Tartrate-resistant Acid Phosphatase Research Articles

Effects of systemic ozone administration on the fresh extraction sockets healing: a histomorphometric and immunohistochemical study in rats.

Studies have highlighted numerous benefits of ozone therapy in the field of medicine and dentistry, including its antimicrobial efficacy against various pathogenic microorganisms, its ability to modulate the immune system effectively, reduce inflammation, prevent hypoxia, and support tissue regeneration. However, its effects on dental extraction healing remain to be elucidated. .Therefore, this study aimed to evaluate the effects of systemically administered ozone (O3) at different doses in the healing of dental extraction sockets in rats. To this end, 72 Wistar rats were randomly divided into four groups after extraction of the right upper central incisor: Group C - control, no systemic treatment; Group OZ0.3 - animals received a single dose of 0.3 mg/kg O3; Group OZ0.7 - a single dose of 0.7 mg/kg O3; and Group OZ1.0 - a single dose of 1.0 mg/kg O3, intraperitoneally. In total, six animals from each group were euthanized at 7, 14, and 21 days after the commencement of treatment. Bone samples were harvested and further analyzed by descriptive histology, histomorphometry, and immunohistochemistry for osteocalcin (OCN) and tartrate-resistant acid phosphatase (TRAP) protein expression. All applied doses of O3 were shown to increase the percentage of bone tissue (PBT) after 21 days compared to group C. After 14 days, the OZ0.7 and OZ1.0 groups showed significantly higher PBT when compared to group C. The OZ1.0 group presented the most beneficial results regarding PBT among groups, which denotes a dose-dependent response. OCN immunostaining was higher in all groups at 21 days. However, after seven and 14 days, the OZ1.0 group showed a significant increase in OCN immunostaining compared to C group. No differences in TRAP+ osteoclasts were found between groups and time points. Therefore, O3 therapy at higher doses might be beneficial for bone repair of the alveolar socket following tooth extraction.

The role of bone turnover markers in diagnosis, monitoring, and pathological fractures of osteoporosis.

We investigated the utility of specific biomarkers-namely, c-terminal telopeptide (CTX), n-telopeptide (NTX), deoxypyridinoline (DPD), and tartrate-resistant acid phosphatase (TRAP)-compared to conventional diagnostic methods. We hy-pothesized that these novel biomarkers could hold substantial value in the diagnosis, treatment, and monitoring of osteoporosis. The study was conducted over a three-year period, from January 1, 2020, to January 1, 2023. We enrolled a total of 520 patients aged 50 years or older who had been diagnosed with osteoporosis. Patients undergoing steroid treatments, which are known to contribute to osteoporosis, were excluded from the study. Additionally, we carefully selected and matched a control group consisting of 500 patients based on demographic characteristics relevant to the diagnosis of osteoporosis. This meticulous selection process resulted in a comprehensive cohort comprising 1,020 patients. Throughout the study, patients were closely monitored for a duration of one year to track the occurrence of pathological fractures and assess their overall prognosis. As a result of our rigorous investigation, we identified CTX, NTX, DPD, and TRAP as pivotal biomarkers that play a crucial role in evaluating bone health, monitoring treatment effectiveness, and detecting pathological fractures in the context of osteoporosis. Our study underscores the significance of these biomarkers in advancing the diagnosis and management of osteo-porosis, offering valuable insights into the disease's progression and treatment outcomes.

Evaluation of in vitro osteoblast and osteoclast differentiation from stem cell: a systematic review of morphological assays and staining techniques.

