Rat Osteoblast-like Cells Research Articles

Abaloparatide Maintains Normal Rat Blood Calcium Level in Part Via 1,25-Dihydroxyvitamin D/osteocalcin Signaling Pathway.

The PTH-related peptide(1-34) analog, abaloparatide (ABL), is the second anabolic drug available for the treatment of osteoporosis. Previous research demonstrated that ABL had a potent anabolic effect but caused hypercalcemia at a significantly lower rate. However, the mechanism by which ABL maintains the stability of blood calcium levels remains poorly understood. Our in vivo data showed that ABL treatment (40 µg/kg/day for 7 days) significantly increased rat blood level of 1,25-dihydroxyvitamin D [1,25-(OH)2D] without raising the blood calcium value. ABL also significantly augmented the carboxylated osteocalcin (Gla-Ocn) in the blood and bone that is synthesized by osteoblasts, and increased noncarboxylated Ocn, which is released from the bone matrix to the circulation because of osteoclast activation. The in vitro data showed that ABL (10 nM for 24 hours) had little direct effects on 1,25-(OH)2D synthesis and Gla-Ocn formation in nonrenal cells (rat osteoblast-like cells). However, ABL significantly promoted both 1,25-(OH)2D and Gla-Ocn formation when 25-hydroxyvitamin D, the substrate of 1α-hydroxylase, was added to the cells. Thus, the increased 1,25-(OH)2D levels in rats treated by ABL result in high levels of Gla-Ocn and transient calcium increase in the circulation. Gla-Ocn then mediates calcium ions in the extracellular fluid at bone sites to bind to hydroxyapatite at bone surfaces. This regulation by Gla-Ocn at least, in part, maintains the stability of blood calcium levels during ABL treatment. We conclude that the signaling pathway of ABL/1,25-(OH)2D/Gla-Ocn contributes to calcium homeostasis and may help understand the mechanism of ABL for osteoporosis therapy.

PD03-01 FGF-23 PRODUCTION COUPLING WITH ALDOSTERONE AND PREDICTING CLINICAL OUTCOMES IN PRIMARY ALDOSTERONISM

You have accessJournal of UrologyCME1 Apr 2023PD03-01 FGF-23 PRODUCTION COUPLING WITH ALDOSTERONE AND PREDICTING CLINICAL OUTCOMES IN PRIMARY ALDOSTERONISM Jeff Chueh, Shuo-Meng Wang, VinCent Wu, and Kang-Yung Peng Jeff ChuehJeff Chueh More articles by this author , Shuo-Meng WangShuo-Meng Wang More articles by this author , VinCent WuVinCent Wu More articles by this author , and Kang-Yung PengKang-Yung Peng More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003220.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Higher fibroblast growth factor-23 (FGF-23) levels are associated with activating renin-angiotensin-aldosterone system (RAAS), more volume overload, and hypertension. The roles of FGF-23 in primary aldosteronism (PA) haven't been studied. METHODS: Unilateral PA (uPA) patients who underwent adrenalectomy were enrolled and followed for their clinical outcomes from Aug 15 to Jan 19. The FGF-23 expression in a rat osteoblast-like cell culture was evaluated after the addition of aldosterone. RESULTS: A total of 121 uPA patients & 69 matched essential hypertension (EH) controls were enrolled. The log [carboxyl-terminal FGF-23 (cFGF-23)] level was higher in uPA patients (p=0.001).] In Cox proportional hazard model, the preoperative Log [plasma cFGF-23]>1.98 was a risk factor for either all-cause mortality and cardiovascular or kidney events (HR, 4.01, p=0.038). Plasma cFGF-23 levels were increased in a nonlinear manner re: plasma aldosterone concentration (PAC). Log [plasma cFGF-23]>1.75 independently predicted post-op remission of hypertension (odds ratio (OR), 3.8, p=0.004). The plasma total FGF-23 level was significantly higher in PA patients (p<0.001), and it was significantly reduced post-op (p<0.001). When osteoblast-like UMR-106 cells were cultured with aldosterone, the cFGF-23 concentration in the culture medium was elevated (p=0.028), but it was abrogated after family with sequence similarity 20, member C (FAM20c) short hairpin RNA was added (p= 0.659). Docking experiments showed that aldosterone may bind to the potential drug binding pocket of FAM20c (Pdb: 5YH3; Figure 1A, B). CONCLUSIONS: Compared with EH controls, uPA patients had higher cFGF-23 levels--associated with a higher risk of cardiovascular composite outcomes, though such higher cFGF-23 levels predicted hypertension remission after adrenalectomy. Their total FGF-23 levels were significantly attenuated after adrenalectomy. An increase in cFGF-23 production among the uPA patients was secondary to increased cleavage of intact FGF-23 evidenced by higher FAM20c activity modulated by aldosterone. Source of Funding: Taiwan (NSC 101-2314-B-002-132-MY3, NSC100-2314-B-002-119, NSC 101-2314-B-002-085-MY3, MOST 104-2314-B-002 -125 -MY3; 111-2314-B-002-232-MY3) and NTUH 100-N1776, 101-M1953, 102-S2097 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e74 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jeff Chueh More articles by this author Shuo-Meng Wang More articles by this author VinCent Wu More articles by this author Kang-Yung Peng More articles by this author Expand All Advertisement PDF downloadLoading ...

