Activating BK channels ameliorates vascular smooth muscle calcification through Akt signaling.

Vascular calcification (VC) is characterized by pathological depositions of calcium and phosphate in the arteries and veins via an active cell-regulated process, in which vascular smooth muscle cells (VSMCs) transform into osteoblast/chondrocyte-like cells as in bone formation. VC is associated with significant morbidity and mortality in chronic kidney disease (CKD) and cardiovascular disease, but the underlying mechanisms remain unclear. In this study we investigated the role of large-conductance calcium-activated potassium (BK) channels in 3 experimental VC models. VC was induced in vascular smooth muscle cells (VSMCs) by ß-glycerophosphate (ß-GP), or in rats by subtotal nephrectomy, or in mice by high-dosage vitamin D3. We showed that the expression of BK channels in the artery of CKD rats with VC and in ß-GP-treated VSMCs was significantly decreased, which was functionally confirmed by patch-clamp recording. In ß-GP-treated VSMCs, BK channel opener NS1619 (20 µM) significantly alleviated VC by decreasing calcium content and alkaline phosphatase activity. Furthermore, NS1619 decreased mRNA expression of ostoegenic genes OCN and OPN, as well as Runx2 (a key transcription factor involved in preosteoblast to osteoblast differentiation), and increased the expression of α-SMA protein, whereas BK channel inhibitor paxilline (10 µM) caused the opposite effects. In primary cultured VSMCs from BK-/- mice, BK deficiency aggravated calcification as did BK channel inhibitor in normal VSMCs. Moreover, calcification was more severe in thoracic aorta rings of BK-/- mice than in those of wild-type littermates. Administration of BK channel activator BMS191011 (10 mg· kg-1 ·d-1) in high-dosage vitamin D3-treated mice significantly ameliorated calcification. Finally, co-treatment with Akt inhibitor MK2206 (1 µM) or FoxO1 inhibitor AS1842856 (3 µM) in calcified VSMCs abrogated the effects of BK channel opener NS1619. Taken together, activation of BK channels ameliorates VC via Akt/FoxO1 signaling pathways. Strategies to activate BK channels and/or enhance BK channel expression may offer therapeutic avenues to control VC.

Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization.

Mineralization-competent cells like osteoblasts and chondrocytes release matrix vesicles (MVs) which accumulate Ca2+ and Pi, creating an optimal environment for apatite formation. The mineralization process requires the involvement of proteins, such as annexins (Anx) and tissue-nonspecific alkaline phosphatase (TNAP), as well as low molecular-weight compounds. Apigenin, a flavonoid compound, has been reported to affect bone metabolism, but there are doubts about its mechanism of action under physiological and pathological conditions. In this report, apigenin potency to modulate annexin A6 (AnxA6)- and TNAP-mediated osteoblast mineralization was explored using three cell lines: human fetal osteoblastic hFOB 1.19, human osteosarcoma Saos-2, and human coronary artery smooth muscle cells HCASMC. We compared the mineralization competence, the morphology and composition of minerals, and the protein distribution in control and apigenin-treated cells and vesicles. The mineralization ability was monitored by AR-S/CPC analysis, and TNAP activity was determined by ELISA assay. Apigenin affected the mineral structure and modulated TNAP activity depending on the concentration. We also observed increased mineralization in Saos-2 cells. Based on TEM-EDX, we found that apigenin influenced the mineral composition. This flavonoid also disturbed the intracellular distribution of AnxA6 and TNAP, especially blocking AnxA6 aggregation and TNAP attachment to the membrane, as examined by FM analysis of cells and TEM-gold analysis of vesicles. In summary, apigenin modulates the mineralization process by regulating AnxA6 and TNAP, as well as through various effects on normal and cancer bone tissues or atherosclerotic soft tissue.

The role of statins in the differentiation and function of bone cells.

