Colorimetric and fluorescent TRAP assays for visualising and quantifying fish osteoclast activity

Histochemical detection of tartrate-resistant acid phosphatase (TRAP) activity is a fundamental technique for visualizing osteoclastic bone resorption and assessing osteoclast activity status in tissues. This approach has mostly employed colorimetric detection, which has limited quantification of activity in situ and co-labelling with other skeletal markers. Here we report simple colorimetric and fluorescent TRAP assays in zebrafish and medaka, two important model organisms for investigating the pathogenesis of bone disorders. We show fluorescent TRAP staining, utilising the ELF97 substrate, is a rapid, robust and stable system to visualise and quantify osteoclast activity in zebrafish, and is compatible with other fluorescence stains, transgenic lines and antibody approaches. Using this approach, we show that TRAP activity is predominantly found around the base of the zebrafish pharyngeal teeth, where osteoclast activity state appears to be heterogeneous.

Synergistic Effect of Biphasic Calcium Phosphate and Platelet-Rich Fibrin Attenuate Markers for Inflammation and Osteoclast Differentiation by Suppressing NF-κB/MAPK Signaling Pathway in Chronic Periodontitis

Background: Periodontitis is characterized by excessive osteoclastic activity, which is closely associated with inflammation. It is well established that MAPK/NF-kB axis is a key signaling pathway engaged in osteoclast differentiation. It is stated that that biphasic calcium phosphate (BCP) and platelet-rich fibrin (PRF) have significant antiostoeclastogenic effects in chronic periodontitis. Objective: We aimed to elucidate the synergetic effect of PRF/BCP involvement of the nuclear factor kappa–light–chain–enhancer of activated B cells (NF-kB) and the mitogen-activated protein kinase (MAPK) signaling pathway in osteoclast differentiation in chronic periodontitis. Methods: We induced osteoclast differentiation in vitro using peripheral blood mononuclear cells (PBMCs) derived from patients with chronic periodontitis. We assessed osteoclast generation by tartrate-resistant acid phosphatase (TRAP) activity, proinflammatory cytokines were investigated by ELISA and NF-κB, and IKB by immunoblot, respectively. MAPK proteins and osteoclast transcription factors were studied by Western blot analysis and osteoclast transcriptional genes were assessed by RT-PCR. Results: The results showed that the potent inhibitory effect of PRF/BCP on osteoclastogenesis was evidenced by decreased TRAP activity and the expression of transcription factors, NFATc1, c-Fos, and the osteoclast marker genes, TRAP, MMP-9, and cathepsin-K were found to be reduced. Further, the protective effect of PRF/BCP on inflammation-mediated osteoclastogenesis in chronic periodontitis was shown by decreased levels of proinflammatory cytokines, NF-kB, IKB, and MAPK proteins. Conclusions: PRF/BCP may promote a synergetic combination that could be used as a strong inhibitor of inflammation-induced osteoclastogenesis in chronic periodontitis.

Inhibitory Effect of Asplenium incisum on Bacterial Growth, Inflammation, and Osteoclastogenesis

Background and Objectives:Asplenium incisum, a natural plant, is known to possess numerous pharmacological and biochemical properties. However, the inhibitory effect of A. incisum against Porphyromonas gingivalis and other factors related to periodontal disease have not yet been demonstrated. This study aimed to investigate the potential of A. incisum extract as a phytotherapeutic candidate for improving periodontal diseases by assessing its antibacterial, anti-inflammatory, and anti-osteoclastogenic activities. Materials and Methods: The inhibition of proliferation of P. gingivalis by A. incisum and the sustainability of its antibacterial activity were evaluated in this study. The production of inflammatory cytokines (tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)) and nitric oxide (NO) from lipopolysaccharide-stimulated RAW 264.7 cells was assessed using an enzyme-linked immunosorbent assay. To identify the anti-osteoclastogenic activity, tartrate-resistant acid phosphatase (TRAP) staining and TRAP activity analyses were performed on bone marrow macrophages. Results: The proliferation of P. gingivalis was significantly inhibited by A. incisum (p < 0.001), and the antibacterial activity was sustained for up to 3 days. A. incisum showed anti-inflammatory activities by significantly decreasing the release of TNF-α, IL-6 (p < 0.05), and NO (p < 0.01). In addition, A. incisum significantly suppressed TRAP-positive cells and TRAP activity (at 30 μg/mL, p < 0.01) without causing any cytotoxicity (p > 0.05). Conclusions:A. incisum showed antibacterial, anti-inflammatory, and anti-osteoclastogenic activities, suggesting it has strong therapeutic potential against periodontal diseases.