Glucocorticoids dexamethasone and prednisolone suppress fibroblast growth factor 23 (FGF23)

Fibroblast growth factor 23 (FGF23) is a hormone mainly secreted by bone cells. Its most prominent effects are the regulation of renal phosphate reabsorption and calcitriol (active vitamin D, 1,25(OH)2D3) formation, effects dependent on its co-receptor αKlotho. Besides these actions, further paracrine and endocrine effects exist. The production of FGF23 is regulated by 1,25(OH)2D3, parathyroid hormone, dietary phosphate intake, iron status, as well as inflammation. Glucocorticoids are hormones with anti-inflammatory properties and are, therefore, widely used for acute and chronic inflammatory diseases, autoimmune disorders, and malignancies. The present study explored whether glucocorticoids influence the production of FGF23 in vitro as well as in mice. Fgf23 transcription was analyzed by semi-quantitative real-time PCR. Serum concentrations of FGF23 and 1,25(OH)2D3 were measured by ELISA. Urinary phosphate and Ca2+ excretion were determined in metabolic cages. As a result, in UMR106 rat osteoblast-like cells and in MC3T3-E1 cells, both, dexamethasone and prednisolone, downregulated Fgf23 transcription and FGF23 protein synthesis. Dexamethasone increased Dmp1 and Phex (encoding FGF23-regulating genes) as well as Nfkbia (encoding NFκB inhibitor IκBα) transcription in UMR106 cells. In mice, a single injection of dexamethasone or prednisolone was followed by a significant decrease of serum C-terminal and intact FGF23 concentration and bone Fgf23 mRNA expression within 12 h. These effects were paralleled by increased renal phosphate excretion and enhanced 1,25(OH)2D3 formation. We conclude that a single glucocorticoid treatment strongly downregulates the FGF23 plasma concentration.Key messagesGlucocorticoids dexamethasone and prednisolone suppress the formation of bone-derived hormone fibroblast growth factor 23 (FGF23) in vitro.The effect is accompanied by an upregulation of Dmp1, Phex, and IκBα, negative regulators of FGF23, in UMR106 osteoblast-like cells.Glucocorticoid receptor antagonist RU-486 attenuates the effect of dexamethasone on FGF23, Dmp1, and Phex.In mice, a single glucocorticoid dose suppresses FGF23 and enhances 1,25(OH)2D3 (active vitamin D).

Total flavonoid fraction of rhizoma drynaria improves bone properties in ovariectomized mice and exerts estrogen-like activities in rat osteoblast-like (UMR-106) cells

Rhizoma Drynaria (RD) has been widely used for healing bone fractures or related diseases in traditional Chinese medicine. The present study was designed to determine if RD total flavonoids (RDTF) could exert estrogen-like protective actions on bone. Young C57/BL6J mice were ovariectomized (OVX) and treated orally with RDTF (0.087, 0.173 or 0.346 mg/g/day), 17β-estradiol (2μg/g/day) or its vehicle for 6 weeks. Bone mineral densities (BMD) was measured by peripheral quantitative computed tomography (pQCT). Rat osteoblast-like UMR-106 cells were co-incubated with ER antagonist ICI 182, 780 to determine if the effects of RDTF on osteoblastic functions were ER-dependent. The functional transactivation of ER-α and ER-β as well as ER-αphosphorylation by RDTF were also studied. RDTF increased trabecular-rich BMD at distal femur and lumbar spine in OVX mice. 0.173 mg/g/day was most effective in improving bone properties in OVX mice. The stimulatory effects of RDTF on osteoblastic functions could be abolished by co-incubation with ICI 182, 780 in UMR-106 cells. Transient transfection study indicated that RDTF dose-dependently stimulated ERE-dependent luciferase activity in UMR-106 cells via ER-α and ER-β. Moreover, 0.2 μg/mL of RDTF significantly induced ER-α phosphorylation at serine 118 in UMR-106 cells.