Understanding human stem cell differentiation into osteoblasts and osteoclasts is crucial for bone regeneration and disease modeling. Numerous morphological techniques have been employed to assess this differentiation, but a comprehensive review of their application and effectiveness is lacking. Guided by the PRISMA framework, we conducted a rigorous search through the PubMed, Web of Science and Scopus databases, analyzing 254 articles. Each article was scrutinized against pre-defined inclusion criteria, yielding a refined selection of 14 studies worthy of in-depth analysis. The trends in using morphological approaches were identified for analyzing osteoblast and osteoclast differentiation. The three most used techniques for osteoblasts were Alizarin Red S (mineralization; six articles), von Kossa (mineralization; three articles) and alkaline phosphatase (ALP; two articles) followed by one article on Giemsa staining (cell morphology) and finally immunochemistry (three articles involved Vinculin, F-actin and Col1 biomarkers). For osteoclasts, tartrate-resistant acid phosphatase (TRAP staining) has the highest number of articles (six articles), followed by two articles on DAPI staining (cell morphology), and immunochemistry (two articles with VNR, Cathepsin K and TROP2. The study involved four stem cell types: peripheral blood monocyte, mesenchymal, dental pulp, and periodontal ligament. This review offers a valuable resource for researchers, with Alizarin Red S and TRAP staining being the most utilized morphological procedures for osteoblasts and osteoclasts, respectively. This understanding provides a foundation for future research in this rapidly changing field.

Effects of fine particulate matter on bone mass and osteogenic differentiation of bone marrow mesenchymal stem cells in mice

To observe the effects of exposure to fine particulate matter(PM_(2.5)) on bone mass, microstructure, biomechanical properties, and osteogenic differentiation ability of bone marrow mesenchymal stem cells(BMSCs) in mice. A total of 16 C57BL/6J mice aged 8 weeks were randomly divided into control group(NS group) and PM_(2.5) exposure group(PM group). NS group was given normal saline, PM group was given 14 mg/kg PM_(2.5) suspension, 50 μL, poisoning every 3 day. After 10 weeks, the lungs of mice were taken for HE staining, and the left tibia was taken for Micro CT detection to analyze parameters related to cancellous and cortical bone. The right tibia was taken for HE staining to observe changes in bone trabeculae. Immunohistochemical staining was used to detect type I collagen(Col I), osteoprotegerin(OPG), and nuclear factor-κB receptor activating factor ligand(RANKL) protein expression, tartrate resistant acid phosphatase(TRAP) staining for detection of osteoclasts. Extract primary BMSCs from bilateral femurs, induce osteogenesis, and then perform alkaline phosphatase(ALP) staining to detect ALP activity, alizarin red staining to detect bone mineralization ability, real-time PCR to detect osteocalcin(OCN), ALP, OPG, and RANKL mRNA expression, and biomechanical testing to test the mechanical properties of the femur. Compared with the NS group, the pulmonary alveolar structure of the PM group mice was disrupted and a large number of inflammatory cells gathered. Prompt for successful PM_(2.5) poisoning operation. Micro CT result showed that the bone mineral density(BMD) and bone volume fraction(BV/TV) of the PM group mice were 276.959±15.152 mg/cm~3 and 0.208%±0.009%, respectively. The NS group had 316.709±28.205 mg/cm~3 and 0.236%±0.019%, respectively. The PM group was lower than the NS group(P<0.05), but the trabecular number(Tb. N) There was no statistically significant difference in parameters such as trabecular thickness(Tb. Th) and trabecular separation(Tb. SP)(P>0.05). The HE staining result of the tibia showed that the trabeculae in the NS group were thick, dense, and uniform. The bone trabeculae in the PM group were slender, with a decrease in number, widened spacing, and sparse arrangement. The expression of Col I(0.023±0.009) and OPG(0.036±0.010) in the PM group increased compared to the NS group(0.079±0.007, 0.059±0.012), while the expression of RANKL(0.036±0.006) decreased compared to the NS group(0.022±0.002)(P<0.05); The number of TRAP positive particles increased in the PM group. The experimental result after osteoinduction of BMSCs in mice showed that compared with the NS group, the PM group had a decrease in the number of ALP positive cells and a decrease in the number of calcium nodules. The relative expression of ALP, OCN, and OPG mRNA in the PM group(0.375±0.021, 0.585±0.088, 0.768±0.112) was significantly reduced compared to the NS group(1.001±0.043, 1.006±0.132, 1.002±0.086), while the relative expression of RANKL mRNA(1.278±0.118) was increased compared to the NS group(1.001±0.057)(P<0.05). The biomechanical experimental result showed that the maximum deflection of the NS group was 0.337±0.031 mm, while the maximum deflection of the PM group was 0.258±0.041 mm. Compared with the NS group, the maximum deflection of the PM group decreased significantly(P<0.05), and the maximum stress and maximum load showed a decreasing trend, but the difference was not statistically significant(P>0.05). After 10 weeks of exposure to PM_(2.5), it can affect the bone health of mice, and its mechanism may be related to increased osteoclast activity and inhibition of the osteogenic differentiation ability of BMSCs.