Use of Therapeutic Pathogen Recognition Receptor Ligands for Osteo-Immunomodulation

Therapeutic pathogen recognition receptor (PRR) ligands are reaching clinical practice following their ability to skew the immune response in a specific direction. We investigated the effects of various therapeutic PRR ligands on bone cell differentiation and inflammation. Following stimulation, alkaline phosphatase (ALP) activity (Day 10), osteocalcin, osteonectin expression (Day 14), and calcium deposition (Day 21) were quantified in bone marrow-derived human mesenchymal stem cells (hMSCs). The osteoclastogenic response was determined by measuring tartrate-resistant acid phosphate (TRAP) activity in human monocytes. TNF-α, IL-6, IL-8, and IL-10 expressions were measured by enzyme-linked immunosorbent assay as an indicator of the ligands’ inflammatory properties. We found that nucleic acid-based ligands Poly(I:C) and CpG ODN C increased early ALP activity in hMSCs by 4-fold without affecting osteoclast formation. These ligands did not enhance expression of the other, late osteogenic markers. MPLA, Curdlan, and Pam3CSK4 did not affect osteogenic differentiation, but inhibited TRAP activity in monocytes, which was associated with increased expression of all measured cytokines. Nucleic acid-based ligands are identified as the most promising osteo-immunomodulators, as they favor early osteogenic differentiation without inducing an exaggerated immune-cell mediated response or interfering in osteoclastogenesis and thus can be potentially harnessed for multifunctional coatings for bone biomaterials.

Keel-bone fractures are associated with bone quality differences in laying hens

This study aimed to investigate the relationship between bone quality in terms of metabolism, homeostasis of elements, bone mineral density (BMD), and microstructure and keel-bone fractures in laying hens (Gallusgallusdomesticus). One hundred and twenty 17 week old Lohmann White laying hens with normal keel bones were individually housed in furnished cages for 25 weeks. Birds were then euthanased and dissected to assess keel-bone status at 42 weeks. Serum and keel-bone samples from normal keel (NK) and fractured keel (FK) hens were collected to determine the previously mentioned bone quality parameters. The results showed FK hens to have higher levels of the components of osteocalcin, greater alkaline phosphatase activity in serum and keel bones, and greater tartrate-resistant acid phosphatase (TRAP) activity in keel bones, compared to NK hens. Additionally, FK hens also had higher concentrations of Li, B, K, Cu, As, Se, Sn, Hg, and Pb, but lower concentrations of Na, P, and Ca. Moreover, FK hens showed decreased bone microstructural parameters including bone volume/tissue volume, trabecular number, degree of anisotropy, connectivity density, and BMD, but increased trabecular separation. Meanwhile, no differences were detected in serum TRAP activity, trabecular thickness, bone surface, or bone surface/bone volume. Results showed laying hens with keel-bone fractures to have differences in bone metabolism, elements of homeostasis, bone microstructure parameters, and BMD. These results suggest that keel-bone fractures may be associated with bone quality.

VILLOUS LYMPHOCYTES IN BLOOD AND BONE MARROW IN SOME FORMS OF B-CELL LYMPHOID MALIGNANCIES

The cytological and immunocytochemical features of the lymphocytes with villous morphology in peripheral blood and bone marrow in some B-lymphoproliferative disorders were studied. The diagnosis of hairy cell leukemia, a hairy cell leukemia variant, splenic marginal zone lymphoma and splenic diffuse red pulp small B-cell lymphoma was ascertained in accordance with the new revision of the WHO classification (2016). The neoplastic cells of hairy cell leukemia were determined by the presence of high tartrate resistant acid phosphatase (TRAP) activity. Cell surface expression of CD19, CD20 and CD21 antigens was detected. Also, the expression of CD25, CD103 and CD200, and in some cases cyclin D1, was found out. CD5, CD10 and CD23 were not detected. The immunophenotype of cells in splenic marginal zone lymphoma with villous processes also corresponded to the mature B cells. The expression of CD19, CD20 and CD21 was observed in all cases, CD11c – in 50% of patients, CD25 or CD5 – in 10% of patients. In 80% of patients, the pathologic cells did not show TRAP activity. In the bone marrow and peripheral blood cells of patients with diffuse red pulp lymphoma, TRAP activity was not detected. An immunophenotype in the hairy cell leukemia variant was different from those of classic HCL (CD19+CD20+CD22+CD103+CD11c+CD5–CD10–CD23–). Characterized immunophenotypical markers, which have differential diagnostic values in several forms of lymphoid tumors of B cell origin, will be important for the choice of treatment methods and prognosis

