Effects of novel lactoferrin peptides on LPS-induced alveolar bone destruction in a rat model.

To develop novel bovine lactoferrin (bLF) peptides targeting bLF-tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6) binding sites, we identified two peptides that could target bLF-TRAF6 binding sites using structural analysis. Moreover, another peptide that could bind to the TRAF6 dimerization area was selected from the bLF sequence. The effects of each peptide on cytokine expression in lipopolysaccharide (LPS)-stimulated osteoblasts (ST2) and on osteoclastogenesis were examined using an LPS-treated co-culture of primary bone marrow cells (BMCs) with ST2 cells and a single culture of osteoclast precursor cells (RAW-D) treated with soluble receptor activator of NF-κB ligand. Finally, the effectiveness of these peptides against LPS-induced alveolar bone destruction was assessed. Two of the three peptides significantly suppressed LPS-induced TNF-α and interleukin-1ß expression in ST2 cells. Additionally, these peptides inhibited and reversed LPS-induced receptor activator of NF-κB ligand (RANKL) upregulation and osteoprotegerin (OPG) downregulation, respectively. Furthermore, both peptides significantly reduced LPS-induced osteoclastogenesis in the BMC-ST2 co-culture and RANKL-induced osteoclastogenesis in RAW-D cells. In vivo, topical application of these peptides significantly reduced the osteoclast number by downregulating RANKL and upregulating OPG in the periodontal ligament. It is indicated that the novel bLF peptides can be used to treat periodontitis-associated bone destruction.

pH-Responsive Mesoporous Silica Nanoparticles Loaded with Naringin for Targeted Osteoclast Inhibition and Bone Regeneration.

Background: It is well-established that osteoclast activity is significantly influenced by fluctuations in intracellular pH. Consequently, a pH-sensitive gated nano-drug delivery system represents a promising therapeutic approach to mitigate osteoclast overactivity. Our prior research indicated that naringin, a natural flavonoid, effectively mitigates osteoclast activity. However, naringin showed low oral availability and short half-life, which hinders its clinical application. We developed a drug delivery system wherein chitosan, as gatekeepers, coats mesoporous silica nanoparticles loaded with naringin (CS@MSNs-Naringin). However, the inhibitory effects of CS@MSNs-Naringin on osteoclasts and the underlying mechanisms remain unclear, warranting further research. Methods: First, we synthesized CS@MSNs-Naringin and conducted a comprehensive characterization. We also measured drug release rates in a pH gradient solution and verified its biosafety. Subsequently, we investigated the impact of CS@MSNs-Naringin on osteoclasts induced by bone marrow-derived macrophages, focusing on differentiation and bone resorption activity while exploring potential mechanisms. Finally, we established a rat model of bilateral critical-sized calvarial bone defects, in which CS@MSNs-Naringin was dispersed in GelMA hydrogel to achieve in situ drug delivery. We observed the ability of CS@MSNs-Naringin to promote bone regeneration and inhibit osteoclast activity in vivo. Results: CS@MSNs-Naringin exhibited high uniformity and dispersity, low cytotoxicity (concentration≤120 µg/mL), and significant pH sensitivity. In vitro, compared to Naringin and MSNs-Naringin, CS@MSNs-Naringin more effectively inhibited the formation and bone resorption activity of osteoclasts. This effect was accompanied by decreased phosphorylation of key factors in the NF-κB and MAPK signaling pathways, increased apoptosis levels, and a subsequent reduction in the production of osteoclast-specific genes and proteins. In vivo, CS@MSNs-Naringin outperformed Naringin and MSNs-Naringin, promoting new bone formation while inhibiting osteoclast activity to a greater extent. Conclusion: Our research suggested that CS@MSNs-Naringin exhibited the strikingly ability to anti-osteoclasts in vitro and in vivo, moreover promoted bone regeneration in the calvarial bone defect.

In Vitro Assessment of 4-Acetyl-Antroquinonol B and Erinacine A in Suppressing Breast Cancer-Induced Osteoclastogenesis.