Peroxisome proliferator-activated receptor α (PPARα)-dependent regulation of fibroblast growth factor 23 (FGF23).

Bone cells secrete fibroblast growth factor 23 (FGF23), a hormone that inhibits the synthesis of active vitamin D (1,25(OH)2D3) and induces phosphate excretion in the kidney. In addition, it exerts paracrine effects on other cells including hepatocytes, cardiomyocytes, and immune cells. The production of FGF23 is controlled by different factors including parathyroid hormone, 1,25(OH)2D3, alimentary phosphate, insulin, inflammation, and AMP-dependent kinase (AMPK) regulation of store-operated Ca2+ entry (SOCE). Peroxisome proliferator-activated receptor α (PPARα) is a transcription factor with anti-inflammatory properties regulating lipid metabolism. Fibrates, PPARα agonists, are used in the treatment of dyslipidemia and activate AMPK. Here, we tested whether PPARα is a regulator of FGF23. Fgf23 gene expression was analyzed in UMR106 rat osteoblast-like cells by qRT-PCR, AMPK phosphorylation by Western blotting, and SOCE assessed by fluorescence optics. PPARα agonists fenofibrate and WY-14643 suppressed, whereas PPARα antagonist GW6471 and siRNA-mediated knockdown of PPARα induced Fgf23 gene expression. Fenofibrate induced AMPK activity in UMR106 cells and lowered SOCE. AMPK inhibitor compound C abrogated the PPARα effect on FGF23 in large part. Silencing of Orai-1 resulted in failure of PPARα to significantly influence Fgf23 expression. Taken together, PPARα is a potent regulator of FGF23. PPARα agonists down-regulate FGF23 formation at least in part through AMPK-mediated suppression of SOCE.

MON-537 Fusion of Parathyroid Hormone to Growth Hormone Binding Protein Delays Clearance Whilst Retaining Biological Activity

Parathyroid hormone (PTH) is an 84 aa peptide and biological activity resides in residues 1-34. PTH 1-84 (Natpara) is licensed for treatment of hypoparathyroidism replacement but requires daily injections and is complicated by fluctuating calcium levels. There is an unmet need for a long acting PTH that provides constant physiological levels of PTH bioactivity. We have tested whether fusions of human PTH 1-34 to growth hormone binding protein (GHBP) could generate long acting PTH molecules. Two PTH fusion molecules were constructed: Fusion-1 consists of PTH (1-34) linked to GHBP (residues 1-238), and Fusion-2 consists of human PTH (1-34) linked to the N-terminal PTH receptor domain (PTHrExt, residues 29-187) and GHBP (residues 1-238). Fusions were expressed as secreted products from a CHO cell line and purified using a combination of ion exchange and affinity chromatography. In vitro bioactivity was measured using a Dual Luciferase Reporter Assay (DLRA), using the rat osteoblast-like cell line, UMR-106. Both fusions showed biological activity when compared to PTH 1-34 with an EC50 nM (mean ± SD) for PTH 1-34 of 40 ± 15.66, Fusion-1, 151 ± 5.85, & Fusion-2, 721 ± 307. PTH was 3.8-fold more active than Fusion-1 and 18-fold more active than Fusion-2; although, Fusion-2 showed a consistently higher maximal fold induction compared to both PTH and Fusion-1. A pharmacokinetic (PK) study for Fusion-1 was conducted in Crl:WI(Han) male rats (n = 6) by administering a single subcutaneous bolus injection at 10 nmol/kg. Serum samples were analysed for the presence of Fusion-1 using an in-house Elisa. A Cmax (mean ± SD) of 1.16 ± 0.36 nM was observed at 8 hrs with a serum half-life of 36 hrs. In conclusion, a PTH fusion molecule with GHBP showed bioactivity and delayed clearance. The reduced bioactivity of Fusion-2 compared to PTH and Fusion-1 is compatible with our hypothesis that there is intra-molecular binding to the extracellular domain of the PTH receptor which may result in a store of bioactivity and may explain the greater maximal fold induction in the bioassay despite a reduced EC50. The published serum half-life of PTH 1-34 in rats is 59 minutes1 and so Fusion-1 had greatly delayed clearance. These results validate the approach of generating a long-acting PTH through protein fusion with GHBP. 1. Kostenuik, PJ et al. Journal of Bone and Mineral Research, Volume 22, Number 10, 2007 Sources of Research Support: MRC P2D