The osteocytic actions of glucocorticoids on bone mass, mechanical properties, or perilacunar remodeling outcomes are not rescued by PTH(1-34).

Glucocorticoids (GC) and parathyroid hormone (PTH) are widely used therapeutic endocrine hormones where their effects on bone and joint arise from actions on multiple skeletal cell types. In osteocytes, GC and PTH exert opposing effects on perilacunar canalicular remodeling (PLR). Suppressed PLR can impair bone quality and joint homeostasis, including in GC-induced osteonecrosis. However, combined effects of GC and PTH on PLR are unknown. Given the untapped potential to target osteocytes to improve skeletal health, this study sought to test the feasibility of therapeutically mitigating PLR suppression. Focusing on subchondral bone and joint homeostasis, we hypothesize that PTH(1-34), a PLR agonist, could rescue GC-suppressed PLR. The skeletal effects of GC and PTH(1-34), alone or combined, were examined in male and female mice by micro-computed tomography, mechanical testing, histology, and gene expression analysis. For each outcome, females were more responsive to GC and PTH(1-34) than males. GC and PTH(1-34) exerted regional differences, with GC increasing trabecular bone volume but reducing cortical bone thickness, stiffness, and ultimate force. Despite PTH(1-34)'s anabolic effects on trabecular bone, it did not rescue GC's catabolic effects on cortical bone. Likewise, cartilage integrity and subchondral bone apoptosis, tartrate-resistant acid phosphatase (TRAP) activity, and osteocyte lacunocanalicular networks showed no evidence that PTH(1-34) could offset GC-dependent effects. Rather, GC and PTH(1-34) each increased cortical bone gene expression implicated in bone resorption by osteoclasts and osteocytes, including Acp5, Mmp13, Atp6v0d2, Ctsk, differences maintained when GC and PTH(1-34) were combined. Since PTH(1-34) is insufficient to rescue GC's effects on young female mouse bone, future studies are needed to determine if osteocyte PLR suppression, due to GC, aging, or other factors, can be offset by a PLR agonist.

Evaluation of osteogenic properties of a novel injectable bone-repair material containing strontium in vitro and in vivo.

Objective: This study aims to develop and evaluate the biocompatibility and osteogenic potential of a novel injectable strontium-doped hydroxyapatite bone-repair material. Methods: The properties of strontium-doped hydroxyapatite/chitosan (Sr-HA/CS), hydroxyapatite/chitosan (HA/CS) and calcium phosphate/chitosan (CAP/CS) were assessed following their preparation via physical cross-linking and a one-step simplified method. Petri dishes containing Escherichia coli and Staphylococcus epidermidis were inoculated with the material for in vitro investigations. The material was also co-cultured with stem cells derived from human exfoliated deciduous teeth (SHEDs), to assess the morphology and proliferation capability of the SHEDs, Calcein-AM staining and the Cell Counting Kit-8 assay were employed. Osteogenic differentiation of SHEDs was determined using alkaline phosphatase (ALP) staining and Alizarin Red staining. For in vivo studies, Sr-HA/CS was implanted into the muscle pouch of mice and in a rat model of ovariectomy-induced femoral defects. Hematoxylin-eosin (HE) staining was performed to determine the extent of bone formation and defect healing. The formation of new bone was determined using Masson's trichrome staining. The osteogenic mechanism of the material was investigated using Tartrate-resistant acid phosphatase (TRAP) staining and immunohistochemical studies. Results: X-ray diffraction (XRD) and energy-dispersive spectroscopy (EDS) showed that strontium was successfully doped into HA. The Sr-HA/CS material can be uniformly squeezed using a syringe with a 13% swelling rate. Sr-HA/CS had a significant antibacterial effect against both E. coli and S. epidermidis (p < 0.05), with a stronger effect observed against E. coli. The Sr-HA/CS significantly improved cell proliferation and cell viability in vitro studies (p < 0.05). Compared to CAP/CS and CS, Sr-HA/CS generated a substantially greater new bone area during osteoinduction experiments (p < 0.05, p < 0.001). The Sr-HA/CS material demonstrated a significantly higher rate of bone repair in the bone defeat studies compared to the CAP/CS and CS materials (p < 0.01). The OCN-positive area and TRAP-positive cells in Sr-HA/CS were greater than those in control groups (p < 0.05). Conclusion: A novel injectable strontium-doped HA bone-repair material with good antibacterial properties, biocompatibility, and osteoinductivity was successfully prepared.