Bone metastasis in metastatic breast cancer commonly results in osteolytic lesions due to osteoclast activity, promoting bone destruction and tumor progression. The bioactive fungal isolates, 4-acetyl-antroquinonol B (4-AAQB) and erinacine A, have diverse pharmacological and biological activities. However, their effects on breast cancer bone metastasis treatment remain unclear. Our study aimed to examine the impact of 4-AAQB or erinacine A on breast cancer metastases in bone. The effects of 4-AAQB and erinacine A on breast cancer-induced osteoclastogenesis, breast cancer migration, production of prometastatic cytokine (TGF-ß) and marker (MMP-9), as well as potential MAPK signaling transductions were assessed. The results revealed that 4-AAQB and erinacine A effectively suppressed breast cancer-induced osteoclastogenesis and migration, and reduced TGF-ß and MMP-9 production via Erk or JNK signaling transductions, specifically in breast cancer cells or in breast cancer cells-induced osteoclasts. Based on these findings, either 4-AAQB or erinacine A showed promise in preventing breast cancer metastases in bone.

Korean Black Goat Extract Exerts Estrogen-like Osteoprotective Effects by Stimulating Osteoblast Differentiation in MC3T3-E1 Cells and Suppressing Osteoclastogenesis in RAW 264.7 Cells.

Postmenopausal osteoporosis, characterized by an imbalance between osteoclast-mediated bone resorption and osteoblast-driven bone formation, presents substantial health implications. In this study, we investigated the role of black goat extract (BGE), derived from a domesticated native Korean goat, estrogen-like activity, and osteoprotective effects in vitro. BGE's mineral and fatty acid compositions were analyzed via the ICP-AES method and gas chromatography-mass spectrometry, respectively. In vitro experiments were conducted using MCF-7 breast cancer cells, MC3T3-E1 osteoblasts, and RAW264.7 osteoclasts. BGE exhibits a favorable amount of mineral and fatty acid content. It displayed antimenopausal activity by stimulating MCF-7 cell proliferation and augmenting estrogen-related gene expression (ERα, ERß, and pS2). Moreover, BGE positively impacted osteogenesis and mineralization in MC3T3-E1 cells through Wnt/ß-catenin pathway modulation, leading to heightened expression of Runt-related transcription factor 2, osteoprotegerin, and collagen type 1. Significantly, BGE effectively suppressed osteoclastogenesis by curtailing osteoclast formation and activity in RAW264.7 cells, concurrently downregulating pivotal signaling molecules, including receptor activator of nuclear factor κ B and tumor necrosis factor receptor-associated factor 6. This study offers a shred of preliminary evidence for the prospective use of BGE as an effective postmenopausal osteoporosis treatment.

[Atorvastatin promotes the healing of alveolar bone defect in rats and its effect on Wnt/ß-catenin signaling pathway].

PURPOSE: To investigate the therapeutic effect of atorvastatin on alveolar bone defect model in rats, and to observe the effect of atorvastatin on Wnt/ß-catenin. METHODS: Thirty rats were randomly divided into normal group (group N), model group (group M) and atorvastatin administration group (group ATV). Except group N, bone defects were made in other rats' alveolar bone to construct alveolar bone defect model. After successful modeling, 20 mg/kg atorvastatin suspension was administered by gavage in group ATV, and the same amount of sodium carboxymethyl cellulose solution was administered by gavage in group N and group M for twenty-one days. After the last administration, tail vein blood was collected to detect the concentrations of serum osteoprotegerin (OPG), alkaline phosphatase (ALP) and osteocalcin (BPG). H-E staining was used to observe the pathological changes of maxillary defect area, and lane Sandhu score was performed. Tartrate resistant acid phosphatase(TRAP) staining was used to detect the number of osteoclasts in the defect area. Real time fluorescence quantitative PCR(RT-qPCR) and Western blot(WB) were used to detect Wnt, ß-catenin and Runx2 mRNA protein expression. Statistical analysis was performed with SPSS 23.0 software package. RESULTS: Compared with group N, the concentrations of OPG, ALP, BGP and Lane Sandhu score in group M decreased, and the number of osteoclasts increased. Compared with group M, the concentrations of OPG, ALP and BGP and lane Sandhu score in group ATV increased, and the number of osteoclasts decreased. After H-E staining, the amount of bone formation in maxillary defect area in group N was more,there was fewer bone tissues in the defect area in group M, the amount of bone tissues in the defect area increased in group ATV. Compared with group N, Wnt, ß-catenin and Runx2 mRNA protein decreased. Compared with group M, Wnt, ß-catenin and Runx2 mRNA protein expression increased. CONCLUSIONS: Atorvastatin can promote the healing of alveolar bone defect and accelerate bone reconstruction in rat models. This effect may be related to the activation of Wnt/ß-catenin signaling pathway.