In Vitro Antiosteoporosis Activity and Hepatotoxicity Evaluation in Zebrafish Larvae of Bark Extracts of Prunus jamasakura Medicinal Plant

Osteoporosis is one of the main health problems in the world today characterized by low bone mass and deterioration in bone microarchitecture. In recent years, the use of natural products approach to treat it has been in the increase. In this study, in vitro antiosteoporosis activity and hepatotoxicity of P. jamasakura bark extracts were evaluated. Methods. Mouse bone marrow macrophage (BMM) cells were incubated with tartrate-resistant acid phosphate (TRAP) buffers and p-nitrophenyl phosphate and cultured with different P. jamasakura bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 μg/ml in the presence of the receptor activator of nuclear factor kappa-Β ligand (RANKL) for 6 days. The osteoclast TRAP activity and cell viability were measured. Nitric oxide (NO) assay was conducted using murine macrophage-like RAW 264.7 cells treated with P. jamasakura ethanolic and methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, 50, 100, and 200 μg/ml. For hepatotoxicity assessment, zebrafish larvae were exposed to P. jamasakura bark extracts, 0.05% dimethyl sulfoxide as a negative control, and 5 μM tamoxifen as a positive control. The surviving larvae were anesthetized and assessed for hepatocyte apoptosis. Results. TRAP activity was significantly inhibited (p < 0.001) at all concentrations of P. jamasakura extracts compared to the control treatment. At 50 μg/ml, both ethanolic and methanolic extracts of P. jamasakura exhibited significant (p < 0.01) BMM cell viability compared to the control treatment. P. jamasakura ethanolic and methanolic extracts had significant inhibitory (p < 0.01) effects on lipopolysaccharide (LPS)-induced NO production at 200 μg/ml and exhibited significant (p < 0.01) and (p < 0.05) stimulative effects, respectively, on RAW 264.7 cell viability. No overt hepatotoxicity was observed in the liver of zebrafish larvae in any of the treatments. Conclusion. The TRAP activity of P. jamasakura bark gives a foundation for further studies to enhance future development of antiosteoporosis drug.

Scopoletin and Coumarin Attenuate Aberrant Bone Remodeling in Glucose-exposed Osteoblasts and Osteoclasts (P06-062-19)

Objectives Diabetes mellitus is a complex disease with harmful effects on the osteoblast and osteoclast activity. Type 2 diabetic patients have normal or high bone mineral density but associated with an increased risk of fractures. Coumarins (1-benzopyran-2-one) are chemical compounds in the benzopyrone class of organic compounds found in many plants. The purpose of this study was to identify that hyperglycemia resulted in impaired bone remodeling and to examine whether coumarins were capable of preventing diabetic osteoporosis. Methods The in vitro study employed osteoblastic MC3T3-E1 cells that were exposed to 33 mM glucose for 6 days in the presence of 20 μM coumarins of scopoletin, aesculetin and coumarin. Alkaline phosphatase (ALP) activity was quantitatively determined in osteoblastic MC3T3-E1 cells by using stable p-nitrophenyl phosphate. In addition, murine macrophage Raw 264.7 cells were differentiated with receptor activator of nuclear factor-κΒ ligand (RANKL) in 33 mM glucose and 20 μM coumarins for 5 days. Tartrate-resistant acid phosphatase (TRAP) activity was measured using an assay kit. Results High glucose attenuated the ALP activity of osteoblastic MC3T3-E1 cells, which was enhanced by treating scopoletin, aesculetin and coumarin to cells. In addition, 33 mM glucose diminished TRAP activity in RANKL-differentiated Raw 264.7 macrophages, indicating that bone remodeling was impaired in diabetic osteoclasts. In contrast, scopoletin and coumarin elevated osteoclastic differentiation and activation. Interestingly, coumarin was highly effective in balancing bone turnover of osteoblasts and osteoclasts. Conclusions These results demonstrate that glucose evoked abnormal bone remodeling leading to diabetic bone disorder. In addition, coumarins such as scopoletin and coumarin ameliorated bone remodeling impaired in diabetic osteoblasts and osteoclasts. These findings suggest the possibility that coumarins might be a potential agent for the treatment of diabetic osteoporosis. Funding Sources This work (Grants No. C0501612) was supported by project for Cooperative R&D between Industry, Academy, and Research Institute funded Korea Ministry of SMEs and Startups in 20.