BACKGROUND: Statins are 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors blocking cholesterol biosynthesis in hepatic cells, thereby causing an increase in low-density lipoprotein (LDL) receptors resulting in enhanced uptake and clearance of atherogenic LDL-cholesterol (LDL-C) from the blood. Accordingly, statins decrease the risk of developing atherosclerosis and its acute complications, such as acute myocardial infarction and ischaemic stroke. Besides the LDL-C-lowering impact, statins also have other so-called pleiotropic effects. Among them, the ability to modulate differentiation and function of bone cells and exert direct effects on osteosynthesis factors. Specifically, earlier studies have shown that statins cause in vitro and in vivo osteogenic differentiation. DESIGN: The most relevant papers on the bone-related 'pleiotropic' effects of statins were selected following literature search in databases and were reveiwed. RESULTS: Statins increase the expression of many mediators involved in bone metabolism including bone morphogenetic protein-2 (BMP-2), glucocorticoids, transforming growth factor-beta (TGF-ß), alkaline phosphatase (ALP), type I collagen and collagenase-1. As a result, they enhance bone formation and improve bone mineral density by modulating osteoblast and osteoclast differentiation. CONCLUSION: This review summarizes the literature exploring bone-related 'pleiotropic' effects of statins and suggests an anabolic role in the bone tissue for this drug class. Accordingly, current knowledge encourages further clinical trials to assess the therapeutic potential of statins in the treatment of bone disorders, such as arthritis and osteoporosis.

Fenugreek seeds attenuate thioacetamide induced liver damage.

The intention to conduct this study was to evaluate the hepatoprotective effects of Fenugreek seeds' extract supplementation in thioacetamide induced liver damage in male Sprague Dawley rats. For this study, 24 male Sprague Dawley rats (200-264gm) were distributed randomly into four groups. Group I remained untreated as control rats, group II received thioacetamide (200mg/Kg b.w i.p, administered on alternative days for 8 weeks), group III received thioacetamide (200mg/Kg b.w i.p administered on alternative days for 8 weeks) as well as 2ml of 2% extract of fenugreek seeds (orally administered daily from 4th week till 8th week of the experiment. Group IV only received 2ml of 2% extract of Fenugreek seeds daily for 4 weeks respectively. At the end of the experiment, blood was sampled to obtain plasma that was used for the analysis of liver markers and liver was used for analysis of antioxidant enzymes (catalase and SOD). Increase in total bilirubin, direct bilirubin, ALT and ALP levels, catalase activity and decrease in SOD activity was found in TAA-treated groups which assured liver damage. Whereas, treatment with Fenugreek seeds extract restored the altered levels of total bilirubin, direct bilirubin, ALT, ALP, catalase and SOD activities in the Test + Supp group. The results of this study confirmed the hepatoprotective role of Fenugreek seeds extract in thioacetamide induced liver damage.

Protective effect of Phaeoporus obliquus polysaccharide against acute liver injury induced by carbon tetrachloride and alcohol in mice.

Studied the optimum extraction process of polysaccharide from Phaeoporus obliquus and the effect of Phaeoporus obliquus polysaccharide on carbon tetrachloride (CCl4)- or alcohol-induced acute liver injury in mice. The main factor in influencing the extraction rate of Phaeoporus obliquus polysaccharide were extraction power and time, which was a kind of pyran glucose by infrared spectroscopy. CCl4 and alcohol were employed respectively to establish CCl4 and alcohol-induced acute liver injury mouse models. Compared with model groups mice, Phaeoporus obliquus polysaccharide treatment at the doses of 100mg/kg and 200mg/kg exhibited an obvious reduction liver index, ALP, ALT, AST levels, MDA content and TNF-α level (p<0.01) and SOD activity was increased, which was in a dose-dependent manner. Compared with the model group, the necrosis degree of hepatocytes was obviously reduced and the small fat droplets were formed in some cytoplasm, especially in high dose group, which the liver cells recovered to the level of normal group. Rt-PCR results showed that the expression of CYP2E1 mRNA in liver tissues of Phaeoporus obliquus polysaccharide groups were significantly reduced, and the difference were statistically significant compared with the model group (p<0.05). These results demonstrated that Phaeoporus obliquus polysaccharide has significantly hepatoprotective effect on CCl4 and alcohol-induced acute liver injury in mice.

17ß-Estradiol improves osteoblastic cell function through the Sirt1/NF-κB/MMP-8 pathway.