Evaluation of Two Anorganic Bovine Xenogenous Grafts in Bone Healing of Critical Defect in Rats Calvaria.

The purpose of this study was to provide an evaluation of two different xenogeneic bone substitutes in bone healing of critical-sized bone defects (Ø =5mm) created in rats calvaria. Thirty animals were randomized into 3 groups with one of the following treatments. In the control group (n=10), the defects were filled with blood clots; BO group (n=10), the defects were filled with bovine medullary bone substitute (Bio-Oss®); BF group (n=10), the defects were filled with bovine cortical bone substitute (Bonefill®). All defects were covered with an absorbable membrane. Five animals from each group were euthanized at 30 and 45 days, subsequently histomorphometrical and immunohistochemical analyses were performed. The histomorphometry was used to measure the percentage of new bone formation in the total area of the defect while the immunohistochemistry evaluated the expression of bone immunomarkers for bone morphogenetic protein 2/4 (BMP2/4), osteocalcin (OCN) and tartrate-resistant acid phosphatase (TRAP). Data was statistically analyzed with a 5% significance level. The results demonstrated that the BO group showed greater bone formation compared to the BF group at 30 days (P<0.05). However, there was no statistically significant difference between the control and BO groups at 30 days (P>0.05). The expression of BMP2/4 and OCN were higher in the BO group at 45 days compared to the BF at 30 and 45 days respectively (P<0.05). In conclusion, even with the higher expression of proteins related to bone formation, there was no difference in new bone formation at 45 days when both anorganic bovine xenogenous grafts were evaluated.

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.

Possible roles of short-chain fatty acids produced by oral bacteria in the development of alveolar osteitis.

Alveolar osteitis (dry sockets) is a painful condition characterized by a limited immune response. It is typically caused by the removal of blood clots from extracted tooth sockets, which leads to the fermentation of trapped food remnants by oral bacteria in the cavities, producing high concentrations of short-chain fatty acids (SCFAs). This study examined the effects of SCFAs on immunity and bone metabolism. Mouse macrophage Raw264.7 cells were treated with oral bacteria supernatants or SCFA mixtures, and inducible nitric oxide synthase (iNOS) levels were determined by western blot. The same cells were treated with SCFA mixtures in the presence of receptor activator of nuclear factor-kappa B ligand (RANKL), and osteoclast-like cells were counted. MC3T3-E1 cells were treated with SCFA mixtures and stained with alizarin red S. Raw264.7 cells treated with oral bacterial culture supernatants of Porphyromonas gingivalis and Fusobacterium nucleatum inhibited lipopolysaccharide (LPS)-induced iNOS production, likely due to SCFA content. SCFA mixtures mimicking these supernatants inhibited the number of RANKL-induced tartrate-resistant acid phosphatase (TRAP)-positive cells and MC3T3-E1 cell mineralization. These data suggest that SCFAs produced by P. gingivalis and F. nucleatum may reduce the inflammatory response and mildly induce mineralization of the alveolar walls. These results may contribute to the understanding of alveolar osteitis.