Ginkgetin attenuates bone loss in OVX mice by inhibiting the NF-κB/IκBα signaling pathway.

Background: Osteoporosis is a disease associated with bone resorption, characterized primarily by the excessive activation of osteoclasts. Ginkgetin is a compound purified from natural ginkgo leaves which has various biological properties, including anti-inflammation, antioxidant, and anti-tumor effects. This study investigated the bone-protective effects of ginkgetin in ovariectomized (OVX) mice and explored their potential signaling pathway in inhibiting osteoclastogenesis in a mouse model of osteoporosis. Methods: Biochemical assays were performed to assess the levels of Ca, ALP, and P in the blood. Micro CT scanning was used to evaluate the impact of ginkgetin on bone loss in mice. RT-PCR was employed to detect the expression of osteoclast-related genes (ctsk, c-fos, trap) in their femoral tissue. Hematoxylin and eosin (H&E) staining was utilized to assess the histopathological changes in femoral tissue due to ginkgetin. The TRAP staining was used to evaluate the impact of ginkgetin osteoclast generation in vivo. Western blot analysis was conducted to investigate the effect of ginkgetin on the expression of p-NF-κB p65 and IκBα proteins in mice. Results: Our findings indicate that ginkgetin may increase the serum levels of ALP and P, while decreasing the serum level of Ca in OVX mice. H&E staining and micro CT scanning results suggest that ginkgetin can inhibit bone loss in OVX mice. The TRAP staining results showed ginkgetin suppresses the generation of osteoclasts in OVX mice. RT-PCR results demonstrate that ginkgetin downregulate the expression of osteoclast-related genes (ctsk, c-fos, trap) in the femoral tissue of mice, and this effect is dose-dependent. Western blot analysis results reveal that ginkgetin can inhibit the expression of p-NF-κB p65 and IκBα proteins in mice. Conclusion: Ginkgetin can impact osteoclast formation and activation in OVX mice by inhibiting the NF-κB/IκBα signaling pathway, thereby attenuating bone loss in mice.

Discovery of TCP-(MP)-caffeic acid analogs as a new class of agents for treatment of osteoclastic bone loss.

Inhibition of LSD1 was proposed as promising and attractive therapies for treating osteoporosis. Here, we synthesized a series of novel TCP-(MP)-Caffeic acid analogs as potential LSD1 inhibitors to assess their inhibitory effects on osteoclastogenesis by using TRAP-staining assay and try to explore the preliminary SAR. Among them, TCP-MP-CA (11a) demonstrated osteoclastic bone loss both in vitro and in vivo, showing a significant improvement in the in vivo effects compared to the LSD1 inhibitor GSK-LSD1. Additionally, we elucidated a mechanism that 11a and its precursor that 11e directly bind to LSD1/CoREST complex through FAD to inhibit LSD1 demethylation activity and influence its downstream IκB/NF-κB signaling pathway, and thus regulate osteoclastic bone loss. These findings suggested 11a or 11e as potential novel candidates for treating osteoclastic bone loss, and a concept for further development of TCP-(MP)-Caffeic acid analogs for therapeutic use in osteoporosis clinics.

Pulmonary osteoclast-like cells in silica induced pulmonary fibrosis.

The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored mechanisms of silica-induced pulmonary fibrosis in human lung samples collected from patients with occupational exposure to silica and in a longitudinal mouse model of silicosis using multiple modalities including whole-lung single-cell RNA sequencing and histological, biochemical, and physiologic assessments. In addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor κΒ ligand (RANKL) in pulmonary lymphocytes, and alveolar type II cells. Anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated pulmonary fibrosis. We conclude that silica induces differentiation of pulmonary osteoclast-like cells leading to progressive lung injury, likely due to sustained elaboration of bone-resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis.

Synthetic high-density lipoprotein (sHDL): a bioinspired nanotherapeutics for managing periapical bone inflammation.

