Tartrate-resistant Alkaline Phosphatase Research Articles

#129 Impact of Ramadan fasting on bone mineral metabolism in hemodialysis: a multi-center prospective study

Abstract Background and Aims Fasting is fundamental in several major religions and cultural traditions worldwide. In Islam, Ramadan fasting is characterized by daily complete eating and drinking abstinence from the dawn to sunset for an entire lunar month. Sick patients, including patients with advanced chronic kidney disease are not required to fast, however the majority of them are still choose to fast. The Impact of Ramadan fasting on bone health has not been studied prospectively. This study aims to examine the effect of Ramadan fasting on parameters of bone mineral metabolism in patients on hemodialysis. Method : This is a multi-center prospective study on 138 patients with ESKD who are maintained on intermittent hemodialysis for at least six months. Ninety-three (67%) were fasting while 45 patients (33%) did not fast. All patients' demographics, clinical and laboratory parameters were compared before and after the holy month of Ramadan. Serum biochemical parameters included serum sodium, potassium, bicarbonate, chloride, albumin, calcium, phosphorus, magnesium, intact parathyroid hormone (iPTH), FGF23, klotho, sclerostin, bone-specific alkaline phosphatase (BSAP), and tartrate-resistant alkaline phosphatase 5b (TRAP5b). Results The two groups exhibited comparable demographics and clinical parameters at baseline, except for the mean age, which was significantly lower in the fasting group (45.5 vs. 54.4 years, p-value = 0.003) and the non-smoker habit was significantly higher in the fasting group (71% vs. 56%, p-value = 0.02). There was no change in the clinical parameters by the end of Ramadan in both groups including intra-dialytic hypotension, inter-dialytic weight gain, and hypoglycemia. The basic biochemical parameters did not change significantly at the end of Ramadan in both groups, except there was an increased serum albumin level (p-value = 0.04). The BSAP levels significantly went down by the end of Ramadan in the fasting group (p-value = 0.01). The other novel serum biomarkers (FGF23, klotho, sclerostin, and TRAP5b) did not change significantly in both groups. Conclusion This multi-center prospective study demonstrates that Ramadan fasting does not significantly alter the clinical and biochemical parameters in hemodialysis patients, highlighting its safety in this population. Moreover, our findings reveal a noteworthy reduction of the higher BSAP towards normal levels in fasting patients, suggesting a potential benefit of fasting in improving high bone turnover. Further research is warranted to elucidate this phenomenon's underlying mechanisms and long-term implications, which may hold promise for managing bone health in hemodialysis patients during Ramadan fasting.

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.

Collagen type I-based recombinant peptide promotes bone regeneration in rat critical-size calvarial defects by enhancing osteoclast activity at late stages of healing

IntroductionWe recently demonstrated the bone-forming potential of medium-cross-linked recombinant collagen peptide (mRCP) in animal models of bone defects. However, these studies were limited to a 4-week observation period; therefore, in the present study, we aimed to further evaluate mRCP as a suitable bone graft material for the alveolar cleft by analyzing its bone-forming potential, osteogenic-inducing ability, and biodegradation over an extended period of 12 weeks, using a rat critical-size calvarial defect model. MethodsUsing Sprague-Dawley rats, we created critical-size calvarial defects through a surgical procedure. The defects were then filled with 3 mg of mRCP (mRCP group) or 18 mg of Cytrans® (CA) granules, which has a carbonate apatite-based composition resembling natural bone, was used as a reference material (CA group). For negative control, the defects were left untreated. Bone volume, total bone volume (bone volume including CA granules), and bone mineral density (BMD) in the defect were assessed using micro-computed tomography (μ-CT) at 0, 4, 8, and 12 weeks after implantation. Using histomorphometric analyses of hematoxylin and eosin (H&E)-stained sections, we measured the amount of newly formed bone and total newly formed bone (new bone including CA granules) in the entire defect site, as well as the amount of newly formed bone in the central side, two peripheral sides (left and right), periosteal (top) side, and dura mater (bottom) side. In addition, we measured the amount of residual bone graft material in the defect. Osteoclasts and osteoblasts in the newly formed bone were detected using tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) staining, respectively. ResultsBone volume in the mRCP group increased over time and was significantly larger at 8 and 12 weeks after surgery than at 4 weeks. The bone volume in the mRCP group was greater than that of the CA and control groups at 4, 8, and 12 weeks after implantation, and while the total bone volume was greater in the CA group after 4 and 8 weeks, the mRCP group had comparable levels of total bone volume to that of the CA group at 12 weeks after implantation. The BMD of the mRCP group reached similar levels to native calvaria bone at the same time point. H&E-stained sections revealed a larger amount of newly formed bone 12 weeks after implantation in the mRCP group compared to that of the CA and control groups. The total newly formed bone at 12 weeks after implantation was on par with that in the CA group. Furthermore, at the defect site, the area of newly formed bone was larger on the peripheral and dura mater sides. Notably, the number of osteoclasts in the mRCP group was higher than in the CA and control groups and peaked 8 weeks after implantation, which coincided with the timing of the greatest resorption of mRCP. Although the ALP-positive area was greater in the mRCP group compared to other groups, we did not detect any significant changes in the number of osteoblasts over time. ConclusionThis study demonstrated the bone-forming potential of mRCP over an extended period of 12 weeks, suggesting that mRCP sufficiently resists resorption to promote bone formation through induction of osteoclast activation in the late stages of the healing period.