Multitargeted combination effects of a triherbal formulation containing ELP against osteoporosis: in vitro evidence.

An anti-osteoporotic herbal formula ELP containing Epimedii Herba (E), Ligustri Lucidi Fructus (L) and Psoraleae Fructus (P) was studied to investigate the herb-herb interaction (or the possible synergistic effect) among each component and to identify the principal herbs in different modes of action. Rat osteoblast-like UMR-106 cells proliferation, rat MSCs-derived osteoblastogenesis and RANKL-induced RAW 264.7 osteoclastogenesis were adopted to investigate the bone-forming activity and bone-degrading activity of the herbal extracts. In the statistical aspect, a modified Tallarida's approach was employed to assess the synergistic effects in herbal combinations. Psoraleae Fructus is the active herb for stimulating osteoblast proliferation, and mild synergy was detected in the pairwise combinations EL, LP and formula ELP. In osteoblastogenesis assay, E and L are the principal herbs for promoting osteoblast differentiation and significant synergy was detected in the pairwise combination EL. For inhibiting osteoclast formation, L is the active herb and significant synergy was detected in the 3-way combination ELP. The presence of E, L and P is essential for ELP formula as a whole to act against osteoporosis via enhancing bone formation and reducing bone reabsorption. An optimal dosage at 150 μg/ml was proposed for ELP based on our findings.

Physico-chemical and in vitro cellular properties of different calcium phosphate-bioactive glass composite chitosan-collagen (CaP@ChiCol) for bone scaffolds.

In the present study, scaffolds for bone tissue engineering applications were made by immersing the inorganic phases of three different calcium phosphate (CaPs) (hydroxyapatite (HA), tricalcium phosphate (TCP), and biphasic calcium phosphate (BCP)) mixing bioactive glass (15Ca:80Si:5P) (BG) with polycaprolactone (PCL) as a binder in an organic phase of chitosan/collagen (ChiCol) matrix (CaPBG@ChiCol). Porous scaffolds were obtained by freeze drying the combinations. The mechanical properties and in vitro growth of rat osteoblast-like UMR-106 cells were investigated. The investigation indicated that the compressive strength was controlled by the types of CaP. The highest compressive modulus of the composites was 479.77MPa (23.84MPa for compressive strength) which is for the BCPBG@ChiCol composite. Compressive modulus of 459.01 and 435.95MPa with compressive strength of 22.73 and 17.89MPa were observed for the HABG@ChiCol and TCPBG@ChiCol composites, respectively. In vitro cell availability and proliferation tests confirmed the osteoblast attachment and growth on the CaPBG@ChiCol surface. Comparing the scaffolds, cells grown on the BCPBG based composite showed the higher cell density. To test its bioactivity, BCPBG@ChiCol was chosen for MTT and ALP assays on UMR-106 cells. The results indicated that the UMR-106 cells were viable and had higher ALP activity as the culturing times were increased. Therefore, ChiCol-fabricated BCPBG scaffold shows promise for bone regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1758-1766, 2017.

Expression of osteoclastogenic factor transcripts in osteoblast-like UMR-106 cells after exposure to FGF-23 or FGF-23 combined with parathyroid hormone.