Apical periodontitis (AP) is a dental-driven condition caused by pathogens and their toxins infecting the inner portion of the tooth (i.e., dental pulp tissue), resulting in inflammation and apical bone resorption affecting 50% of the worldwide population, with more than 15 million root canals performed annually in the United States. Current treatment involves cleaning and decontaminating the infected tissue with chemo-mechanical approaches and materials introduced years ago, such as calcium hydroxide, zinc oxide-eugenol, or even formalin products. Here, we present, for the first time, a nanotherapeutics based on using synthetic high-density lipoprotein (sHDL) as an innovative and safe strategy to manage dental bone inflammation. sHDL application in concentrations ranging from 25 µg to 100 µg/mL decreases nuclear factor Kappa B (NF-κB) activation promoted by an inflammatory stimulus (lipopolysaccharide, LPS). Moreover, sHDL at 500 µg/mL concentration markedly decreases in vitro osteoclastogenesis (P < 0.001), and inhibits IL-1α (P = 0.027), TNF-α (P = 0.004), and IL-6 (P < 0.001) production in an inflammatory state. Notably, sHDL strongly dampens the Toll-Like Receptor signaling pathway facing LPS stimulation, mainly by downregulating at least 3-fold the pro-inflammatory genes, such as Il1b, Il1a, Il6, Ptgs2, and Tnf. In vivo, the lipoprotein nanoparticle applied after NaOCl reduced bone resorption volume to (1.3 ± 0.05) mm3 and attenuated the inflammatory reaction after treatment to (1 090 ± 184) cells compared to non-treated animals that had (2.9 ± 0.6) mm3 (P = 0.012 3) and (2 443 ± 931) cells (P = 0.004), thus highlighting its promising clinical potential as an alternative therapeutic for managing dental bone inflammation.

Modulation of osteoclastogenesis by macrogeometrically designed hydrophilic dual acid-etched titanium surfaces.

The aim of this study was to evaluate the influence of implant macrodesign and surface hydrophilicity on osteoclast (OC) differentiation, activation, and survival in vitro. Titanium disks were produced with a sandblasted, dual acid-etched surface, with or without additional chemical modification for increasing hydrophilicity (SAE-HD and SAE, respectively) and different macrodesign comprising trapezoidal (HLX) or triangular threads (TMX). This study evaluated 7 groups in total, 4 of which were experimental: HLX/SAE-HD, HLX-SAE, TMX/SAE-HD, and TMX/SAE; and 3 control groups comprising OC differentiated on polystyrene plates (CCPC): a positive CCPC (+), a negative CCPC (-), and a lipopolysaccharide-stimulated assay positive control group, CCPC-LPS. Murine macrophage RAW264.7 cells were seeded on the disks, differentiated to OC (RAW-OC) by receptor activator of nuclear factor-κB ligand (RANKL) treatment and cultured for 5 days. Osteoclast differentiation and cell viability were respectively assessed by specific enzymatic Tartrate-Resistant Acid Phosphatase (TRAP) activity and MTT assays. Expression levels of various OC-related genes were measured at the mRNA level by quantitative polymerase chain reaction (qPCR). HLX/SAE-HD, TMX/SAE-HD, and HLX/SAE significantly suppressed OC differentiation when compared to CCPC (+). Cell viability was significantly increased in TMX/SAE and reduced in HLX/SAE-HD. In addition, the expression of Interleukin (IL)-6 and Tumour Necrosis Factor (TNF)-α was upregulated in TMX/SAE-HD compared to CCPC (+). Hydrophilic surfaces negatively modulate macrophage/osteoclast viability. Specifically, SAE-HD with double triangular threads increases the cellular pro-inflammatory status, while surface hydrophilicity and macrodesign do not seem to have a distinct impact on osteoclast differentiation, activation, or survival.

Gallium-containing mesoporous nanoparticles influence in-vitro osteogenic and osteoclastic activity.