Alveolar Bone Preservation Using a Combination of Nanocrystalline Hydroxyapatite and Injectable Platelet-Rich Fibrin: A Study in Rats.

Alveolar bone resorption is a post-extraction complication wherein there is a reduction in the dimensions and quality of the alveolar bone. This study aimed to examine the effects of implantation of a combination of nanocrystalline hydroxyapatite (nHA) and injectable platelet-rich fibrin (IPRF) on the expression of tartrate-resistant acid phosphatase (TRAP), alkaline phosphatase (ALP), osteocalcin (OCN), and new bone formation. A total of 32 male rats had their upper right incisors extracted under general anesthesia and were then divided into a control group, nHA group, IPRF group, and nHA-IPRF group. Decapitation was carried out on day 14 and day 28 in each group and the jaws of each rat were subjected to immunohistochemical and histological analysis. The results showed a decrease in TRAP expression in the nHA-IPRF group compared with the control group on day 14 (p = 0.074) and day 28 (p = 0.017). The study also showed an increase in ALP and OCN in the HA-IPRF group on day 14 and day 28 compared with the control group. New bone formation suggested a significant increase in the nHA-IPRF group compared with the control group on day 14 (p = 0.001) and day 28 (p = 0.001). nHA-IPRF implantation can suppress alveolar bone resorption, which is indicated by decreased TRAP expression, and it can increase bone growth, as indicated by increased expression of ALP, OCN, and new bone formation.

Molecular Hydrogen Prevents Osteoclast Activation in a Glucocorticoid-Induced Osteoporosis Zebrafish Scale Model.

Antioxidants represent a powerful tool for many human diseases and, in particular, molecular hydrogen has unique characteristics that make it a very promising therapeutic agent against osteoporosis. Zebrafish scales offer an innovative model in which new therapeutic approaches against secondary osteoporosis are tested. Scale bone loss obtained by prednisolone (PN) treatment is characterized by increased osteoclast activity and decreased osteoblast activity highlighted with bone enzymatic assays. We used this read-out system to test the therapeutic effects of hydrogen-rich water (HRW), an innovative antioxidant approach. HRW prevented osteoclast activation and bone loss in PN-treated fish scales, as verified by both biochemical and histochemical tartrate-resistant alkaline phosphatase assays. On the other hand, HRW treatment did not prevent PN-dependent osteoblast suppression, as measured by alkaline phosphatase activity. Moreover, HRW treatment did not facilitate the reparation of resorption lacunae induced in scales by PN. Our study highlighted a specific effect of HRW on adult osteoclast activity but not in osteoblasts, introducing an intriguing new antioxidant preventive approach against osteoporosis.

Iodine-125 brachytherapy suppresses tumor growth and alters bone metabolism in a H1299 xenograft mouse model.

The present study aimed to investigate the efficacy of Iodine-125 (I-125) brachytherapy in a mouse model of non-small cell lung cancer, to further explore the efficacy and appropriate method of implantation of the I-125 radioactive seed. This study also aimed to determine the impact of brachytherapy on bone metabolism. A total of 18 mice were used to establish H1299 xenograft models, and were randomly assigned to three groups. These included non-radioactive seed implantation (Sham IM), fractionated I-125 seed implantation (Fractionated IM) and single I-125 seed implantation (Single IM) groups. Mice were euthanized after 28days of implantation. H&E staining, Ki67 immunohistochemistry, CD31 morphometric analysis and TUNEL immunofluorescence assays were respectively used to determine the histopathological changes, proliferation, micro-angiogenesis and apoptosis of tumors. In addition, bone volume and microstructure were evaluated using trabecular bone area (Tb.Ar), trabecular thickness (Tb.Th), trabecular number (Tb.N) and cortical thickness. Bone metabolic status was analyzed using histomorphometric staining of tartrate-resistant acid phosphate (TRAP) and alkaline phosphatase (ALP) expression in the femur, and using an ELISA assay to determine the expression of C-telopeptide of type 1 collagen (CTX-1) and procollagen type 1 n-terminal propeptide (P1NP) in the serum. Moreover, reverse transcription-quantitative PCR and western blotting were carried out for the analysis of bone remodeling-related gene expression in the bone tissue. Results of the present study demonstrated that compared with the Sham IM group, both the I-125 seed implantation groups, including Fractionated IM and Single IM, demonstrated significant therapeutic effects in both tumor volume and weight. More specifically, the most significant therapeutic effects on tumor inhibition were observed in the Fractionated IM group. Results of Ki67 and CD31 immunohistochemical staining suggested a notable reduction in tumor cell proliferation and micro-angiogenesis, and results of the TUNEL assay demonstrated an increase in tumor cell apoptosis. Although the cortical bone appeared thinner and more fragile in both I-125 seed implantation groups, no notable adverse changes in the morphology of the cancellous bone were observed, and the index of Tb.Ar, Tb.Th and Tb.n was not significantly different among Sham IM and I-125 implantation groups. However, alterations in bone metabolism were characterized by a decrease in CTX-1 and P1NP expression, accompanied by an increase in TRAP activity and a decrease in ALP activity. Results of the present study also demonstrated the notable suppression of osteocalcin and runt-related transcription factor 2. I-125 seed implantation may be an effective and safe antitumor strategy. Moreover, the use of fractionated implantation patterns based on tumor shape exhibited improved therapeutic effect on tumor suppression when the total number of I-125 seeds was equivalent along with reduced complications associated with bone loss.