Histological Analyses of Arthritic Joints in Collagen-Induced Arthritis Model Mice.

Histological analysis is a morphological technique and an effective method for understanding the pathology of rheumatoid arthritis (RA). RA is an inflammatory disease characterized by increased synovial tissue and osteoclasts, angiogenesis, infiltration of inflammatory cells, and pannus formation. These pathologies can be observed in a collagen-induced arthritis model mouse using formaldehyde-fixated paraffin-embedded (FFPE) samples. For the preparation of FFPE samples, the conditions of the fixation and decalcification process significantly affect tissue staining results. Since the lesion sites include bone tissue, a decalcification process is necessary when preparing an FFPE sample. Therefore, selecting an optimal condition for the fixating and decalcifying solution is important. In this chapter, we describe the procedures of preparing paraffin samples, including fixation, decalcification, embedding, and sectioning from the RA model mouse, as well as different staining methods (hematoxylin and eosin, tartrate-resistant acidphosphatase).

Cinobufagin Suppresses Lipid Peroxidation and Inflammation in Osteoporotic Mice by Promoting the Delivery of miR-3102-5p by Macrophage-Derived Exosomes.

Cinobufagin, the primary active compound in toad venom, is commonly used for anti-tumor, anti-inflammatory, and analgesic purposes. However, its specific bone-protective effects remain uncertain. This research aims to ascertain the bone-protective properties of cinobufagin and investigate underlying mechanisms. Mice were ovariectomized to establish an osteoporosis model, followed by intraperitoneal injections of cinobufagin and cinobufagin-treated RAW.264.7-derived exosomes for therapy. MicroCT, HE staining, and TRAP staining were employed to evaluate bone mass and therapeutic outcomes, while mRNA sequencing and immunoblotting were utilized to assess markers of bone metabolism, inflammation, and lipid peroxidation. Osteoblast and osteoclast precursor cells were differentiated to observe the impact of cinobufagin-treated exosomes derived from RAW264.7 cells on bone metabolism. Exosomes characteristics were studied using transmission electron microscopy and particle size analysis, and miRNA binding targets in exosomes were determined by luciferase reporting. In ovariectomized mice, cinobufagin and cinobufagin-treated exosomes from RAW264.7 cells increased trabecular bone density and mass in the femur, while also decreasing inflammation and lipid peroxidation. The effect was reversed by an exosomes inhibitor. In vitro experiments revealed that cinobufagin-treated exosomes from RAW264.7 cells enhanced osteogenic and suppressed osteoclast differentiation, possibly linked to Upregulated miR-3102-5p in RAW-derived exosomes. MiR-3102-5p targets the 3'UTR region of alox15, thereby suppressing its expression and reducing the lipid peroxidation process in osteoblasts. Overall, this study clarified cinobufagin's bone-protective effects and revealed that cinobufagin can enhance the delivery of miR-3102-5p targeting alox15 through macrophage-derived exosomes, demonstrating anti-lipid peroxidation and anti-inflammatory effects.

The autocrine action of salidroside on osteoclast during osteoclastogenesis via hypoxia-inducible factor-1α pathway.

The objective of this study was to investigate the potential of salidroside (SAL) (a major active compound in Rhodiola rosea L.) in regulating osteoclast differentiation and function by modulating the HIF-1α pathway and its downstream target genes. The expression of HIF-1α and its downstream target genes was examined at both mRNA and protein levels in osteoclasts treated with SAL. Immunofluorescence analysis was performed to assess the nuclear translocation and transcriptional activity of HIF-1α in response to SAL. MTT, flow cytometry, qPCR, TRAP staining and bone resorption assays were used to evaluate the potential effect of salidroside on osteoclasts. SAL enhanced the expression of HIF-1α and its downstream target genes in osteoclasts. Immunofluorescence analysis confirmed the facilitation of HIF-1α nuclear translocation and transcriptional activity by SAL. In addition, SAL enhanced osteoclast viability, differentiation and bone resorption activity in an autocrine manner through HIF-1α/VEGF, IL-6 and ANGPTL4 pathways. SAL promotes osteoclast proliferation, differentiation and bone resorption through HIF-1α/VEGF, IL-6 and ANGPTL4 pathways.