Mesoporous silica nanoparticles were synthesized using a microemulsion-assisted sol-gel method, and calcium, gallium or a combination of both, were used as dopants. The influence of these metallic ions on the physicochemical properties of the nanoparticles was investigated by scanning and transmission electron microscopy, as well as N2 adsorption-desorption methods. The presence of calcium had a significant impact on the morphology and textural features of the nanoparticles. The addition of calcium increased the average diameter of the nanoparticles from 80 nm to 150 nm, while decreasing their specific surface area from 972 m2/g to 344 m2/g. The nanoparticles of all compositions were spheroidal, with a disordered mesoporous structure. An ion release study in cell culture medium demonstrated that gallium was released from the nanoparticles in a sustained manner. In direct contact with concentrations of up to 100 µg/mL of the nanoparticles, gallium-containing nanoparticles did not exhibit cytotoxicity towards pre-osteoblast MC3T3-E1 cells. Moreover, in vitro cell culture tests revealed that the addition of gallium to the nanoparticles enhanced osteogenic activity. Simultaneously, the nanoparticles disrupted the osteoclast differentiation of RAW 264.7 macrophage cells. These findings suggest that gallium-containing nanoparticles possess favorable physicochemical properties and biological characteristics, making them promising candidates for applications in bone tissue regeneration, particularly for unphysiological or pathological conditions such as osteoporosis.

Attenuating bone loss in osteoporosis: the potential of corylin (CL) as a therapeutic agent.

The global prevalence of osteoporosis is being exacerbated by the increasing number of aging societies and longer life expectancies. In response, numerous drugs have been developed in recent years to mitigate bone resorption and enhance bone density. Nonetheless, the efficacy and safety of these pharmaceutical interventions remain constrained. Corylin (CL), a naturally occurring compound derived from the anti-osteoporosis plant Psoralea corylifolia L., has exhibited promising potential in impeding osteoclast differentiation. This study aims to evaluate the effect and molecular mechanisms of CL regulating osteoclast differentiation in vitro and its potential as a therapeutic agent for osteoporosis treatment in vivo. Our investigation revealed that CL effectively inhibits osteoclast formation and their bone resorption capacity by downregulating the transcription factors NFATc1 and c-fos, consequently resulting in the downregulation of genes associated with bone resorption. Furthermore, it has been observed that CL can effectively mitigate the migration and fusion of pre-osteoclast, while also attenuating the activation of mitochondrial mass and function. The results obtained from an in vivo study have demonstrated that CL is capable of attenuating the bone loss induced by ovariectomy (OVX). Based on these significant findings, it is proposed that CL exhibits considerable potential as a novel drug strategy for inhibiting osteoclast differentiation, thereby offering a promising approach for the treatment of osteoporosis.

Prolonged Cadmium Exposure and Osteoclastogenesis: A Mechanistic Mouse and in Vitro Study.

BACKGROUND: Cadmium (Cd) is a highly toxic and widespread environmental oxidative stressor that causes a myriad of health problems, including osteoporosis and bone damage. Although nuclear factor erythroid 2-related factor 2 (NRF2) and its Cap 'n' Collar and basic region Leucine Zipper (CNC-bZIP) family member nuclear factor erythroid 2-related factor 1 (NRF1) coordinate various stress responses by regulating the transcription of a variety of antioxidant and cytoprotective genes, they play distinct roles in bone metabolism and remodeling. However, the precise roles of both transcription factors in bone loss induced by prolonged Cd exposure remain unclear. OBJECTIVES: We aimed to understand the molecular mechanisms underlying Cd-induced bone loss, focusing mainly on the roles of NRF2 and NRF1 in osteoclastogenesis provoked by Cd. METHODS: Male wild-type (WT), global Nrf2-knockout (Nrf2-/-) and myeloid-specific Nrf2 knockout [Nrf2(M)-KO] mice were administered Cd (50 or 100 ppm) via drinking water for 8 or 16 wk, followed by micro-computed tomography, histological analyses, and plasma biochemical testing. Osteoclastogenesis was evaluated using bone marrow-derived osteoclast progenitor cells (BM-OPCs) and RAW 264.7 cells in the presence of Cd (10 or 20 nM) with a combination of genetic and chemical modulations targeting NRF2 and NRF1. RESULTS: Compared with relevant control mice, global Nrf2-/- or Nrf2(M)-KO mice showed exacerbated bone loss and augmented osteoclast activity following exposure to 100 ppm Cd in drinking water for up to 16 wk. In vitro osteoclastogenic analyses suggested that Nrf2-deficient BM-OPCs and RAW 264.7 cells responded more robustly to low levels of Cd (up to 20 nM) with regard to osteoclast differentiation compared with WT cells. Further mechanistic studies supported a compensatory up-regulation of long isoform of NRF1 (L-NRF1) and subsequent induction of nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1 (NFATc1) as the key molecular events in the Nrf2 deficiency-worsened and Cd-provoked osteoclastogenesis. L-Nrf1 silenced (via lentiviral means) Nrf2-knockdown (KD) RAW cells exposed to Cd showed dramatically different NFATc1 and subsequent osteoclastogenesis outcomes compared with the cells of Nrf2-KD alone exposed to Cd, suggesting a mitigating effect of the Nrf1 silencing. In addition, suppression of reactive oxygen species by exogenous antioxidants N-acetyl-l-cysteine (2 mM) and mitoquinone mesylate (MitoQ; 0.2µM) mitigated the L-NRF1-associated effects on NFATc1-driven osteoclastogenesis outcomes in Cd-exposed Nrf2-KD cells. CONCLUSIONS: This in vivo and in vitro study supported the authors' hypothesis that Cd exposure caused bone loss, in which NRF2 and L-NRF1 responded to Cd and osteoclastogenic stimuli in a cooperative, but contradictive, manner to coordinate Nfatc1 expression, osteoclastogenesis and thus bone homeostasis. Our study suggests a novel strategy targeting NRF2 and L-NRF1 to prevent and treat the bone toxicity of Cd. https://doi.org/10.1289/EHP13849.

