Osteocalcin mRNA Levels Research Articles

Dietary Calcium Deficiency and Excess Both Impact Bone Development and Mesenchymal Stem Cell Lineage Priming in Neonatal Piglets

Background: Optimizing calcium nutrition to maximize bone accretion during growth to prevent fragility fractures later in life has spurred greater interest in calcium nutrition in neonates.Objective: The aim of this study was to determine the effect of dietary calcium, from deficiency through excess, on bone growth, and the in vivo and in vitro behavior of mesenchymal stem cells (MSCs) in neonatal pigs.Methods: Twenty-four male and female piglets (24 ± 6 h old) were fed either a calcium-deficient [Ca-D; 0.6% Ca on a dry matter (DM) basis], a calcium-adequate diet (Ca-A; 0.9% Ca on a DM basis), or a calcium-excessive diet (Ca-E; 1.3% Ca on a DM basis) for 14 d to assess the impact of dietary calcium on calcium homeostasis and on the behavior of MSCs.Results: Growth rate was not affected by the Ca-E diet, although bone ash content was 16% higher (P < 0.05) and urinary calcium excretion was 5-fold higher, when normalized to creatinine, compared with the Ca-A group at trial completion. Serum parathyroid hormone (PTH) concentrations were elevated (P < 0.05) in Ca-D piglets in comparison with other groups at both 7 and 14 d. In vivo proliferation of MSCs was 30% higher (P < 0.05) in Ca-E piglets than the other groups. MSCs from both Ca-D– and Ca-E–fed piglets had greater adipogenic potential based on increased gene expression (P < 0.05) of peroxisome proliferator-activated receptor γ (Pparg) and adipocyte fatty acid–binding protein (Ap2) than MSCs from Ca-A piglets. Interestingly, only MSCs from Ca-E–fed piglets had greater (P < 0.05) gene expression of lipoprotein lipase (Lpl) during adipocytic differentiation than those from Ca-A piglets. To assess alterations in lineage allocation and priming, the most and least osteogenic (O+ and O−, respectively) and adipogenic (A+ and A−, respectively) colonies from each MSC isolation were selected on the basis of functional staining. The O+ colonies from Ca-D piglets expressed lower (P < 0.05) levels of osteocalcin (OC) mRNA than did those from other groups, whereas the O− colonies from Ca-E piglets expressed higher (P < 0.05) levels of mRNA of Pparg, Ap2, and Lpl than did those from other groups.Conclusions: Neonatal calcium deficiency appears to reduce the osteogenic priming of MSCs while enlarging a subpopulation of potentially adipogenic cells, and excess dietary calcium appears to allow greater multipotency of MSCs. These programming alterations of MSCs could have long-term consequences for bone health.

Primary human osteoblasts in response to 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3.

The most biologically active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D3 (25(OH)D3) can affect osteoblast function via conversion to 1,25(OH)2D3, however, it is largely unknown whether 25(OH)D3 can affect primary osteoblast function on its own. Furthermore, 25(OH)D3 is not only converted to 1,25(OH)2D3, but also to 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH)D3 itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R,25(OH)2D3 can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH)D3 and 1,25(OH)2D3 by reducing their proliferation and enhancing their differentiation by the increase of alkaline phosphatase, osteocalcin and osteopontin expression. Osteoblasts expressed CYP27B1 and CYP24 and synthesized 1,25(OH)2D3 and 24R,25(OH)2D3 dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)2D3 synthesis, but we observed no significant differences in mRNA levels of differentiation markers in CYP27B1-silenced cells compared to control cells after treatment with 25(OH)D3. We demonstrated that 24R,25(OH)2D3 increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R,25(OH)2D3 strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH)D3, 1,25(OH)2D3 and 24R,25(OH)2D3 can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)2D3, but also to 24R,25(OH)2D3 by enhancing osteoblast differentiation. This suggests that 25(OH)D3 can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)2D3, but also via conversion to 24R,25(OH)2D3. Whether 25(OH)D3 has direct actions on osteoblast function needs further investigation.

Bone morphogenetic protein-2-encapsulated grafted-poly-lactic acid-polycaprolactone nanoparticles promote bone repair.