Effects of Soft Tissue Sarcoma and Doxorubicin on Bone Metabolism in Mice.

This study aimed to evaluate the effects of doxorubicin (Dox) on bone microstructure and metabolism in a mouse model of soft tissue sarcoma. CCRF S-180II cells (2-4×105 cells/0.2 ml) were injected subcutaneously into the back of mice. The mice were divided into four groups according to tumor and treatment status and were reared and sacrificed after 2 or 4 weeks. Micro-computed tomography (CT) was performed to calculate the architecture of the femoral bone. The proximal tibia was double stained with tartrate-resistant acid phosphatase (TRACP) and alkaline phosphatase (ALP), and bone morphometry was performed. Trabecular bone mass was significantly reduced in the Sarcoma and Sarcoma+Dox groups. Cortical bone thickness was reduced in the DOX group, with a stronger effect observed in the Sarcoma+Dox group. In bone morphometry, osteoclast number at the bone surface (Oc.N/BS) was significantly lower in the Dox, Sarcoma, and Sarcoma+Dox groups than in the Control group at 2 weeks. The osteoblast surface at the bone surface (Ob.S/BS) was significantly lower in the Dox and Sarcoma groups than in the Control group at 2 weeks. At 4 weeks, the differences were smaller for both Oc.N/BS and Ob.S/BS. The use of doxorubicin alone worsened the cortical bone structure; however, the presence of both soft-tissue sarcoma and doxorubicin use worsened both cortical and trabecular bone structures from an early stage.

Analysis of the femoral neck from rats in the periestropause treated with oxytocin and submitted to strength training.

Among the interventions used to prevent osteoporosis in female organisms, strength training (ST) and oxytocin (OT) stand out, as a promising hormone with anabolic action on bone. This study aimed to verify whether the combined action of OT and ST, compared to isolated interventions, potentiates the bone remodeling process of the femoral neck of Wistar rats during periestropause. Forty Wistar rats (18months) with irregular estrous cycle were randomly distributed into groups: 1-Vehicle (Veh; NaCl 0.15mol/L ip); 2-Oxytocin (Ot; 134μg/kg/ip); 3-Strength training (St); 4-Ot+St. The animals of the 1, 2 and 4 groups received two intraperitoneal injections with an interval of 12h every 30days, totaling 8 injections at the end of the experimental period (18 to 21months). The animals in the St and Ot+St groups performed ST on a ladder 3 times a week, maximal voluntary carrying capacity (MVCC) test monthly. After 120days, the animals were euthanized; the femur was collected for analysis of biomechanical testing, densitometry, bone microtomography, Raman spectroscopy, tissue PCR, and blood for analysis of bone biomarkers, liver damage, and oxidative stress. The main effects in the Ot group were observed in the maximum load and energy in the compression testing (femoral head), and stiffness and energy in the three-points bending testing (femur diaphysis). In addition, the main effects occurred on the bone mineral density (BMD), cortical thickness (Ct.Th), number of pores (Po.N), polar moment of inertia (J), trabecular thickness (Tb.Th), and connectivity density (Conn.Dn), Bone alkaline phosphatase (Alp), Tumor necrosis factor receptor superfamily member 11b (Opg), Tumor necrosis factor ligand superfamily member 11 (Rankl) and Cathepsin K (Ctsk) expression. There was an effect in the tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP). In the St group, the main effect was observed on the energy (compression and the three-points bending), stiffness, aBMD, BMD, cortical bone area (Ct.Ar), Po.N, trabecular bone volume (BV/TV), Tb.Th and in the mineralization ratio (ѵ1PO4/proline), Runt-related transcription factor 2 (Runx2), Bone morphogenetic protein 2 (Bmp2), Alp, Osteopontin/secreted phosphoprotein 1 (Opn/Spp1), Opg, Tumor necrosis factor receptor superfamily member 11ª (Rank), Rankl, Ctsk expression. There was an effect in the TRAP and ALP. The interaction in the combination of therapies in the Ot+St group was verified in energy to maximum load (compression and three-points bending testing), stiffness, BMD, Ct.Th, J, Tb.Th and ѵ1PO4/proline. In the gene analysis there was interaction in the Runx2, Osterix/Sp7 transcription factor (Osx/Sp7), Bmp2, Alp, Osteocalcin/Bone gamma-carboxyglutamate protein (Ocn/Bglap), Opg, Rankl and Acid phosphatase 5, tartrate resistant (Trap/Acp5) expression. In addition, the combination of OT and ST resulted in a higher maximum load compared to the Veh group, with higher BV/TV than the Ot group, higher Rankl and Ctsk expression than Veh and Ot groups, and lower Po.N and lower activity of TRAP than the other groups. In oxidative stress, total antioxidant capacity (TAC) was lower. These results showed that the combination of interventions is a promising anabolic strategy for the prevention of osteoporosis in the period of periestropause, standing out from the effects of isolated interventions.