The Role of Traditional Chinese Medicines in the Treatment of Osteoporosis.

Osteoporosis (OP) represents a substantial public health issue and is associated with increasing rates of morbidity and mortality. It is characterized by reduced bone mineral density, deterioration of bone tissue quality, disruption of the microarchitecture of bones, and compromised bone strength. These changes may be attributed to the following factors: intercellular communication between osteoblasts and osteoclasts; imbalanced bone remodeling; imbalances between osteogenesis and adipogenesis; imbalances in hormonal regulation; angiogenesis; chronic inflammation; oxidative stress; and intestinal microbiota imbalances. Treating a single aspect of the disease is insufficient to address its multifaceted nature. In recent decades, traditional Chinese medicine (TCM) has shown great potential in the treatment of OP, and the therapeutic effects of Chinese patent drugs and Chinese medicinal herbs have been scientifically proven. TCMs, which contain multiple components, can target the diverse pathogeneses of OP through a multitargeted approach. Herbs such as XLGB, JTG, GSB, Yinyanghuo, Gusuibu, Buguzhi, and Nvzhenzi are among the TCMs that can be used to treat OP and have demonstrated promising effects in this context. They exert their therapeutic effects by targeting various pathways involved in bone metabolism. These TCMs balance the activity of osteoblasts (bone-forming cells) and osteoclasts (bone-resorbing cells), and they exhibit anti-inflammatory, immunomodulatory, anti-oxidative, and estrogen-like functions. These multifaceted mechanisms underlie the efficacy of these herbs in the management and treatment of OP. Herein, we examine the efficacy of various Chinese herbs and Chinese patent drugs in treating OP by reviewing previous clinical trials and basic experiments, and we examine the potential mechanism of these therapies to provide evidence regarding the use of TCM for treating OP.

Dietary Puerarin Translocates to Femur and Suppresses Osteoclast Differentiation in Ovariectomized Mice.

Osteoporosis is characterized by bone loss and deterioration in bone microstructure, leading to bone fragility. It is strongly correlated with menopause in women. Previously, we reported that diets supplemented with a kudzu (Pueraria lobata) vine extract suppressed bone resorption in ovariectomized (OVX) mice, a postmenopausal model. The main isoflavone in kudzu is puerarin (daidzein-8-C-glycoside). Puerarin (daidzein-8-C-glycoside), which is main isoflavone of kudzu, probably contributes to the beneficial effect. However, the underlying mechanism is unclear. Therefore, the nutrikinetics of puerarin and the comparison with the suppressive effects of kudzu isoflavones on osteoclast differentiation was examined in this study. We demonstrated that orally administered puerarin was absorbed from the gut and entered the circulation in an intact form. In addition, puerarin accumulated in RAW264.7 pre-osteoclast cells in a time-dependent manner. Tartrate-resistant acid phosphatase activity was decreased by puerarin treatment in a concentration-dependent manner in RAW264.7 cells stimulated with the receptor activator of nuclear factor kappa-B ligand. Ovariectomy-induced elevated bone resorption was suppressed, and the fragile bone strength was improved by puerarin ingestion in the diet. These findings suggested that orally administered puerarin was localized in bone tissue and suppressed bone resorption and osteoclastogenesis in ovariectomized mice.