As a bone-derived hormone, fibroblast growth factor-23 (FGF-23) negatively regulates phosphate and calcium metabolism, while retaining growth-promoting action for mesenchymal cell differentiation. Elevated FGF-23 levels, together with hyperparathyroidism, are often observed in chronic kidney disease, which is associated with impaired bone mineralization and enhanced bone resorption. Although overexpression of osteoblast-derived osteoclastogenic cytokines might contribute to this metabolic bone disease, whether FGF-23 alone and FGF-23 plus parathyroid hormone (PTH) directly modulated the expression of osteoblast-derived osteoclastogenic genes remained elusive. Herein, we demonstrated the direct effects of FGF-23 on proliferation and mRNA expression of osteoblast-specific differentiation and osteoclastogenic markers in rat osteoblast-like UMR-106 cells in the presence or absence of PTH. FGF-23 was found to suppress UMR-106 cell proliferation, while increasing FGF-23 expression, the latter of which suggested the presence of positive feedback regulation of FGF-23 expression in osteoblasts. FGF-23 also upregulated the mRNA expression of osteoblast differentiation markers (e.g., Runx2, osterix, AJ18, Dlx5, alkaline phosphatase, and osteopontin), osteoclastogenic factors (e.g., MCSF, MCP-1, IL-6, and TNF-α), and bone resorption regulators (RANKL and osteoprotegerin). However, combined PTH and FGF-23 exposure did not alter the levels of FGF-23-induced transcripts, suggesting that both hormones had no additive effect. In conclusion, FGF-23 directly suppressed osteoblast proliferation, while inducing osteoclastogenic gene expression in UMR-106 cells, and the FGF-23-induced transcripts were not altered by long-standing PTH exposure.

Differentiation of deer tendons from cattle tendons by a loop-mediated isothermal amplification (LAMP) test and bone remodeling bioassays.

BackgroundDeer tendons are believed more effective than cattle tendons in tonifying kidney yang (shen yang) and enhancing bone and tendons. This study aims to differentiate the two types of tendons by a loop-mediated isothermal amplification (LAMP) test and bone remodeling bioassays.MethodsInternal control primers to detect both types of tendons and specific primers for deer tendons were designed according to a sequence analysis. The LAMP test was set up and the results were analyzed by conventional gel electrophoresis, real-time fluorescence observation, and colorimetric detection. Crude tendon extracts were prepared by water extraction to compare their effects on bone. The anti-osteoclastic effects were investigated on mouse pre-osteoclast Raw264.7 cells by cell viability determination and tartrate-resistant acid phosphatase staining. The osteogenic effects were examined using rat osteoblast-like UMR106 cells by evaluation of cell proliferation, alkaline phosphatase activity, and calcium deposition. The relative gene expressions of bone remodeling-related markers, including nuclear factor of activated T-cells cytoplasmic 1, tartrate-resistant acid phosphatase, cathepsin K, and osteoprotegerin/receptor activator of NF-κB ligand, were determined by real-time PCR.ResultsIn the LAMP test, both deer and cattle tendons were detected in the control reactions, while only deer tendons were amplified by the specific LAMP test. In the bioassays, both tendons inhibited the viability and differentiation of pre-osteoclast Raw264.7 cells, and promoted the proliferation and mineralization of osteoblast-like UMR106 cells. The mRNA expressions of bone remodeling-related markers were consistent with the results of the bioassays.ConclusionThis study demonstrated that the isothermal LAMP test can distinguish between deer tendons and cattle tendons. Both types of tendons exhibited similar beneficial effects on bone remodeling according to the bioassay findings.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-015-0065-6) contains supplementary material, which is available to authorized users.

The Effects of Different Titanium Surfaces on the Behaviour of Osteoblast-Like Cells

This study investigated the influence of different titanium surfaces on the differentiation of rat osteoblast-like cells (osteo-1). Osteo-1 cells were cultured on the following titanium surfaces: 1) pretreated, smooth surface (PT); 2) sandblasted and acid etched surface (SLA); and 3) sandblasted and acid-etched surface rinsed under nitrogen protection to prevent exposure to air and preserved in isotonic saline solution (modSLA). Cell metabolism, total protein content, collagen content and alkaline phosphatase (AP) activity and the formation of calcified nodules were analyzed. The titanium surface did not influence cell metabolism, total protein content and collagen content. The SLA surface influenced cell differentiation, with the observation of a significant reduction of AP activity and formation of calcified nodules after 21 days compared to the PT surface. No difference was observed between the PT and modSLA surfaces. All titanium surfaces tested permitted the full expression of the osteoblast phenotype by osteo-1 cells, including matrix mineralization.

Parathyroid hormone activates the orphan nuclear receptor Nurr1 to induce FGF23 transcription.

