation on pre-osteoblast and osteoblast activities in vitro. Pre-osteoblasts (MC3T3) and osteoblasts were treated with bisphosphonate (alendronate) at concentrations of 1, 5, and 10 µM. Alendronate was then withdrawn at different time points. The negative control consisted of untreated cells (0 µM), while the positive control consisted of cells incubated with alendronate throughout the experiment. Cell viability, cell adhesion, cell cytoskeleton, mineralization, and gene expressions were investigated. Pre-osteoblasts and osteoblasts showed a decrease in cell viability after treatment with 5-10 μM alendronate for 4 days or longer. Two days of alendronate discontinuation significantly increased cell viability compared with the positive control. However, these levels did not reach those of the negative control. Bone nodule formation was reduced by alendronate. Discontinuation of alendronate regained bone nodule formation. Longer periods of discontinuation were more effective in restoring nodule formation than shorter periods. Addition of alendronate resulted in an increase in the percentage of dead cells, which, in turn, decreased when alendronate was discontinued. Alendronate affected the cell cytoskeleton by disassembling actin stress fibers. Cell adhesion and cell morphological parameters were also affected by alendronate. Discontinuation of alendronate restored cell adhesion and these parameters. Overall, the highest improvement after alendronate discontinuation was seen at 10 µM. However, alendronate treatment and discontinuation did not affect osteoblast gene expression. Discontinuation of alendronate helps to reverse the negative effects of the drug on cell viability, cell adhesion, and mineralization by restoring the cell cytoskeleton. Our data suggest the benefits of drug holiday and/or intermittent strategies for alendronate administration at the cellular level.

PINK1 restrains periodontitis-induced bone loss by preventing osteoclast mitophagy impairment

The oral colonization of periodontal pathogens onto gingival tissues establishes hypoxic microenvironment, often disrupting periodontal homeostasis in conjunction with oxidative stress. The association between reactive oxygen species (ROS) and osteolytic periodontitis have been suggested by recent studies. PTEN-induced kinase 1 (PINK1), a mitochondrial serine/threonine kinase, is an essential protein for mitochondrial quality control as it protects cells from oxidative stress by promoting degradation of damaged mitochondria through mitophagy. However, the pathophysiological roles of PINK1 in osteoclast-mediated bone loss have not been explored. Here we aimed to determine whether PINK1 plays a role in the regulation of osteoclastogenesis and alveolar bone resorption associated with periodontitis. C57BL/6 wild type (WT) and Pink1 knockout (KO) mice were subjected to ligature-induced periodontitis (LIP), and alveolar bones were evaluated by μCT-analysis and tartrate-resistant acid phosphatase (TRAP) staining. The μCT-analysis showed that bone volume fraction and travecular thickness were lower in Pink1 KO compared to WT mice. The number of TRAP-positive osteoclasts was markedly increased in the periodontal tissues of Pink1 KO mice with LIP. The genetic silencing or deletion of Pink1 promoted excessive osteoclast differentiation and bone resorption in vitro, as respectively indicated by TRAP staining and resorption pits on dentin slices. PINK1 deficiency led to mitochondrial instabilities as indicated by confocal microscopy of mitochondrial ROS, mitochondrial oxygen consumption rate (OCR) analysis, and transmission electron microscopy (TEM). Consequently, a significant increase in Ca2+-nuclear factor of activated T cells 1 (NFATc1) signaling was also found. On the other hand, restoration of mitophagy and autophagy by spermidine (SPD) treatment and the resolution of oxidative stress by N-acetyl-l-cysteine (NAC) treatment protected PINK1 deficiency-induced excessive generation of osteoclasts. Taken together, our findings demonstrate that PINK1 is essential for maintaining mitochondrial homeostasis during osteoclast differentiation. Therefore, targeting PINK1 may provide a novel therapeutic strategy for severe periodontitis with fulminant osteolysis.

Farrerol suppresses osteoclast differentiation and postmenopausal osteoporosis by inhibiting the nuclear factor kappa B signaling pathway

Excessive bone resorption caused by upregulated osteoclast activity is a key factor in osteoporosis pathogenesis. Farrerol is a typical natural flavanone and exhibits various pharmacological actions. However, the role and mechanism of action of farrerol in osteoclast differentiation regulation remain unclear. This study aimed to evaluate the effects and mechanism of farrerol on the inhibition of osteoclastogenesis. Tartrate-resistant acid phosphatase staining, F-actin staining, and the pit formation assay were performed to examine the differentiation and functions of osteoclasts in vitro. The expression of proteins associated with the nuclear factor kappa B and mitogen-activated protein kinase signaling pathways was analyzed by western blotting. Dual X-ray absorptiometry, microcomputed tomography, and histopathological and immunohistochemical analyses were performed to determine the therapeutic effect of farrerol in vivo bone loss prevention. The effects of farrerol on osteoblastic bone formation were assessed using alkaline phosphatase, alizarin red S staining, and calcein-alizarin red S double labeling. Farrerol inhibited osteoclastogenesis and bone resorption in osteoclasts by suppressing nuclear factor kappa B signaling rather than mitogen-activated protein kinase signaling in vitro. Farrerol protected mice against ovariectomy-induced bone loss by inhibiting osteoclast-mediated bone resorption, instead of promoting osteoblast-mediated bone formation in vivo. The findings of the current study revealed that farrerol is a potential therapeutic agent for osteoporosis.