Therapeutic Potential of Ginger Exosome-Like Nanoparticles for Alleviating Periodontitis-Induced Tissue Damage.

Periodontitis is a chronic inflammatory oral disease that causes defects in periodontal tissue. Conventional therapies are limited, and often lead to high recurrence rates. The emerging concept of medicinal food homology has shed light on the potential of ginger as a therapeutic adjuvant for periodontitis, given its antioxidant and anti-inflammatory properties. However, fresh ginger exhibits poor stability and bioavailability. Ginger exosome-like nanoparticles (GELNs), a derivative of ginger, have not been reported to exert therapeutic effects in periodontitis. This study aimed to explore the therapeutic effects of GELNs on tissue damage caused by periodontitis and their underlying mechanisms of action. The GELNs composition was analyzed using a widely targeted metabolome. Stability was assessed using nanoparticle tracking analysis (NTA) and zeta potential measurements, flavor was evaluated using an electronic nose, and membrane penetration was studied using confocal microscopy. A periodontitis model was established in SD rats, periodontal clinical indicators were monitored, and histological changes were assessed using H&E and TRAP staining. Co-culture experiments investigate the antioxidant and reparative abilities of GELNs on periodontal ligament fibroblasts (PDLFs) in inflammatory environment. NF-κB protein expression was examined by immunofluorescence and immunohistochemistry. The findings revealed that GELNs demonstrated good stability in different environments and mitigated the pungent taste of the raw ginger. In vivo experiments showed that GELNs improved periodontal clinical parameters and pathology compared with ginger juice. In vitro data suggested that GELNs enhanced the proliferation and migration of PDLFs while reducing the reactive oxygen species (ROS) levels by inhibiting the NF-κB signaling pathway in an inflammatory setting. This study is the first to demonstrate that GELNs have a potential therapeutic effect on periodontitis. GELNs can alleviate oxidative stress (OS) and inflammatory reactions by inhibiting the NF-κB signaling pathway. These findings provide a promising method for the treatment of periodontitis by regulating an unbalanced OS state.

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.

Enzymatic activity of bone markers on Lithobates catesbeianus (Shaw, 1802) growth during the ossification process.

In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.

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.

The essential role of Mkx in periodontal ligament on the metabolism of alveolar bone and cementum

IntroductionThe periodontium is a connective tissue which consists of periodontal ligament, alveolar bone, cementum and gingiva. Periodontal ligament (PDL) is a specialized connective tissue that connects the cementum – coating the surface of the tooth – to the alveolar bone. Mohawk homeobox (Mkx) is a transcription factor that is expressed in PDL, that is known to play a vital role in the development and homeostasis of PDL. A detailed functional analysis of Mkx in the periodontal ligament for alveolar bone and cementum metabolism has not yet been conducted. Materials and methodsAlveolar bone height, bone mineral density (BMD) and bone volume fractions (Bone volume/Total volume: BV/TV) were measured and analyzed using micro-computed tomography (Micro-CT) and 3DBon on 7-week-old male wild-type (WT) (Mkx+/+) (n = 10) and Mkx-knockout (Mkx−/−) (n = 6) rats. Hematoxylin and Eosin (H&E), tartrate-resistant acid phosphatase (TRAP), alkaline phosphatase (ALP) and Masson Trichrome staining were performed on 5, 6, and 7-week-old Mkx+/+ and Mkx−/− rats. Cementum surface area and the number of TRAP-positive osteoclasts/mm were quantified, measured, and compared for 5,6 and 7-week-old Mkx+/+ and Mkx−/− rats (n = 3 each). ResultsThe level of alveolar bone height was significantly higher in Mkx−/− rats than in Mkx+/+ rats. On the other hand, there was significantly less BMD in Mkx−/− alveolar bone. A significant increase in cellular cementum could be observed as early as 5 weeks in Mkx−/− rats when compared with Mkx+/+ rats of the same age. More TRAP-positive osteoclasts were observed in Mkx−/− rats. ConclusionOur findings further reveal the essential roles of Mkx in the homeostasis of the periodontal tissue. Mkx was found to contribute to bone and cementum metabolism and may be essential to the prevention of diseases such as periodontitis, and could show potential in regenerative treatments.