Parathyroid hormone (PTH) increases FGF23 mRNA and protein levels in vivo and in vitro. Here we tested whether the increased FGF23 expression by PTH is mediated by the orphan nuclear receptor Nurr1. PTH increased Nurr1 mRNA levels prior to elevation of FGF23 mRNA levels in UMR-106 rat osteoblast-like cells. Activation of PKA increased both FGF23 and Nurr1 mRNA levels. Modification of Nurr1 expression showed that Nurr1 is essential for the PTH-mediated increase in FGF23 and luciferase reporter gene experiments identified a functional promoter region containing several potential Nurr1 binding sites. Chromatin immunoprecipitation assays confirmed the binding of Nurr1 to these regions in the FGF23 promoter. In vivo, Nurr1 mRNA and protein levels were increased in calvaria from rats with experimental CKD together with high PTH and FGF23 expression. Calcimimetics decrease PTH and FGF23 levels in CKD patients. Importantly, in rats with experimental CKD, the calcimimetic R568 decreased PTH expression, calvaria Nurr1 mRNA and protein levels, and FGF23 mRNA. Immunohistochemistry for Nurr1 showed an increase in the number of Nurr1 expressing osteocytes in the femurs of rats with CKD and this was decreased by R568. Thus, the effect of PTH to increase FGF23 transcription is mediated by Nurr1 in vitro and in vivo. In CKD, calcimimetics decrease PTH, which in turn decreases Nurr1 and consequently FGF23.

Mechanical properties, biological activity and protein controlled release by poly(vinyl alcohol)–bioglass/chitosan–collagen composite scaffolds: A bone tissue engineering applications

In the present study, composite scaffolds made with different weight ratios (0.5:1, 1:1 and 2:1) of bioactive glass (15Ca:80Si:5P) (BG)/polyvinyl alcohol (PVA) (PVABG) and chitosan (Chi)/collagen (Col) (ChiCol) were prepared by three mechanical freeze–thaw followed by freeze-drying to obtain the porous scaffolds. The mechanical properties and the in vitro biocompatibility of the composite scaffolds to simulated body fluid (SBF) and to rat osteoblast-like UMR-106 cells were investigated. The results from the studies indicated that the porosity and compressive strength were controlled by the weight ratio of PVABG:ChiCol. The highest compressive modulus of the composites made was 214.64MPa which was for the 1:1 weight ratio PVABG:ChiCol. Mineralization study in SBF showed the formation of apatite crystals on the PVABG:ChiCol surface after 7days of incubation. In vitro cell availability and proliferation tests confirmed the osteoblast attachment and growth on the PVABG:ChiCol surface. MTT and ALP tests on the 1:1 weight ratio PVABG:ChiCol composite indicated that the UMR-106 cells were viable. Alkaline phosphatase activity was found to increase with increasing culturing time. In addition, we showed the potential of PVABG:ChiCol drug delivery through PBS solution studies. 81.14% of BSA loading had been achieved and controlled release for over four weeks was observed. Our results indicated that the PVABG:ChiCol composites, especially the 1:1 weight ratio composite exhibited significantly improved mechanical, mineral deposition, biological properties and controlled release. This made them potential candidates for bone tissue engineering applications.

Advanced oxidation protein products accelerate bone deterioration in aged rats

Advanced oxidation protein products (AOPPs) are novel markers of oxidation-mediated protein damage, and accumulation of AOPPs is involved in many pathophysiological conditions. Our previous studies demonstrated that the serum level of AOPPs negatively correlated with the age-related change in bone mineral density (BMD) in rats and that AOPPs inhibited rat osteoblast-like cell proliferation and differentiation in vitro. However, whether AOPPs are involved in senile osteoporosis is still largely unknown. The present study aimed to test the hypothesis that accumulation of AOPPs might accelerate bone deterioration in aged rats. Seventy 18-month-old male Sprague Dawley (SD) rats were randomized to intravenous injection of vehicle, native rat serum albumin (RSA), AOPPs-modified RSA (AOPPs-RSA) with or without oral administration of apocynin (a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor), or apocynin alone. After treatment for 8weeks or 16weeks, seven rats in each group were sacrificed. Bone and blood samples were harvested for BMD measurement, micro-computed tomographic (micro-CT) imaging, and biochemical analysis of circulating bone biomarkers. Compared to RSA- or vehicle-treated rats, AOPPs-RSA-treated animals displayed significantly decreased total vertebral BMD and deteriorated microstructure in both the tibias and the lumbar vertebral bodies, which were associated with down-regulated plasma bone-specific alkaline phosphatase concentration and up-regulated tartrate-resistant acid phosphatase 5b concentration. These AOPPs-induced perturbations in aged rats could be prevented by the oral administration of apocynin. However, no significant differences in BMD were detected in the femurs or the biomechanical parameters tested between the different treatment groups. These data suggest that accumulation of AOPPs accelerates bone deterioration in aged rats, likely via the activation of NADPH oxidase. This study provides new information toward understanding the pathogenic basis of senile osteoporosis and may provide targets for intervention.