In vitro and in vivo studies on exogenous polyamines and α-difluoromethylornithine to enhance bone formation and suppress osteoclast differentiation.

Exogenous polyamines, including putrescine (PUT), spermidine (SPD), and spermine (SPM), and the irreversible inhibitor of the rate-limiting enzyme ornithine decarboxylase (ODC) of polyamine biosynthesis, α-difluoromethylornithine (DFMO), are implicated as stimulants for bone formation. We demonstrate in this study the osteogenic potential of exogenous polyamines and DFMO in human osteoblasts (hOBs), murine monocyte cell line RAW 264.7, and an ovariectomized rat model. The effect of polyamines and DFMO on hOBs and RAW 264.7 cells was studied by analyzing gene expression, alkaline phosphatase (ALP) activity, tartrate-resistant acid phosphatase (TRAP) activity, and matrix mineralization. Ovariectomized rats were treated with polyamines and DFMO and analyzed by micro computed tomography (micro CT). The mRNA level of the early onset genes of osteogenic differentiation, Runt-related transcription factor 2 (Runx2) and ALP, was significantly elevated in hOBs under osteogenic conditions, while both ALP activity and matrix mineralization were enhanced by exogenous polyamines and DFMO. Under osteoclastogenic conditions, the gene expression of both receptor activator of nuclear factor-κB (RANK) and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) was reduced, and TRAP activity was suppressed by exogenous polyamines and DFMO in RAW 264.7 cells. In an osteoporotic animal model of ovariectomized rats, SPM and DFMO were found to improve bone volume in rat femurs, while trabecular thickness was increased in all treatment groups. Results from this study provide in vitro and in vivo evidence indicating that polyamines and DFMO act as stimulants for bone formation, and their osteogenic effect may be associated with the suppression of osteoclastogenesis.

Preclinical evaluation of ELP-004 in mice.

This study provides a detailed understanding of the preclinical pharmacokinetics and metabolism of ELP-004, an osteoclast inhibitor in development for the treatment of bone erosion. Current treatments for arthritis, including biological disease-modifying antirheumatic drugs, are not well-tolerated in a substantial subset of arthritis patients and are expensive; therefore, new treatments are needed. Pharmacokinetic parameters of ELP-004 were tested with intravenous, oral, and subcutaneous administration and found to be rapidly absorbed and distributed. We found that ELP-004 was non-mutagenic, did not induce chromosome aberrations, non-cardiotoxic, and had minimal off-target effects. Using in vitro hepatic systems, we found that ELP-004 is primarily metabolized by CYP1A2 and CYP2B6 and predicted metabolic pathways were identified. Finally, we show that ELP-004 inhibits osteoclast differentiation without suppressing overall T-cell function. These preclinical data will inform future development of an oral compound as well as in vivo efficacy studies in mice.

Biological and mechanical performance of calcium phosphate cements modified with phytic acid.

Calcium phosphate cements, primarily brushite cements, require the addition of setting retarders to ensure adequate processing time and processability. So far, citric acid has been the primary setting retarder used in this context. Due to the poor biocompatibility, it is crucial to explore alternative options for better processing. In recent years, the setting retarder phytic acid (IP6) has been increasingly investigated. This study investigates the biological behaviour of calcium phosphate cements with varying concentrations of IP6, in addition to their physical properties. Therefore cytocompatibility in vitro testing was performed using osteoblastic (MG-63) and osteoclastic (RAW 264.7 differentiated with RANKL) cells. We could demonstrate that the physical properties like the compressive strength of specimens formed with IP6 (brushite_IP6_5 = 11.2 MPa) were improved compared to the reference (brushite = 9.8 MPa). In osteoblast and osteoclast assays, IP6 exhibited significantly better cytocompatibility in terms of cell activity and cell number for brushite cements up to 11 times compared to the brushite reference. In contrast, the calcium-deficient hydroxyapatite (CDHA) cements produced similar results for IP6 (CDHA_IP6_0.25 = 27.0 MPa) when compared to their reference (CDHA = 21.2 MPa). Interestingly, lower doses of IP6 were found to be more effective than higher doses with up to 3 times higher. Additionally, IP6 significantly increased degradation in both passive and active resorption. For these reasons, IP6 is emerging as a strong new competitor to established setting retarders such as citric acid. These cements have potential applications in bone augmentation, the stabilisation of non-load bearing fractures (craniofacial), or the cementation of metal implants.