Objective: This study aims to investigate the beneficial effects of 17ß-estradiol supplementation on the function of osteoblastic cells through the Sirtuin-1/nuclear transcription factor-κB/matrix metalloproteinase-8 (Sirt1/NF-κB/MMP-8) pathway.Methods: Mouse primary osteoblasts were obtained from neonatal mouse calvaria, and the cells were treated with or without 17ß-estradiol. We first detected the effect of 17ß-estradiol on the function of osteoblastic cells. Then, the changes in estrogen receptor-α (ERα), Sirt1, NF-κB, and MMP-8 were determined after the osteoblasts were treated with 17ß-estradiol. During supplementation with 17ß-estradiol, knockdown of Sirt1 in osteoblasts was used to further measure the changes of NF-κB and MMP-8 and observe the cell function.Results: In primary osteoblastic cells, exposure to 17ß-estradiol improved cell viability and increased the levels of bone formation biomarkers, including osteocalcin, osteoprotegerin (OPG), procollagen type 1 N-terminal propeptide (P1NP), and alkaline phosphatase (ALP). In addition, 17ß-estradiol supplement activated ERα and Sirt1 expression and inhibited NF-κB and MMP-8 expression. Moreover, these effects induced by 17ß-estradiol were reversed by knockdown of Sirt1 in mouse primary osteoblasts.Conclusion: 17ß-Estradiol replacement therapy may treat postmenopausal osteoporosis by improving osteoblastic cell function via the Sirt1/NF-κB/MMP-8 pathway.

Induction of osteogenic differentiation by demineralized and decellularized bovine extracellular matrix derived hydrogels associated with barium titanate.

Bone tissue-derive biomaterials have become of great interest to treat diseases of the skeletal system. Biological scaffolds of demineralized and decellularized extracellular matrices (ECM) have been developed and one of these options are ECM hydrogels derived from bovine bone. Nanomaterials may be able to regulate stem cell differentiation due to their unique physical-chemical properties. The present work aimed to evaluate the osteoinductive effects of ECM hydrogels associated with barium titanate nanoparticles (BTNP) on dental pulp cells derived from exfoliated teeth. The addition of BTNP in the ECM derived hydrogel did not affect cell proliferation and the formation of bone nodules. Furthermore, it increased the expression of bone alkaline phosphatase. The results demonstrated that the nanobiocomposites were able to promote the osteogenic differentiation, even in the absence of chemical inducing factors for osteogenic differentiation. In conclusion, bovine bone ECM hydrogel combined with BTNP presented and increased expression of markers of osteogenic differentiation in the absence of chemical inducing factors.

The PERK-EIF2α-ATF4 signaling branch regulates osteoblast differentiation and proliferation by PTH.

Parathyroid hormone (PTH) and its related peptide (PTH-related peptide 1-34) are two of the Food and Drug Administration-approved bone-promoting drugs for age-related osteoporosis. Treatment with PTH stimulates bone formation. However, the molecular mechanisms of PTH-mediated osteoblast differentiation and cell proliferation are still not completely understood. In this study, we showed that PTH induced endoplasmic reticulum (ER) stress in osteoblasts through the PKR-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2α (EIF2α)-activating transcription factor 4 (ATF4)-signaling pathway. After separately blocking PERK-EIF2α-ATF4 signaling with two different inhibitors [AMG'44 and integrated stress response inhibitor (ISRIB)] or specific small interfering RNA for PERK and ATF4, the following targets were all downregulated: expression of osteoblast differentiation markers [runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), type I collagen (Col1a1), and osteocalcin (Ocn)], cell proliferation markers (CyclinE, CyclinD, and CDC2), amino acid import (Glyt1), and metabolism-related genes (Asns). Additionally, Alp-positive staining cells, Alp activity, matrix mineralization, Ocn secretion, and cell proliferation indexes were inhibited. Interestingly, we found that salubrinal enhanced PTH-induced osteoblast differentiation and proliferation by maintenance of phosphorylation of EIF2α. Furthermore, we observed that PTH increased the association between heat shock protein 90 (HSP90) and PERK and maintained PERK protein stabilization in the early stages of PTH-induced ER stress. Treatment of MC3T3-E1 cells with geldanamycin, an HSP90 inhibitor, decreased PERK protein expression and inhibited osteoblast differentiation and cell proliferation upon PTH treatment. Taken together, our data demonstrate that PTH regulates osteoblast differentiation and cell proliferation, partly by activating the HSP90-dependent PERK-EIF2α-ATF4 signaling pathway.