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.

Collagen-like Osteoclast-Associated Receptor (OSCAR)-Binding Motifs Show a Co-Stimulatory Effect on Osteoclastogenesis in a Peptide Hydrogel System.

Osteoclastogenesis, one of the dynamic pathways underlying bone remodelling, is a complex process that includes many stages. This complexity, while offering a wealth of therapeutic opportunities, represents a substantial challenge in unravelling the underlying mechanisms. As such, there is a high demand for robust model systems to understand osteoclastogenesis. Hydrogels seeded with osteoclast precursors and decorated with peptides or proteins mimicking bone's extracellular matrix could provide a useful synthetic tool to study pre-osteoclast-matrix interactions and their effect on osteoclastogenesis. For instance, fibrillar collagens have been shown to provide a co-stimulatory pathway for osteoclastogenesis through interaction with the osteoclast-associated receptor (OSCAR), a regulator of osteoclastogenesis expressed on the surface of pre-osteoclast cells. Based on this rationale, here we design two OSCAR-binding peptides and one recombinant OSCAR-binding protein, and we combine them with peptide-based hydrogels to study their effect on osteoclastogenesis. The OSCAR-binding peptides adopt the collagen triple-helical conformation and interact with OSCAR, as shown by circular dichroism spectropolarimetry and surface plasmon resonance. Furthermore, they have a positive effect on osteoclastogenesis, as demonstrated by appropriate gene expression and tartrate-resistant acid phosphatase staining typical of osteoclast formation. Combination of the OSCAR-binding peptides or the OSCAR-binding recombinant protein with peptide-based hydrogels enhances osteoclast differentiation when compared to the non-modified hydrogels, as demonstrated by multi-nucleation and by F-actin staining showing a characteristic osteoclast-like morphology. We envisage that these hydrogels could be used as a platform to study osteoclastogenesis and, in particular, to investigate the effect of costimulatory pathways involving OSCAR.

Delayed Reaction of Radiation on the Central Nervous System and Bone System in C57BL/6J Mice.

The aim of this study was to provide a suitable mouse model of radiation-induced delayed reaction and identify potential targets for drug development related to the prevention and treatment of radiation injury. C57BL/6J mice were subjected to singular (109 cGy/min, 5 Gy*1) and fractional (109 cGy/min, 5 Gy*2) total body irradiation. The behavior and activity of mice were assessed 60 days after ionizing radiation (IR) exposure. After that, the pathological changes and mechanism of the mouse brain and femoral tissues were observed by HE, Nissl, Trap staining micro-CT scanning and RNA sequencing (RNA-Seq), and Western blot. The results show that singular or fractional IR exposure led to a decrease in spatial memory ability and activity in mice, and the cognitive and motor functions gradually recovered after singular 5 Gy IR in a time-dependent manner, while the fractional 10 Gy IR group could not recover. The decrease in bone density due to the increase in osteoclast number may be relative to the down-regulation of RUNX2, sclerostin, and beta-catenin. Meanwhile, the brain injury caused by IR exposure is mainly linked to the down-regulation of BNDF and Tau. IR exposure leads to memory impairment, reduced activity, and self-recovery, which are associated with time and dose. The mechanism of cognitive and activity damage was mainly related to oxidative stress and apoptosis induced by DNA damage. The damage caused by fractional 10 Gy TBI is relatively stable and can be used as a stable multi-organ injury model for radiation mechanism research and anti-radiation medicine screening.