Physicochemical and biochemical properties of iron-loaded silicon substituted hydroxyapatite (FeSiHAp)

We have synthesized scaffolding materials based on iron-loaded silicon substituted hydroxyapatite (SiHAp) (FeSiHAp). The compounds, Ca10−x(Fex)(PO4)5-(SiO4)(OH)2−δ (x = 0.1, 0.3, 0.5), were synthesized using the stoichiometric amounts of the chemical constituents. Both physicochemical properties (structure, surface charge and hardness) and in vitro biochemical properties (Bovine serum albumin (BSA) adsorption and rat osteoblast-like UMR-106 cells response) of the products were measured. The synthesized powders still exhibited the apatite structure for iron contents up to x = 0.5. The surface charge became less negative, from −21.3 to −13.5 mV as the iron loading was increased. Compounds of FeSiHAp whisker showed a micro hardness in rang of 80–84 MPa for x = 0.1–0.5. BSA protein adsorption by FeSiHAp particles was able to fit to the energetically homogeneous behaviors in the Freundlich and Langmuir models. The release of BSA from the iron-loaded SiHAp particles was slow when compared to the release from the non iron loaded particles. Mineralization study showed the formation of apatite crystals on the FeSiHAp surface after modified simulated body fluid (m-SBF) incubation. In vitro cell growth on the FeSiHAp surface, the density of cells, the spread and production of calcium nodules on the substrate were seen to be higher for iron content x = 0.1 (FeSiHAp01). MTT tests on the FeSiHAp indicated that the UMR-106 cells were viable and there was a confluence of cells on the substrate.

Reactive oxygen species induce cell death via Akt signaling in rat osteoblast-like cell line ROS 17/2.8

In bones, osteoblasts are responsible for bone formation. The cell death of osteoblasts may cause a series of bone diseases and lead to bone loss, such as osteoarthrosis, hyperparathyroidism, and Paget's disease. Reactive oxygen species (ROS) are reported as a main factor for osteoblast cell death and further several bone diseases. However, the detailed mechanism is still largely unknown. Here, we found that ROS could induce cell death of rat osteoblast-like cell line ROS 17/2.8 via Akt (protein kinase B). Also, the mammalian target of rapamycin signaling was involved in this process. Our findings could help to reveal the cellular mechanism of osteoblast cell death, which is served for the pursuit of clinical treatment targets of relative bone diseases.

Drynaria fortunei-derived total flavonoid fraction and isolated compounds exert oestrogen-like protective effects in bone

Drynaria fortunei (Kunze) J. Sm. (DF), a Chinese herb commonly used for the treatment of bone fracture, was previously shown to exert anabolic effects on bone. However, its active ingredients as well as the mechanisms of action are far from clear. The present study aimed to characterise the bone anabolic effects of DF flavonoid fraction (DFTF) in ovariectomised (OVX) mice and to determine if DFTF and its isolated compounds exert oestrogen-like effects in rat osteoblast-like UMR-106 cells. Young OVX C57/BL6J mice were treated orally with DFTF (0·087, 0·173 or 0·346 mg/g per d), 17b-oestradiol (2 mg/g per d) or its vehicle for 6 weeks. Serum and urine samples were collected for biochemical marker analysis. Bones were collected for computed tomography analysis. UMR-106 cells were treated with DFTF and isolated compounds naringin, (2S)-5,7,30,50-tetrahydroxy-flavonone 7-O-neohesperidoside (compound 1) and 5,7-dihydroxychromone 7-O-neohesperidoside (compound 2). DFTF exerted dose-dependent effects in improving bone mineral densities as well as bone strength at the femur, tibia and lumbar spine L1 in OVX mice. DFTF and the three isolated compounds stimulated osteoblastic cell proliferation and alkaline phosphatase activities in a dose-dependent manner. In addition, they stimulated the ratio of osteoprotegrin and receptor-activator NF-kB ligand mRNA expression, suggesting their involvement in inhibiting osteoclastogenesis. These stimulatory effects on osteoblastic functions were abolished in the presence of oestrogen receptor (ER) antagonist, ICI 182780. The present results suggested that DFTF is effective in protecting against OVX-induced bone loss in mice, and its actions in regulating osteoblastic activities appear to be mediated by ER.