Linagliptin in Combination With Metformin Ameliorates Diabetic Osteoporosis Through ModulatingBMP-2 and Sclerostin in the High-Fat Diet Fed C57BL/6 Mice.

BackgroundDiabetic osteoporosis is a poorly managed serious skeletal complication, characterized by high fracture risk, increased bone resorption, reduced bone formation, and disrupted bone architecture. There is a need to investigate drugs that can improve bone health along with managing glycemic control. DPP-4 inhibitors and metformin have proven benefits in improving bone health. Here, we investigated the effects of linagliptin, a DPP inhibitor, and metformin alone and in combination to treat diabetic osteoporosis in high-fat-fed mice.MethodsC57BL/6 mice were kept on the high-fat diet (HFD) for 22 weeks to induce diabetic osteoporosis. Linagliptin (10mg/Kg), metformin (150mg/Kg), and their combination were orally administered to the diabetic mice from the 18th-22nd week. Femur and tibial bone microarchitecture together with bone mineral density (BMD) were evaluated using µCT and histopathological changes were assessed. Further, bone turnover biomarkers namely bone morphogenetic protein-2 (BMP-2), sclerostin, tartrate-resistant acid phosphatase (TRAP), osteocalcin, alkaline phosphatase (ALP), calcium, and pro-inflammatory cytokines were assessed. Additionally, metabolic parameters including body weight, fasting blood glucose (FBG), glucose & insulin tolerance, lipids profile, and leptin were measured.ResultsHFD feeding resulted in impaired bone microarchitecture, reduced BMD, distorted bone histology, and altered bone turnover biomarkers as indicated by the significant reduction in bone ALP, BMP-2, osteocalcin, and an increase in sclerostin, TRAP, and serum calcium. Interestingly, treatment with linagliptin and its combination with metformin significantly reverted the impaired bone architecture, BMD, and positively modulated bone turnover biomarkers, while metformin alone did not exhibit any significant improvement. Further, HFD induced diabetes and metabolic abnormalities (including an increase in body weight, FBG, impaired glucose and insulin tolerance, leptin, triglycerides, cholesterol), and pro-inflammatory cytokines (TNF-alpha and IL-1β) were successfully reversed by treatment with linagliptin, metformin, and their combination.ConclusionLinagliptin and its combination with metformin successfully ameliorated diabetic osteoporosis in HFD-fed mice possibly through modulation of BMP-2 and sclerostin. The study provides the first evidence for the possible use of linagliptin and metformin combination for managing diabetic osteoporosis.

The Impact of Hempseed Consumption on Bone Parameters and Body Composition in Growing Female C57BL/6 Mice.

Optimizing peak bone mass is critical to healthy aging. Beyond the established roles of dietary minerals and protein on bone integrity, fatty acids and polyphenols modify bone structure. This study investigated the effect of a diet containing hempseeds (HS), which are rich in polyunsaturated fatty acids and polyphenols, on bone mineral density, bone cell populations and body composition. Groups (n = 8 each) of female C57BL/6 mice were fed one of three diets (15% HS by weight; 5% HS; 0% HS (control)) from age 5 to 30 weeks. In vivo whole-body composition and bone mineral density and content were measured every 4 weeks using dual-energy X-ray absorptiometry. Ex vivo humeri cell populations in the epiphyseal plate region were determined by sectioning the bone longitudinally, mounting the sections on slides and staining with tartrate-resistant acid phosphatase and alkaline phosphatase stain to identify osteoclasts and osteoblasts, respectively. Mixed models with repeated measures across experimental weeks showed that neither body weight nor body weight gain across weeks differed among groups yet mice fed the 15% HS diet consumed significantly more food and more kilocalories per g body weight gained than those fed the 5% HS and control diets (p < 0.0001). Across weeks, fat mass was significantly higher in the 5% HS versus the control group (p = 0.02). At the end point, whole-body bone mineral content was significantly higher in the control compared to the 5% HS group (p = 0.02). Humeri from both HS groups displayed significantly lower osteoblast densities compared to the control group (p < 0.0001). No relationship was seen between osteoblast density and body composition measurements. These data invite closer examination of bone cell activity and microarchitecture to determine the effect of habitual HS consumption on bone integrity.