The aim of this study is to test the efficacy of a novel tissue-engineered bone in repairing bone defects, using poly-lactic-acid-polycaprolactone (PLA-PCL) scaffolding seeded with PEG-bone morphogenetic protein-2 (BMP-2)-transfected rBMSCs (rabbit bone marrow stromal cells). The rBMSCs were transfected with PEG/BMP-2 or liposome/BMP-2, and then implanted into a PLA-PCL tissue-engineered bone. The protein level of BMP-2 was assessed by Western blot analysis and immunohistochemistry. ELISA was used to measure the amount of BMP-2 secreted in the culture media. The mRNA level of BMP-2 and osteocalcin was assayed quantitatively by real-time PCR. The middle portion of the bilateral radius in New Zealand rabbits was excised and implanted with tissue-engineered bone, and the modified areas were monitored by X-ray, hematoxylin-eosin staining, and immunohistochemistry staining of BMP-2. PEG-BMP-2 nanoparticles (NPs) and BMP-2-loaded PEG-PLA-PCL tissue-engineered bones were successfully constructed. The novel PEG-PLA-PCL NPs/DNA complex was a superior option for transfecting BMP-2 in rBMSCs compared to normal liposomes Moreover, the mRNA level of osteocalcin and alkaline phosphatase activity was also elevated upon transfection of BMP-2-encapsulated NPs. In vivo implants with BMP-2-carried tissue-engineered bone exhibited dramatic augmentation of BMP-2 and effective bone formation in the rabbit ectopic model. The PEG-PLA-PCL NPs/BMP-2 complex had an advantageous effect on bone repair, which provided an important theoretic basis for potential clinical treatments.

Effects of PEMF exposure at different pulses on osteogenesis of MC3T3-E1 cells

ObjectivePulsed electromagnetic fields (PEMFs) were considered to be a factor which may affect osteogenesis of osteoblasts, but the effects were diverse with different PEMF parameters. The aim of the current study is to explore the effects of exposure to PEMFs at different pulse number on osteogenesis of osteoblasts. DesignThe mouse osteoblast-like MC3T3-E1 cells were exposed to 0, 400 or 2800 pulses 400kV/m PEMF and the proliferation, differentiation and mineralization of cells were observed after PEMF exposure by the methods of MTT, biochemical measurement, real-time PCR and Alizarin Red assay. ResultsCompared with 0 pulses groups, the growth curve, alkaline phosphatase (ALP) activity, mRNA level of osteocalcin (OCN) and mineralized nodule formation of MC3T3-E1 cells did not change after 400 pulses PEMF exposure, but decreased after 2800 pulses PEMF exposure. It suggested that under our experimental conditions, only 2800 pulses 400kV/m PEMF exposure can suppress the proliferation, differentiation and mineralization of MC3T3-E1 cells, but 400 pulses 400kV/m PEMF exposure cannot. ConclusionsPulse number is another involved parameter which may influence the effects of PEMF on osteogenesis of osteoblasts.

Deposition of superparamagnetic nanohydroxyapatite on iron–fibrin substrates: Preparation, characterization, cytocompatibility and bioactivity studies

In the present study, nanosized hydroxyapatite (nHAp) was formed on iron–fibrin substrates and its physico-chemical properties were characterized. The prepared iron–fibrin–nanohydroxyapatite (IF–nHAp) composite was needle shaped with an average width of about 30nm and length of 80nm. The vibrating sample magnetometer (VSM) was used to evaluate the superparamagnetic behavior of the nanocomposite, IF–nHAp. Hemolysis and ELISA (enzyme linked immunosorbent assay) were performed to evaluate the its bio/immunocompatibility and MTT (3-(4,5-dimethylazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay using osteoblast cells was performed to scrutinize its proliferative potential. Alkaline phosphatase activity (ALP) and calcium deposition were studied to investigate the osteogenic property of the nanocomposite. RT-PCR (real time-polymerase chain reaction) was used to quantify the mRNA levels of ALP, OC (osteocalcin), and OPN (osteopontin) genes involved in the osteogenic differentiation and matrix mineralization. Further, the bone bonding ability of IF–nHAp was observed by the deposition of apatite layers on the composite incubated in simulated body fluid (SBF).