The GLP-1 analog liraglutide attenuates acute liver injury in mice.

INTRODUCTION AND OBJECTIVES: Acute liver injury is a current health problem with few effective treatments. The present study investigated the hepatoprotective and curative potential of the glucagon-like peptide-1 analog liraglutide against carbon tetrachloride (CCl4)-induced hepatotoxicity. MATERIALS AND METHODS: Male Swiss mice were subjected to two protocols. The first protocol (Pretreatment) consisted of intraperitoneal (i.p.) treatment with liraglutide (0.057 and 0.118mgkg-1) or vehicle (distilled water) once daily for 7 days. On days 6 and 7, the animals were challenged with 2% CCl4 (5mgkg-1, i.p.). The second protocol (Late treatment) began with an injection of 5% CCl4 (5mgkg-1, i.p.) and subsequent treatment with liraglutide (0.057mgkg-1) or vehicle (distilled water) for 1 day. In both protocols, 24h after the last administration, blood and bile were collected from anesthetized animals, followed by euthanasia and liver collection. Plasma and bile underwent biochemical analyses, and histological, oxidative stress, and metabolic parameters were evaluated in the liver. RESULTS: Both liraglutide treatment protocols attenuated hepatotoxicity that was induced by CCl4, decreasing plasma levels of hepatic enzymes, stimulating the hepatic antioxidant system, and decreasing centrilobular necrosis, hepatic glycogen, and lipid accumulation. CCl4 tended to reduce bile lipid excretion, but liraglutide did not influence this parameter. CONCLUSIONS: The present results demonstrated the hepatoprotective and therapeutic effects of liraglutide, which may be attributable to a decrease in liver oxidative stress and the preservation of metabolism. Liraglutide may have potential as a complementary therapy for acute liver injury.

Tumor necrosis factor-alpha inhibits osteogenic differentiation of pre-osteoblasts by downregulation of EphB4 signaling via activated nuclear factor-kappaB signaling pathway.

BACKGROUND AND OBJECTIVE: The majority of experiments show that tumor necrosis factor-alpha (TNF-α) inhibits osteogenic differentiation of mesenchymal stem cells and pre-osteoblasts by activated nuclear factor-kappaB (NF-κB) signaling. However, the underlying mechanisms by which NF-κB signaling inhibits osteogenic differentiation are not fully understood. The aim of the present study was to investigate whether EphB4 signaling inhibition mediates the effects of TNF-α-activated NF-κB signaling on osteogenic differentiation of pre-osteoblasts. MATERIAL AND METHODS: Murine MC3T3-E1 pre-osteoblasts were treated with 10 ng/mL of TNF-α. NF-κB inhibitor, pyrrolidine dithiocarbamate, was used to achieve NF-κB signaling inhibition. EphB4 signaling was activated using ephrinB2-fc. The mRNA expressions of runt related transcription factor 2 (Runx2), bone sialoprotein (BSP) and EphB4 were determined using reverse transcription-polymerase chain reaction. The protein levels of Runx2, BSP, Col Ia1, osteopontin, EphB4, p-NF-κB p65 and NF-κB p65 were evaluated using western blot assays. Alkaline phosphatase (ALP) activity in MC3T3-E1 cells was evaluated by ALP activity kit, and mineral nodule formation was evaluated by Alizarin Red S staining. RESULTS: TNF-α inhibited EphB4 expression, while it suppressed Runx2, BSP expression from gene and protein levels as well as ALP activity and mineral nodule formation in MC3T3-E1 cells. Activation of EphB4 signaling by ephrinB2-fc promoted osteogenic differentiation of MC3T3-E1 cells, whereas TNF-α impaired the osteogenic differentiation enhanced by ephrinB2-fc. Pyrrolidine dithiocarbamate blocked the activation of NF-κB signaling induced by TNF-α, while it prevented the downregulation of Runx2, BSP and EphB4, induced by TNF-α. CONCLUSION: TNF-α inhibits osteogenic differentiation of pre-osteoblasts by downregulation of EphB4 signaling via activated NF-κB signaling pathway.