7-Ketocholesterol-Induced Micro-RNA-107-5p Increases Number and Activity of Osteoclasts by Targeting MKP1.

Osteoclasts (OCs), which are responsible for bone resorption, play a critical role in cholesterol-induced bone loss and recent studies have suggested that various micro-RNAs (miRs) contribute to modulating OCs. We hypothesized that 7-ketocholesterol (7-KC), a metabolite responsible for cholesterol-induced bone loss, induces miR-107-5p, which affects OCs. Overexpression and knock-down of miR-107-5p were performed using miR-107-5p mimic and anti-miR-107-5p, respectively. The effects of miR-107-5p on OCs were analyzed by tartrate-resistant alkaline phosphatase staining, qPCR, and Western blot. MiR-107-5p was upregulated after 7-KC exposure in receptor activator of nuclear factor kappa-Β ligand-stimulated OCs. Furthermore, miR-107-5p upregulation was also observed in tibiae from an atherogenic diet-fed mice compared with mice fed with a normal diet. MiR-107-5p overexpression enhanced the area and number of OCs, whereas inhibiting the endogenous expression of miR-107-5p generated by 7-KC had the opposite effect. Among the possible candidates, mitogen-activated protein kinase phosphatase-1, a stress-responsive dual-specificity phosphatase that inactivates mitogen-activated protein kinase (MKP1), has been proven to be a target gene of miR-107-5p, as demonstrated by the direct interaction between miR-107-5p and the 3′-untranslated region of MKP1. Collectively, our findings demonstrate that 7-KC-induced miR-107-5p promotes differentiation and function of OCs by downregulating MKP1.

Association of High Serum Chemerin with Bone Mineral Density Loss and Osteoporotic Fracture in Elderly Chinese Women.

BackgroundChemerin has been suggested to be a risk factor for osteoporosis; however, its relationship with osteoporotic fracture is poorly understood. Herein, we intend to explore the association between serum chemerin and osteoporotic fracture.MethodsA total of 111 elderly women patients diagnosed with osteoporotic fracture were selected as the observation group, and 40 healthy subjects were enrolled as controls. Dual-energy X-ray absorptiometry, enzyme-linked immunosorbent assay, electrochemiluminescence immunoassay, and biochemical analysis were separately performed to determine body bone mineral density (BMD), chemerin levels, bone turnover markers, and other parameters. Pearson's correlation analysis was conducted to examine a relationship between chemerin and laboratory parameters. Moreover, the levels of chemokine-like receptor 1 (CMKLR), C-C motif chemokine receptor-like 2 (CCRL2), collagen type I alpha (COLA1), and runt-related transcription factor-2 (RUNX2) were confirmed by quantitative polymerase chain reaction, and the effect of chemerin on osteogenic differentiation of hFOB1.19 cells was indicated by tartrate-resistant acid phosphatase and alkaline phosphatase double staining.ResultsA higher level of chemerin was generally detected in patients with osteoporotic fracture compared with those without (P<0.05). Compared with controls, lower BMD levels and higher β-CTx and P1NP levels were detected in patients with osteoporotic fracture (all P<0.05). Interestingly, chemerin level was negatively correlated to BMD, but positively related to P1NP and β-CTx. Risk of osteoporotic fracture was 2.75-fold higher in subjects with each standard deviation increment of chemerin. Compared with controls, there were no significant differences in CMKLR1 and CCRL2 mRNA after incubation with osteogenic differentiation medium (all P>0.05), whereas there was a remarkable decrease of COLA1 and RUNX2 after incubation with chemerin for nine days (all P<0.05). Furthermore, prolonged incubation with chemerin enhanced osteoclast differentiation and maturation, consequently contributing to an increased risk of fracture.ConclusionChemerin is a strong and independent risk factor for osteoporosis-related fracture among elderly Chinese women.