Periodontal Fibroblasts Modulate Proliferation and Osteogenic Differentiation of Embryonic Stem Cells Through Production of Fibroblast Growth Factors

Periodontal ligament fibroblasts (PLFs) maintain homeostasis of periodontal ligaments by producing paracrine factors that affect various functions of stem-like cells. It is hypothesized that PLFs induce proliferation and differentiation of stem cells more effectively than gingival fibroblasts (GFs) and skin fibroblasts (SFs). PLFs and GFs were isolated from extracted teeth and cultured in the presence and absence of osteogenesis-inducing factors. Mouse embryonic stem (mES) cells and SFs were purchased commercially. mES cells were incubated with culture supernatants of these fibroblasts or cocultured directly with the cells. Proliferation and mineralization in mES cells were determined at various times of incubation. Immunostaining and polymerase chain reaction were performed. The activity of mitogen-activated protein kinase and alkaline phosphatase (ALP) was also measured. In cocultures, PLFs stimulated proliferation of mES cells more effectively than GFs or SFs. Similarly, the addition of culture supernatant of PLFs induced the most prominent proliferation of mES cells, and this was significantly inhibited by treatment with antibody against fibroblast growth factor (FGF)4 or the c-Jun N-terminal kinase inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one). Supplementation with culture supernatant from the fibroblasts induced osteogenic differentiation of mES cells in the order PLFs > GFs > SFs. These activities of PLFs were related to their potential to produce osteogenic markers, such as ALP and runt-related transcription factor-2 (Runx2), and to secrete FGF7. Pretreatment of mES cells with the extracellular signal-regulated kinase inhibitor PD98059 [2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one] or SP600125 clearly attenuated mineralization induced by culture supernatant of PLF with attendant decreases in mRNA levels of Runx2, bone sialoprotein, osteocalcin, and osteopontin. PLFs regulate the proliferation and osteogenic differentiation of mES cells more strongly than GFs and SFs via the secretion of FGF through a mechanism that involves mitogen-activated protein kinase-mediated signaling.

Plasminogen Activator Inhibitor-1 Is Involved in Impaired Bone Repair Associated with Diabetes in Female Mice

Previous studies suggest that fracture healing is impaired in diabetes; however, the underlying mechanism remains unclear. Here, we investigated the roles of plasminogen activator inhibitor-1 (PAI-1) in the impaired bone repair process by using streptozotocin (STZ)-induced diabetic female wild-type (PAI-1 +/+) and PAI-1-deficient (PAI-1 −/−) mice. Bone repair and the number of alkaline phosphatase (ALP)-positive cells at the site of a femoral bone damage were comparable in PAI-1 +/+ and PAI-1 −/− mice without STZ treatment. Although the bone repair process was delayed by STZ treatment in PAI-1 +/+ mice, this delayed bone repair was blunted in PAI-1 −/− mice. The reduction in the number of ALP-positive cells at the site of bone damage induced by STZ treatment was attenuated in PAI-1 −/− mice compared to PAI-1 +/+ mice. On the other hand, PAI-1 deficiency increased the levels of ALP and type I collagen mRNA in female mice with or without STZ treatment, and the levels of Osterix and osteocalcin mRNA, suppressed by diabetic state in PAI-1 +/+ mice, were partially protected in PAI-1 −/− mice. PAI-1 deficiency did not affect formation of the cartilage matrix and the levels of types II and X collagen and aggrecan mRNA suppressed by STZ treatment, although PAI-1 deficiency increased the expression of chondrogenic markers in mice without STZ treatment. The present study indicates that PAI-1 is involved in the impaired bone repair process induced by the diabetic state in part through a decrease in the number of ALP-positive cells.

Role of perlecan in chondrogenic, osteogenic and adipogenic differentiation of synovial mesenchymal calls

Role of perlecan in chondrogenic, osteogenic and adipogenic differentiation of synovial mesenchymal calls

Expression of core-binding factor α1 and osteocalcin in fluoride-treated fibroblasts and osteoblasts