Alkaline phosphatase in metastatic castration-resistant prostate cancer: reassessment of an older biomarker.

Since most patients with metastatic castration-resistant prostate cancer (mCRPC) have bone metastases, it is important to understand the potential impact of therapies on prognostic biomarkers, such as ALP. Clinical studies involving mCRPC life-prolonging agents (i.e., sipuleucel-T, abiraterone, enzalutamide, docetaxel, cabazitaxel, and radium-223) have shown that baseline ALP level is prognostic for overall survival, and may be a better prognostic marker for overall survival than prostate-specific antigen in patients with bone-dominant mCRPC. Mechanism of action differences between therapies may partly explain ALP dynamics during treatment. ALP changes can be interpreted within the context of other parameters while monitoring disease activity to better understand the underlying pathology. This review evaluates the current role of ALP in mCRPC.

Biocalcite and Carbonic Acid Activators.

Based on evolution of biomineralizing systems and energetic considerations, there is now compelling evidence that enzymes play a driving role in the formation of the inorganic skeletons from the simplest animals, the sponges, up to humans. Focusing on skeletons based on calcium minerals, the principle enzymes involved are the carbonic anhydrase (formation of the calcium carbonate-based skeletons of many invertebrates like the calcareous sponges, as well as deposition of the calcium carbonate bioseeds during human bone formation) and the alkaline phosphatase (providing the phosphate for bone calcium phosphate-hydroxyapatite formation). These two enzymes, both being involved in human bone formation, open novel not yet exploited targets for pharmacological intervention of human bone diseases like osteoporosis, using compounds that act as activators of these enzymes. This chapter focuses on carbonic anhydrases of biomedical interest and the search for potential activators of these enzymes, was well as the interplay between carbonic anhydrase-mediated calcium carbonate bioseed synthesis and metabolism of energy-rich inorganic polyphosphates. Beyond that, the combination of the two metabolic products, calcium carbonate and calcium-polyphosphate, if applied in an amorphous form, turned out to provide the basis for a new generation of scaffold materials for bone tissue engineering and repair that are, for the first time, morphogenetically active.

The Influence of Nitrogen on the Biological Properties of Soil Contaminated with Zinc.

This study analyzed the relationship between nitrogen fertilization and the biological properties of soil contaminated with zinc. The influence of various concentrations of zinc and nitrogen on the microbiological and biochemical activity of soil was investigated. In a laboratory experiment, loamy sand with pHKCl 5.6 was contaminated with zinc (ZnCl2) and fertilized with urea as a source of nitrogen. The activity of acid phosphatase, alkaline phosphatase, urease and ß-glucosidase, and microbial counts were determined in soil samples after 2 and 20 weeks of incubation. Zinc generally stimulated hydrolase activity, but the highest zinc dose (1250 mg kg-1) led to the inhibition of hydrolases. Nitrogen was not highly effective in neutralizing zinc's negative effect on enzyme activity, but it stimulated the growth of soil-dwelling microorganisms. The changes in soil acidity observed after the addition of urea modified the structure of microbial communities.

[A novel tissue-engineered bone constructed by using human adipose-derived stem cells and biomimetic calcium phosphate scaffold coprecipitated with bone morphogenetic protein-2].