Effects of I-125 seeds combined with anlotinib on tumor growth and bone metabolism in A549 tumor-bearing mice

Purpose This study aimed to investigate the therapeutic potential of tumor suppression and mechanism for different implantation modes of iodine-125 (I-125) seeds irradiation in a mice xenograft model, and its skeletal complications. Materials and methods A total of 24 mice carrying A549 lung tumor-derived xenografts were randomly assigned to four groups, including non-radioactive (sham) seeds implantation, I-125 seeds fractional implantation, I-125 seeds single implantation and I-125 seeds single implantation combined with anlotinib. Ki67 immunohistochemistry, TUNEL immunofluorescence and CD31 morphometric analysis were used to determine the proliferation index, rate of apoptotic cells and microvessel density, respectively. Additionally, the side effects on the skeletal system in mice treated with I-125 seeds implantation were evaluated by histomorphometric staining with tartrate-resistant acid phosphate (TRAP) and alkaline phosphatase (ALP) expression in femur, tartrate-resistant acid phosphatase 5b (TRACP-5b) and procollagen type I N-terminal propeptide (PINP) levels in serum were evaluated by enzyme-linked immunosorbent assay (ELISA). Results The I-125 seeds single and fractionated implantation had similar therapeutic effects and complications when the total number of I-125 seeds was the same. A single implantation of I-125 seeds with or without anlotinib could analogously inhibit the tumor growth in xenografts mice, while the single implantation combined with anlotinib had more effective in tumor inhibition. The results of Ki67, TUNEL and CD31 staining confirmed an evident reduction in tumor cell proliferation and angiogenesis, as well as an increase in apoptosis. A relatively integrated bone metabolism was indicated after I-125 seeds single implantation with or without anlotinib, and the results were similar in I-125 seeds fractional implantation, including a reduction in the number of TRAP-positive cells and an increase in ALP expression level. Additionally, the serum TRACP-5b activity was decreased and the serum PINP concentration was increased following I-125 seeds implantation. Conclusions Single and fractionated implantation pattern of I-125 radioactive seeds had similar therapeutic efficacy against tumor growth, while brachytherapy with I-125 seeds implantation may be an effective and safe treatment strategy for its potential protection against cancer treatment-induced bone loss.

Suplemen Calcitriol Dosis Tinggi Mencegah Osteoporosis dan Memicu Urolitiasis pada Tikus Ovariektomi

Calcitriol supplementation in ovariectomized rats for 8 weeks reduce osteoporosis risk and safe for kidney depend a proper dose. The objective of the research was to study the efectivity of high dose calcitriol for osteoporosis prevention and safe for kidney in ovariectomized Wistar rats. Fifteen female Wistar rats at 8 weeks of age were randomly divided into three groups of five which were sham operated rats (KN), ovariectomized rats (KOV), and ovariectomized rats+calcitriol supplementation 200ng/day (OVD200). After 8 weeks of treatment, blood samples were taken from plexus orbitalis medialis for estrogen analysis. All rats were then euthanized, left tibia and left kidney were taken for histopathological examination using hematoxylin and eosin stain. Immunohistochemistry using anti TRAP5b monoclonal antibody was also done for left tibia by streptavidin-biotin. The results showed that estradiol level of KOV was significantly decreased compared with KN (P&lt;0.05), meanwhile, estradiol levels of OVD200 were not significantly different compared with KOV. Histopathologic figure of kidney in KOV was not different compared with KN. Glomerulus was surrounded by Bowman‘s capsule, proximal and distal convoluted tubulus were lined with typical cuboidal epithelium. Immunohistochemistry result of proximal tibial epiphysis in KOV were shown more tartrate resistant alkaline phosphatase 5b (TRAP5b) expression in trabecular bone, which was located in bone marrow space, and trabecular speculum surface as well. Meanwhile, proximal tibia epiphysis in OVD200 had less tartrate resistant alkaline phosphatase 5b (TRAP5b) expression compared with KOV. Histopathologic figure of proximal tibia epiphysis in group OVD200 was shown more trabecular bone speculum and less adipocyte in the bone marrow compared with KOV. Histopathologic figure of kidney in OVD200 was showed glomerular hypertrophy and atrophy, as well as protein deposits in proximal convoluted tubules epithelium. In conclusion, calcitriol supplementation 200ng/day in ovariectomized rats for 8 weeks prevented osteoporosis risk but trigger urolithiasis. Key words : : Calcitriol, osteoporosis, ovariectomized rats, urolithiasis

FAM20C plays a critical role in the development of mouse vertebra

Background ContextFamily with sequence similarity 20-member C (FAM20C) is a protein kinase that is responsible for the phosphorylation of many secretory proteins; however, its roles in spine or vertebra development have not be studied. PurposeThe aim of this investigation is to analyze the roles of FAM20C in vertebra development. Study Design/SettingA mouse study of the Fam20c gene using conditional knockout to assess the effects of its inactivation on vertebra development. MethodsBy breeding Sox2-Cre mice with Fam20cflox/flox mice, Sox2-Cre;Fam20cflox/flox mice (abbreviated as cKO mice) are created. X-ray radiography, resin-casted scanning electron microscopy, Hematoxylin and Eosin staining, safranin O staining, Goldner's Masson trichrome staining, Von Kossa staining, tartrate-resistant alkaline phosphatase staining, immunohistochemistry staining, Western Immunoblotting and real-time PCR were employed to characterize the vertebrae of cKO mice compared to the normal control mice. ResultsInactivation of Fam20c in mice results in remarkable spine deformity, severe morphology and mineralization defects, altered levels of osteoblast differentiation markers, reduction of activity of the Wnt/β-catenin signaling pathway and reduced level of osteoclastogenesis in the vertebrae. ConclusionsFAM20C plays an essential role in vertebral development; it may regulate vertebral formation through the Wnt/β-catenin signaling pathway. Clinical significanceMutations in the human FAM20C gene are associated with Raine syndrome. The findings of this study provide valuable clues for the clinical management of Raine syndrome regarding spine manifestations in patients.