Inhibition of Osteoclast Activation and Bone Resorption by Korean Thistle Ethanol Extracts in Receptor Activator of NF‐κB Ligand-differentiated Murine Macrophages (P06-061-19)

Objectives Enhanced bone resorption due to osteoclast activation cause skeletal diseases such as osteoporosis. Botanical antioxidants are being increasingly investigated for their health-promoting effects on bone. Korean thistle (Cirsium setidens Nakai), a wild perennial, is widely consumed as a food and traditional medicine in Korea and contains flavone-type flavonoids of luteolin, apigenin and acacetin with anti-inflammatory and antioxidant activities. This study examined whether Korean thistle extracts and their component acacetin inhibited osteoclast activation in receptor activator of NF‐κB ligand (RANKL)-differentiated murine macrophages. Methods RAW 264.7 murine macrophages were incubated with 10–20 μg/ml Korean thistle extracts and 20 μM acacetin for 5 days in the presence of 50 ng/ml RANKL. Tartrate-resistance acid phosphatase (TRAP) staining and its activity measurement were performed. Western blotting was done with target proteins involved in the osteoclast activation. Results Non-toxic Korean thistle extracts inhibited RANKL-induced formation of multinucleated osteoclasts and diminished TRAP activity enhanced in the osteoclast differentiation process. When Korean thistle extracts were treated to RANKL-exposed macrophages, the bone-resorbing activity was highly attenuated. The treatment of murine macrophages with 20 μg/ml Korean thistle extracts reduced cellular expression of carbonic anhydrase II, vacuolar-type H(+)-ATPase D2 and integrin αvβ3 elevated by RANKL, all involved in the bone resorption. Additionally, Korean thistle extracts reduced the expression of paxillin and cortactin elevated by RANKL, being concurrent with inhibition of induction of TRAF6, Src and PI3K. Korean thistle extracts blocked the formation of actin rings of osteoclasts enhanced by RANKL. Furthermore, the Korean thistle component, acacetin, diminished RANKL-promoted TRAP activity. Conclusions Korean thistle extracts and acacetin deterred preosteoclasts from the cell-cell fusion and the organization of the cytoskeleton for bone resorption. Korean thistle and its effective flavonoid acacetin could be natural therapeutic agents combating osteoclastogenesis. Funding Sources This work (Grants No. C0501612) was supported by project for Cooperative R&D between Industry, Academy, and Research Institute funded Korea Ministry of SMEs and Startups in 20.

Lycopus lucidus Turcz Inhibits the Osteoclastogenesis in RAW 264.7 Cells and Bone Loss in Ovariectomized Rat Model

Lycopus lucidus (LL) is a perennial herb that is traditionally used in Asia to treat edema, wound healing, and gynecological diseases such as irregular menstruation and menstrual pain. We hypothesized that LL would decrease the risk of developing osteoporosis, which is a condition related to gynecological diseases. In this study, we aimed to investigate the effect of a water extract of LL on osteoclastogenesis in vitro and osteoporosis in vivo. In vitro study, we used RAW 264.7 cells as osteoclast precursor cell. Osteoclast differentiation was induced by receptor activator nuclear factor-kappa B ligand (RANKL). We investigated the effect of LL on RANKL-induced osteoclastogenesis, tartrate-resistant acid phosphatase (TRAP) activity, and osteoclast-related genes. In vivo study, we used ovariectomized- (OVX-) induced osteoporosis rat model. OVX-induced Sprague-Dawley rats were randomly separated into sham, OVX, 17β-estradiol (100 μg/kg), wLL-L (15.2 mg/kg), and wLL-H (152 mg/kg) groups. Drugs were administered orally once daily for 9 weeks. wLL inhibited the formation of TRAP-positive osteoclasts; TRAP activity; pit formation; transcription factors (the nuclear factor of activated T-cell cytoplasmic 1 and c-fos); and osteoclast-related genes such as TRAP, carbonic anhydrase II, cathepsin K, osteoclast-associated receptor, and the d2 isoform of the vacuolar ATPase Vo domain. Also, wLL prevented loss of the trabecular area in the OVX femur without change of estrogen level. These results indicate that wLL is able to inhibit osteoclastogenesis and protect bone loss in the OVX-induced osteoporosis model without the influence of hormones like estrogen.