To study the effects and importance of fluoride on FBs in the development of extraperiosteal calcification and the ossification of skeletal fluorosis, the presence of the osteogenic phenotype, which is indicated by the expression of core-binding factor α1 (Cbfa1) and osteocalcin (OCN), in an FB cell line (L929) and in osteoblasts (OBs) exposed to fluoride was determined. Fibroblasts and osteoblasts were exposed to different concentrations of fluoride (0, 0.0001, 0.001, 0.1, 1.0, 10.0 and 20.0mg/L F−). By using RT-PCR and ELISA, the mRNA levels of Cbfa1 and OCN were measured at 48h, and the protein levels of Cbfa1 and OCN were measured at 2, 4, 24, 48 and 72h. The data demonstrated the following: (1) The Cbfa1 protein level in fluoride-treated fibroblasts clearly increased at 48h in the groups treated with 0.0001, 0.001, 0.1, 1.0 and 20.0mg/L F−. The Cbfa1 protein level of the group treated with 10mg/L F− at 72h was higher than that of the control group. The level of Cbfa1 mRNA in the fibroblasts was much higher at 48h in the group treated with 10.0mg/L F− than in the control group. (2) The OCN protein level in fluoride-treated fibroblasts was significantly higher than that of the control group in the 0.0001, 0.1, 1.0, 10.0 and 20.0mg/L F− groups at 2h, and in the 0.001 and 0.1 F− groups at 4h. A slightly higher level of OCN mRNA in fluoride-treated fibroblasts was also found in the 1.0 and 20.0mg/L F− groups compared to the control group. (3) The expressions of Cbfa1 and OCN in osteoblasts treated with the same experimental conditions as the fibroblasts were up-regulated by fluoride following the same trend as in the fibroblasts. Our results showed an increase in the expression of Cbfa1 and OCN in fibroblasts and osteoblasts exposed to fluoride and suggested that the osteogenic function of fibroblasts induced by fluoride could play an important role in the development of extraperiosteal ossification during skeletal fluorosis.

The Thiocarbamate Disulphide Drug, Disulfiram Induces Osteopenia in Rats by Inhibition of Osteoblast Function Due to Suppression of Acetaldehyde Dehydrogenase Activity

Dithiocarbamates (DTC), a sulfhydryl group containing compounds, are extensively used by humans that include metam and thiram due to their pesticide properties, and disulfiram (DSF) as an alcohol deterrent. We screened these DTC in an osteoblast viability assay. DSF exhibited the highest cytotoxicity (IC50 488nM). Loss in osteoblast viability and proliferation was due to induction of apoptosis via G1 arrest. DSF treatment to osteoblasts reduced glutathione (GSH) levels and exogenous addition of GSH prevented DSF-induced reactive oxygen species generation and osteoblast apoptosis. DSF also inhibited osteoblast differentiation in vitro and in vivo, and the effect was associated with inhibition of aldehyde dehydrogenase (ALDH) activity. Out of various ALDH isozymes, osteoblasts expressed only ALDH2 and DSF downregulated its transcript as well as activity. Alda-1, a specific activator of ALDH2, stimulated osteoblast differentiation. Subcutaneous injection of DSF over the calvarium of new born rats reduced the differentiation phenotype of calvarial osteoblasts but increased the mRNA levels of Runx-2 and osteocalcin. DSF treatment at a human-equivalent dose of 30 mg/kg p.o. to adult Sprague Dawley rats caused trabecular osteopenia and suppressed the formation of mineralized nodule by bone marrow stromal cells. Moreover, DSF diminished bone regeneration at the fracture site. In growing rats, DSF diminished growth plate height, primary and secondary spongiosa, mineralized osteoid and trabecular strength. Substantial decreased bone formation was also observed in the cortical site of these rats. We conclude that DSF has a strong osteopenia inducing effect by impairing osteoblast survival and differentiation due to the inhibition of ALDH2 function.

Regeneration of Calvarial Defects with Escherichia coli-Derived rhBMP-2 Adsorbed in PLGA Membrane