Human osteoclast formation and resorptive function on biomineralized collagen

Biomineralized collagen composite materials pose an intriguing alternative to current synthetic bone graft substitutes by offering a biomimetic composition that closely resembles native bone. We hypothesize that this composite can undergo cellular resorption and remodeling similar to natural bone. We investigate the formation and activity of human osteoclasts cultured on biomineralized collagen and pure collagen membranes in comparison to cortical bone slices. Human monocytes/macrophages from peripheral blood differentiate into multinucleated, tartrate-resistant alkaline phosphatase (TRAP)-positive osteoclast-like cells on all substrates. These cells form clear actin rings on cortical bone, but not on biomineralized collagen or pure collagen membranes. Osteoclasts form resorption pits in cortical bone, resulting in higher calcium ion concentration in cell culture medium; however, osteoclast resorption of biomineralized collagen and collagen membranes does not measurably occur. Activity of osteoclast enzymes – TRAP, carbonic anhydrase II (CA-II), and cathepsin-K (CTS-K) – is similar on all substrates, despite phenotypic differences in actin ring formation and resorption. The mesh-like structure, relatively low stiffness, and lack of RGD-containing binding domains are likely the factors responsible for preventing formation of stable actin rings on and resorption of (biomineralized) collagen membranes. This insight helps to guide further research toward the optimized design of biomineralized collagen composites as a more biomimetic bone-graft substitute.

Boeravinone B promotes fracture healing in ovariectomyinduced osteoporotic rats via the regulation of NF-κB p65/IκB-α/SIRT-1 signaling pathway

Purpose: To investigate the fracture-healing effect of boeravinone B in ovariectomy-induced (OVX) osteoporotic rats.&#x0D; Methods: Adult female Wistar rats (n = 30) were ovariectomized and after three months, the unilateral cross-tibial fractures were fixed with intramedullary nails. The rats were then randomly assigned to three groups of 10 rats each: normal control group, OVX group and 100 mg/kg body weight boeravinone B group. Boeravinone B was orally administered for a period of 5 weeks. The effect of boeravinone B on indices of bone formation and resorption was assessed. Levels of inflammatory cytokines including tumor necrosis factor- α (TNF-α) and interleukin-1β (IL-1β) were determined using enzyme-linked immunosorbent assay (ELISA). Western blotting was used to determine the expression levels of NF-κB p65, IкB-α and SIRT1 proteins.&#x0D; Results: There were significant increases in the activities of tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP), and collagen type I fragment (CTX) level and serum osteocalcin (OC) of OVX group, when compared with normal control group (p &lt; 0.05). However, treatment with boeravinone B significantly reduced the activities and levels of these parameters, relative to OVX group (p &lt; 0.05). The levels of TNF-α and IL-1β significantly increased in OVX group, relative normal control group, but were significantly lower following treatment with boeravinone B (p &lt; 0.05). Bone mineral content (BMC) was not significantly altered in OVX and boeravinone B-treated groups, when compared with normal control group (p &gt; 0.05). There was significant reduction in bone mineral density (BMD) of OVX group relative to normal control group (p &lt; 0.05). However, treatment with boeravinone B significantly increased the BMD, when compared with OVX group (p &lt; 0.05). After Week 5 of treatment, boeravinone B significantly enhanced bone remodeling and formation of callus. Treatment with boeravinone B significantly reduced the expression levels of NF-κB p65 and IκB-α proteins, and significantly upregulated the expression of SIRT-1 (p &lt; 0.05).&#x0D; Conclusion: The results obtained in this study suggest that boeravinone B promotes the healing of fracture caused by osteoporosis via a mechanism involving NF-κB p65/IκB-α/SIRT-1 signaling pathway.

Novel fluorescent staining protocol for thick sections of human osteochondral tissues to facilitate correlation with MRI and CT.