OBJECTIVE: To construct a novel biomimetic calcium phosphate (BioCaP) scaffold loaded with bone morphogenetic protein-2 (BMP-2), and to investigate its role in the osteogenesis of human adipose-derived stem cells (hASCs) in vitro and in vivo. METHODS: The BioCaP scaffold coprecipitated with BMP-2 (BMP-2-BioCaP) was constructed in this study. Field emission scanning electron microscopy (SEM) was used to analyze the morphology of the surfaces. The release kinetics was measured to evaluate the slow-release characteristics in vitro. BMP-2-BioCaP was immersed in proliferation medium (PM) or osteogenic medium (OM), respectively. The supernatants were collected and used to culture hASCs in vitro. Cell numbers were determined using the cell-counting kit-8 (CCK-8) to assess the cell proliferation. After 7 and 14 days, alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP. After 14 and 21 days, the calcification deposition was determined by alizarin red S (ARS) staining and quantification. The expressions of the osteoblast-related genes were tested on day 4 and day 14. In the in vivo study, 6 nude mice were used and implanted subcutaneously into the back of the nude mice for 4 groups: (1) BioCaP scaffold only, (2) BioCaP scaffold+hASCs, (3) BMP-2-BioCaP scaffold, (4) BMP-2-BioCaP scaffold+hASCs (test group). After 4 weeks of implantation, hematoxylin-eosin (HE) staining was performed to evaluate the in vivo osteogenesis of hASCs. RESULTS: SEM observations showed that BioCaP and BMP-2-BioCaP scaffold were entirely composed of straight, plate-like and sharp-edged crystal units, and the length of the crystal units varied between 5 and 10 µm. Release kinetics analysis demonstrated that BMP-2 incorporated with BioCaP could be released at certain concentration and last for more than 21 days, and the accumulative protein release could reach 20%. CCK-8 assays showed that cell proliferation was not significantly affected by BMP-2-BioCaP. ALP activity was higher by the induction of OM+BMP-2-BioCaP than of the other groups (P<0.01). More mineralization deposition and more expressions of osteoblast-related genes such as Runt-related transcription factor 2 (RUNX2), ALP, osteopontin (OPN) and osteocalcin (OC) were determined in the OM+BMP-2-BioCaP group at different time points (P<0.01). HE staining showed that, in the test group and BMP-2-BioCaP scaffold group, the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs, and newly-formed bone-like tissues could be found in ECM around the scaffold materials. Moreover, compared with the BMP-2-BioCaP scaffold group, more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test groups. No obvious positive results were found in the other groups. CONCLUSION: BMP-2-BioCaP scaffold could achieve slow-release of BMP-2 and promote the osteogenic differentiation of hASCs in vitro and in vivo. The novel tissue-engineered bone composed of hASCs and BMP-2-BioCaPis promising for the repair of bone defect.

Medicinal role of papaya seeds on thrombocyte count tested on healthy rabbits.

The main objective of the current study is to evaluate the medicinal role of Papaya seeds on thrombocyte count and hepatic parameter on healthy rabbits. Experimental and Interventional study, at the Department of pharmacology Baqai Medical University Karachi. Rabbits (18 in number different age and both sex) were included, subsequently subdivided into three group (n=6). Group A (Control), B and C (sample fed dose 250 & 500mg OD, oral route). Blood were drawn 0 time, subsequently samples were drawn at 15, 30, 45 days. Data was analyzed by using SPSS 19.0. Analysis of results showed increase in the Platelet level 19.2%, 65.5%. No significant change seen in the SGPT, Alkaline Phosphatase, WBC, Neutrophil, Lymphocytes, Eosinophils and Monocytes as compared with the controls. It can be concluded that the administration of Papaya seeds powder dose rapidly increase platelet count and may play an important therapeutic role in thrombocytopenia.

MiR-26a regulates vascular smooth muscle cell calcification in vitro through targeting CTGF.

Vascular calcification is one of the most important factors for high morbidity and mortality from cardiovascular and cerebrovascular diseases. The aim of this study is to investigate the effect and mechanism of miR-26a on vascular smooth muscle cell calcification. First, the VSMCs were induced by ß-glycerol phosphate (ß-GP) for 7d and 14d, and Alizarin Red S staining was performed to examine the mineralized nodule change; then real time RT-PCR and western blotting were performed to explore the expression of miR-26a, CTGF, OPG, RANKL and ALP in un-induced and ß-GP-induced VSMCs; next, the VSMCs were transfected with miR-26a mimics, and Alizarin Red S staining was performed to examine the mineralized nodule change; finally, real time RT-PCR and western blotting were performed to explore the expression of miR-26a, CTGF, OPG, RANKL and ALP in un-transfected and miR-26a mimics transfected VSMCs. After ß-GP treatment, ß-GP promoted clear mineralized nodule changes, and miR-26a and OPG expression were significantly decreased and CTGF, RANKL and ALP expression were increased in VSMCs. Overexpression of miR-26a inhibited VSMCs calcification induced by ß-GP, and regulated the expression of CTGF, OPG, RANKL and ALP. Our findings suggested that up-regulation of miR-26a before ß-GP treatment inhibits VSMCs calcification through targeting CTGF (Fig. 4, Ref. 18).