Engineered Niobium Carbide MXenzyme-Integrated Self-Adaptive Coatings Inhibiting Periprosthetic Osteolysis by Orchestrating Osteogenesis-Osteoclastogenesis Balance.

Periprosthetic osteolysis induced by the ultrahigh-molecular-weight polyethylene (UHMWPE) wear particles is a major complication associated with the sustained service of artificial joint prostheses and often necessitates revision surgery. Therefore, a smart implant with direct prevention and repair abilities is urgently developed to avoid painful revision surgery. Herein, we fabricate a phosphatidylserine- and polyethylenimine-engineered niobium carbide (Nb2C) MXenzyme-coated micro/nanostructured titanium implant (PPN@MNTi) that inhibits UHMWPE particle-induced periprosthetic osteolysis. The specific mechanism by which PPN@MNTi operates involves the bioresponsive release of nanosheets from the MNTi substrate within an osteolysis microenvironment, initiated by the cleavage of a thioketal-dopamine molecule sensitive to reactive oxygen species (ROS). Subsequently, functionalized Nb2C MXenzyme could target macrophages and escape from lysosomes, effectively scavenging intracellular ROS through its antioxidant nanozyme-mimicking activities. This further achieves the suppression of osteoclastogenesis by inhibiting NF-κB/MAPK and autophagy signaling pathways. Simultaneously, based on the synergistic effect of MXenzyme-integrated coatings and micro/nanostructured topography, the designed implant promotes the osteogenic differentiation of bone mesenchymal stem cells to regulate bone homeostasis, further achieving advanced osseointegration and alleviable periprosthetic osteolysis in vivo. This study provides a precise prevention and repair strategy of periprosthetic osteolysis, offering a paradigm for the development of smart orthopedic implants.

Destruxin E backbone modification effects on osteoclast Morphology: Synthesis and SAR study of N-Desmethyl and N-Methyl analogs.

The design and synthesis of N-desmethyl and N-methyl destruxin E analogs have been demonstrated. The X-ray single crystal structure of destruxin E (1a) revealed a stable three-dimensional (3D) structure, including a s-cis amide bond at the MeVal-MeAla moiety and two intramolecular hydrogen bonds between NH(ß-Ala) and OC(Ile) and between NH(Ile) and OC(ß-Ala). N-Desmethyl analogs 2a (MeAla â†’ Ala) and 2b (MeVal â†’ Val) were synthesized through macrolactonization similar to our previously reported synthesis of 1a. Conversely, for the synthesis of N-methyl analogs 2c (Ile â†’ MeIle) and 2d (ß-Ala â†’ Meß-Ala), macrolactonization did not proceed; therefore, cyclization precursors 10c and 10d were designed to maintain the intramolecular hydrogen bonds described above during their cyclization. The macrolactamization proceeded despite the presence of a less reactive N-methylamino group at the N-terminus in both cases. Analog 2a, which exhibits multiple conformers in solutions, was inactive at 50 µM, whereas analog 2b, which exhibits a conformation similar to that of 1a in solutions, exhibited morphological changes against osteoclast-like multinuclear cells at 1.6 µM. The activity of the MeIle analog 2c, which cannot take the intramolecular hydrogen bond (Ile)NH•••OC(ß-Ala) in 1a, was markedly diminished compared with that of 1a, and that of the Meß-Ala analog 2d, which cannot take the intramolecular hydrogen bond (ß-Ala)NH•••OC(Ile) in 1a, was further reduced to one-fourth of that of 2c. The overall results indicate that both the s-cis amide bond at the MeVal-MeAla moiety and two intramolecular hydrogen bonds (ß-Ala)NH•••OC(Ile) and (Ile)NH•••OC(ß-Ala) are important for constraining the conformation of the macrocyclic peptide backbone in destruxin E, thereby exhibiting its potent biological activity.