To describe a novel fluorescent histochemical protocol to visualize osteoclasts, vasculature, and nerves in thick sections of human osteochondral tissues and to demonstrate its feasibility for use in radiologic-pathologic research correlation studies. Consecutive patients scheduled for total knee arthroplasty surgeries underwent pre-operative MRI. CT imaging was performed after tissue collection, and abnormal osteochondral regions were sectioned to 1-2mm in thickness and decalcified. Fluorescent labeling of osteoclasts was performed by staining for tartrate-resistant alkaline phosphatase activity with a fluorescent substrate. Vascular structure was visualized with fluorescently labeled lectin Ulex europaeus Agglutinin I (UEA-I). Immunostaining was performed for proteins including smooth muscle actin expressed in smooth muscle cells surrounding arterioles and fibrotic myofibroblasts, as well as for neuropeptide Y expressed in sympathetic nerves. Sections were then recut at 5μm and stained with hematoxylin and eosin (H&E). Edema-like and cyst-like regions identified with MRI and CT were easily located in fluorescent images and appeared to have increased osteoclast activity. Fibrotic regions were identified with thickened arterioles and increased myofibroblasts. Sympathetic nerve fibers traveled alongside arborizing blood vessels. Stained sections became transparent in a water-based refractive index-matched medium, permitting deep 3D visualization of the elaborate neurovascular network in bone. Sequential staining procedures were successfully performed with the same sections, demonstrating the potential to compare multiple cellular markers from the same locations. Routine H&E staining could be performed after the fluorescent staining protocol. We have developed a multimodal framework to facilitate comparisons between histology and clinical MRI and CT.

Cobalt-containing calcium phosphate induces resorption of biomineralized collagen by human osteoclasts

BackgroundBiomineralized collagen, consisting of fibrillar type-I collagen with embedded hydroxyapatite mineral, is a bone-mimicking material with potential application as a bone graft substitute. Despite the chemical and structural similarity with bone extracellular matrix, no evidence exists so far that biomineralized collagen can be resorbed by osteoclasts. The aim of the current study was to induce resorption of biomineralized collagen by osteoclasts by a two-fold modification: increasing the calcium phosphate content and introducing cobalt ions (Co2+), which have been previously shown to stimulate resorptive activity of osteoclasts.MethodsTo this end, we produced biomineralized collagen membranes and coated them with a cobalt-containing calcium phosphate (CoCaP). Human osteoclasts, derived from CD14+ monocytes from peripheral blood, were differentiated directly on the membranes. Upon fluorescent staining of nuclei, F-actin and tartrate-resistant alkaline phosphatase, the cells were analyzed by laser confocal microscopy. Their resorption capacity was assessed by scanning electron microscopy (SEM), as well as indirectly quantified by measuring the release of calcium ions into cell culture medium.ResultsThe CoCaP coating increased the mineral content of the membranes by 4 wt.% and their elastic modulus from 1 to 10 MPa. The coated membranes showed a sustained Co2+ release in water of about 7 nM per 2 days. In contrast to uncoated membranes, on CoCaP-coated biomineralized collagen membranes, osteoclasts sporadically formed actin rings, and induced formation of resorption lacunae, as observed by SEM and confirmed by increase in Ca2+ concentration in cell culture medium. The effect of the CoCaP layer on osteoclast function is thought to be mainly caused by the increase of membrane stiffness, although the effect of Co2+, which was released in very low amounts, cannot be fully excluded.ConclusionsThis work shows the potential of this relatively simple approach to induce osteoclast resorption of biomineralized collagen, although the extent of osteoclast resorption was limited, and the method needs further optimization. Moreover, the coating method is suitable for incorporating bioactive ions of interest into biomineralized collagen, which is typically not possible using the common biomineralization methods, such as polymer-induced liquid precursor method.

Osteoclast formation, inflammation, and matrix metalloproteinase-9 are downregulated in bone repair following root canal treatment in dogs teeth.

The aim of this study was to investigate the inflammatory infiltrate, osteoclast formation, and expression of MMP-9 during the healing phase following root canal treatment in teeth with apical periodontitis. Apical periodontitis was induced in dogs teeth, and root canal treatment was performed in a single visit or using calcium hydroxide as intracanal medication. One hundred and eighty days following treatment the presence of inflammation was examined, and the tissues were stained to detect osteoclasts by means of a tartrate resistant alkaline phosphatase (TRAP) assay. Synthesis of MMP-9 was detected using Western blotting and immunohistochemistry. Teeth with apical periodontitis that had root canal therapy performed in a single visit presented a higher synthesis of MMP-9 compared with root canal treatment using calcium hydroxide. Treatment with calcium hydroxide resulted in a reduced amount of inflammatory cells and MMP-9 positive cells. Osteoclast formation, the number of MMP-9 positive osteoclasts and cementocytes, was reduced following root canal treatment, regardless of the root canal treatment protocol used. Root canal treatment reduced the amount of inflammatory cells and osteoclasts in periapical area. The use of calcium hydroxide as intracanal medication resulted in a lower synthesis of MMP-9, though the number of osteoclasts and MMP-9 positive osteoclasts were similar between the groups. Periapical bone repair following root canal treatment is impacted by therapy performed either in single visit or using calcium hydroxide dressing measured by inflammatory cell recruitment, osteoclast formation, and MMP-9 synthesis.