Inhibitory effects of advanced glycation end-products and Porphyromonas gingivalis lipopolysaccharide on the expression of osteoblastic markers of rat bone marrow cells in culture.

BACKGROUND AND OBJECTIVES: Diabetes is a major risk factor for periodontitis and there is a close relationship between the degree of hyperglycemia and the severity of periodontitis. Advanced glycation end-products (AGEs) accumulate in various tissues under diabetic conditions. AGEs in the periodontal tissues probably play a role in upregulating periodontal inflammation; however, the association of AGEs with the severity of periodontitis has not been fully clarified. Lipopolysaccharide from Porphyromonas gingivalis (P-LPS) is a potent pathogenic factor in periodontitis. Although the independent effect of AGE or P-LPS on osteoblastic cells has been reported in vitro, the effect of adding both has not been clearly elucidated. In this study, to explore factors aggravating diabetic periodontitis, we investigated the effects of AGE and P-LPS on the expression of osteoblastic markers and the expression of inflammation-related markers in vitro. MATERIAL AND METHODS: Rat bone marrow cells were cultured, and alkaline phosphatase activity and bone nodule formation were evaluated as osteoblastic markers. Reverse transcription-polymerase chain reaction was performed to determine the mRNA expression of molecules associated with bone and inflammation. Protein levels of osteocalcin and interleukin-1ß (IL-1ß) were measured using enzyme-linked immunosorbent assay. RESULTS: AGEs and P-LPS independently reduced alkaline phosphatase activity and bone nodule formation. The addition of both AGE and P-LPS (AGE+P-LPS) further decreased these markers. Reverse transcription-polymerase chain reaction analysis revealed that AGE+P-LPS markedly decreased the mRNA expression of osteoblast-related molecules such as type 1 collagen, osteocalcin and Cbfa1, and markedly increased that of inflammation-related molecules such as IL1ß and S100A8. AGE and P-LPS decreased the protein level of osteocalcin and increased that of IL-1ß, and a further increase of IL-1ß was detected for AGE+P-LPS. CONCLUSION: AGEs and P-LPS inhibited the expression of osteoblastic markers and increased the levels of inflammatory markers in rat bone marrow cells, suggesting that both AGE and P-LPS may be important factors associated with the aggravation of diabetic periodontitis.

Cytocompatibility of HEMA-free resin-based luting cements according to application protocols on dentine surfaces.

AIM: To evaluate the transdentinal cytotoxicity of resin-based luting cements (RBLCs), with no HEMA in their composition, to odontoblast-like cells. METHODOLOGY: Human dentine discs 0.3 mm thick were adapted to artificial pulp chambers (APCs) and placed in wells of 24-well plates containing 1 mL of culture medium (DMEM). Two categories of HEMA-free RBLCs were evaluated: group 1, self-adhesive Rely X Unicem (RU; 3M ESPE), applied directly to the dentine substrate; and group 2, Rely X ARC (RARC; 3M ESPE), applied to dentine previously acid-etched and treated with a bonding agent. In group 3 (control), considered as representing 100% cell metabolic activity, no treatment was performed on dentine. The APC/disc sets were incubated for 24 h or 7 days at 37 °C and 5% CO2 . Then, the extracts (DMEM + dental materials components that diffused through dentine) were applied to cultured odontoblast-like MDPC-23 cells for 24 h. After that, the cell viability (MTT assay), cell morphology (SEM), total protein production (TP) and alkaline phosphatase (ALP) activity were assessed. Data from MTT assay and TP production were analysed by Kruskal-Wallis and Mann-Whitney tests (α = 5%). Data from ALP activity were analysed by one-way anova and Tukey's test (α = 5%). RESULTS: In group 1, a slight reduction in cell viability (11.6% and 16.8% for 24-h and 7-day periods, respectively) and ALP activity (13.5% and 17.9% for 24-h and 7-day periods, respectively) was observed, with no significant difference from group 3 (control) (P > 0.05). In group 2, a significant reduction in cell viability, TP production and ALP activity compared with group 3 (control) occurred (P < 0.05), regardless of incubation time. Alteration in MDPC-23 cell morphology was observed only in group 2. CONCLUSIONS: HEMA-free Rely X ARC cement caused greater toxicity to odontoblast-like MDPC-23 cells than did Rely X Unicem cement when both resin-based luting materials were applied to dentine as recommended by the manufacturer.