In vitro study of vancomycin release and osteoblast-like cell growth on structured calcium phosphate-collagen

A drug delivery vehicle consisting of spherical calcium phosphate-collagen particles covered by flower-like (SFCaPCol) blossoms composed of nanorod building blocks and their cellular response is studied. The spherical structure was achieved by a combination of sonication and freeze-drying. The SFCaPCol blossoms have a high surface area of approximately 280m2g−1. The blossom-like formation having a high surface area allows a drug loading efficiency of 77.82%. The release profile for one drug, vancomycin (VCM), shows long term sustained release in simulated body fluid (SBF), in a phosphate buffer saline (PBS, pH 7.4) solution and in culture media over 2weeks with a cumulative release ~53%, 75% and 50%, respectively, over the first 7days. The biocompatibility of the VCM-loaded SFCaPCol scaffold was determined by in vitro cell adhesion and proliferation tests of rat osteoblast-like UMR-106 cells. MTT tests indicated that UMR-106 cells were viable after exposure to the VCM loaded SFCaPCol, meaning that the scaffold (the flower-like blossoms) did not impair the cell's viability. The density of cells on the substrate was seen to increase with increasing cultured time.

Role of rhBMP-2 and rhBMP-7 in the metabolism and differentiation of osteoblast-like cells cultured on chemically modified titanium surfaces.

This study analyzed the role of recombinant human bone morphogenetic protein 2 (rhBMP-2) and recombinant human bone morphogenetic protein 7 (rhBMP-7) in the adhesion and differentiation of rat osteoblast-like (osteo-1) cells cultured on chemically modified titanium surfaces. Osteo-1 cells were cultured on chemically modified (modified sandblasted and acid-etched) titanium surfaces in 3 different types of medium: control, medium supplemented with 20 ng/mL rhBMP-2, and medium supplemented with 20 ng/mL rhBMP-7. The following parameters were evaluated: cell adhesion after 24 hours; total protein content; collagen content and alkaline phosphatase (AP) activity after 7, 14, and 21 days; and calcified nodule formation after 21 days. The addition of rhBMP-2 or rhBMP-7 did not influence cell adhesion (P = .1175). Cell differentiation was influenced by rhBMP-2, as demonstrated by a significant increase in collagen content after 7 days of culture (P < .0001) and a significant decrease in AP activity after 21 days (P < .0001). The addition of rhBMP-7 only influenced AP activity, and a significant increase was observed after 21 days (P < .0001). Within the limitations of the study, we conclude that the presence of rhBMP-2 or rhBMP-7 did not influence cell adhesion to chemically modified titanium surfaces but provided an additional stimulus during the differentiation of rat osteo-1 cells cultured on this type of surface.

Biocomposite of hydroxyapatite-titania rods (HApTiR): Physical properties and in vitro study

The aim in this research is to study the physical and biocompatible properties of hydroxyapatite (HAp) composites (HApTiR) having different amounts of titania rod (TiR) in them (10–90wt.%). The HAp and TiR were produced using hydrothermal and co-precipitation under reflux methods, respectively. The physical properties and the in vitro biocompatibility of the composites to simulated body fluid (SBF) were investigated. They were also cultured with rat osteoblast-like UMR-106 cells. The synthesized powder showed a core-shell structure with the titania rod as the core and the apatite as the shell. The hardness of the composites of HApTiR's whisker increased from 74.8 to 92.9MPa as the TiR content was increased from 10 to 90wt.%. Mineralization study in SBF showed the formation of apatite crystals on the HApTiR's surface after 7days of incubation. In vitro cell adhesion tests confirmed the osteoblast attachment and growth on the HApTiR's surface. The density of cells, spread and the production of calcium nodules on the substrate were seen to increase with increasing TiR contents except for HApTiR90 (TiR=90wt.%) which exhibited lesser growth. MTT tests on HApTiR70 indicated that UMR-106 cells were viable and the density of cells on the substrate was seen to increase with increasing culturing time.