Alendronate alters osteoblast activities.

OBJECTIVE: Due to accumulation in the bone matrix and a half-life of at least 10 years, it is important to understand the cellular impact of bisphosphonates (BPs). This study assessed the effects of alendronate (ALN) on human primary osteoblasts. MATERIAL AND METHODS: Osteoblasts were incubated with ALN (5, 20 and 100 µM), and both cells and cell culture media were harvested after d 1, 3, 7 or 14. Proliferation was evaluated by 3H-thymidine incorporation and tetrazolium dye (MTT) colorimetric assay, and viability by the lactate dehydrogenase (LDH) activity in the medium. Differentiation was evaluated using protein Luminex multiplex assays and RT-PCR. RESULTS: ALN had no significant effects on cell viability. The lower concentrations enhanced the proliferation, whereas 100 µM diminished the proliferation. mRNA expression of osteocalcin (OC), alkaline phosphatase (ALP) and α-1 type 1 collagen were reduced, whereas ALN enhanced the expression of leptin mRNA and the secretion of interleukin-8 (IL-8) and regulated on activation normal T cell expressed and secreted (RANTES). CONCLUSIONS: ALN enhanced the secretion of immune factors from human osteoblasts. Combined with a lower rate of proliferation and a decline in differentiation, this indicates that higher dosages or accumulation may cause undesirable local changes in bone.

Combined effects of mineral trioxide aggregate and human placental extract on rat pulp tissue and growth, differentiation and angiogenesis in human dental pulp cells.

OBJECTIVE: The aim of this study was to evaluate the combined effects of mineral trioxide aggregate (MTA) and human placental extract (HPE) on cell growth, differentiation and in vitro angiogenesis of human dental pulp cells (HDPCs) and to identify underlying signal transduction mechanisms. In vivo dental pulp responses in rats for a pulp-capping agent were examined. MATERIALS AND METHODS: MTS assay. ALP activity test, alizarin red S staining and RT-PCR for marker genes were carried out to evaluate cell growth and differentiation. HUVEC migration, mRNA expression and capillary tube formation were measured to evaluate angiogenesis. Signal transduction was analysed using Western blotting and confocal microscopy. The pulps of rat maxillary first molars were exposed and capped with either MTA or MTA plus HPE. Histologic observation and scoring were performed. RESULTS: Compared to treatment of HDPCs with either HPE or MTA alone, the combination of HPE and MTA increased cell growth, ALP activity, mineralized nodules and expression of marker mRNAs. Combination HPE and MTA increased migration, capillary tube formation and angiogenic gene expression compared with MTA alone. Activation of Akt, mammalian target of rapamycin (mTOR), p38, JNK and ERK MAPK, Akt, and NF-κB were significantly increased by combining HPE and MTA compared with MTA alone. Pulp capping with MTA plus HPE in rats showed superior dentin bridge formation, odontoblastic layers and dentinal tubules and lower inflammatory cell response, compared to the MTA alone group. CONCLUSIONS: This study demonstrates for the first time that the use of MTA with HPE promotes cell growth, differentiation and angiogenesis in HDPCs, which were associated with mTOR, MAPK and NF-κB pathways. Direct pulp capping with HPE plus MTA showed superior results when compared with MTA alone. Thus, the combination of MTA and HPE may be useful for regenerative endodontics.