Objective:Escherichia coli-derived recombinant human bone morphogenetic protein-2 (E-BMP-2) has been shown to be as effective as mammalian cell-derived BMP-2. However, several in vitro and in vivo experiments are still necessary to validate the effectiveness of E-BMP-2 due to the difference in synthesis process, mainly related to protein nonglycosylation. The objective of this study was to investigate whether biodegradable polylactide-co-glycolide (PLGA) membrane is a suitable carrier for E-BMP-2 delivery for bone regeneration of critical-sized defects in rat calvaria. Materials and Methods: First, the osteoinductive effect of E-BMP-2 was confirmed in vitro in mouse bone marrow stromal cells by analysis of osteocalcin mRNA levels, and calcium deposition was detected by alizarin red staining. Before in vivo experiments, the release profile of E-BMP-2 from PLGA membranes was determined by ELISA. E-BMP-2 (0, 1, 5 and 10 μg/μl) was applied for ectopic and orthotopic bone formation and was analyzed by X-ray, micro-CT and histology. Results: Release-profile testing showed that PLGA membrane could retain 94% of the initially applied E-BMP-2. Ectopic bone formation assay revealed that combination of E-BMP-2/PLGA membrane strongly induced bone formation. Stronger osteoinductivity with complete repair of critical-sized defects was observed only with PLGA membranes adsorbed with 5 and 10 μg/μl of E-BMP-2, whereas no bone formation was observed in the groups that received no membrane or 0-μg/μl dose of E-BMP-2. Conclusion: PLGA membrane was shown to be a suitable carrier for sustained release of E-BMP-2, and the E-BMP-2/PLGA membrane combination was demonstrated to be efficient in bone regeneration in a model of critical-sized defects.

Characteristics and osteogenic effect of zirconia porous scaffold coated with β-TCP/HA.

PURPOSEThe purpose of this study was to evaluate the properties of a porous zirconia scaffold coated with bioactive materials and compare the in vitro cellular behavior of MC3T3-E1 preosteoblastic cells to titanium and zirconia disks and porous zirconia scaffolds.MATERIALS AND METHODSTitanium and zirconia disks were prepared. A porous zirconia scaffold was fabricated with an open cell polyurethane disk foam template. The porous zirconia scaffolds were coated with β-TCP, HA and a compound of β-TCP and HA (BCP). The characteristics of the specimens were evaluated using scanning electron microscopy (SEM), energy dispersive x-ray spectrometer (EDX), and x-ray diffractometry (XRD). The dissolution tests were analyzed by an inductively coupled plasma spectrometer (ICP). The osteogenic effect of MC3T3-E1 cells was assessed via cell counting and reverse transcriptase-polymerase chain reaction (RT-PCR).RESULTSThe EDX profiles showed the substrate of zirconia, which was surrounded by the Ca-P layer. In the dissolution test, dissolved Ca2+ ions were observed in the following decreasing order; β-TCP > BCP > HA (P<.05). In the cellular experiments, the cell proliferation on titanium disks appeared significantly lower in comparison to the other groups after 5 days (P<.05). The zirconia scaffolds had greater values than the zirconia disks (P<.05). The mRNA level of osteocalcin was highest on the non-coated zirconia scaffolds after 7 days.CONCLUSIONZirconia had greater osteoblast cell activity than titanium. The interconnecting pores of the zirconia scaffolds showed enhanced proliferation and cell differentiation. The activity of osteoblast was more affected by microstructure than by coating materials.

Rho-kinase limits BMP-4-stimulated osteocalcin synthesis in osteoblasts: Regulation of the p38 MAP kinase pathway

AimWe previously reported that bone morphogenetic protein-4 (BMP-4) stimulates the synthesis of osteocalcin via p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells, whereas p44/p42 MAP kinase plays as a negative regulator in the synthesis. In the present study, we investigated whether Rho-kinase is involved in BMP-4-stimulated osteocalcin synthesis in MC3T3-E1 cells. Main methodsThe levels of osteocalcin were measured by ELISA. The phosphorylation of each protein kinase was analyzed by Western blotting. The mRNA levels of osteocalcin were determined by real-time RT-PCR. Key findingsBMP-4 induced the phosphorylation of myosin phosphatase targeting subunit-1 (MYPT-1), a substrate of Rho-kinase. Y27632 or fasudil, specific inhibitors of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly amplified the BMP-4-stimulated osteocalcin synthesis in a dose-dependent manner. The osteocalcin mRNA expression levels induced by BMP-4 were enhanced by Y27632 or fasudil. BMP-4-stimulated osteocalcin release was significantly up-regulated in Rho-knocked down cells with Rho A-siRNA. Y27632 or fasudil failed to affect the BMP-4-induced phosphorylation of SMAD1 or p44/p42 MAP kinase. On the other hand, Y27632 or fasudil markedly strengthened the phosphorylation levels of p38 MAP kinase induced by BMP-4. SignificanceThese results strongly suggest that Rho-kinase negatively regulates BMP-4-stimulated osteocalcin synthesis via the p38 MAP kinase pathway in osteoblasts.

GPNMB enhances bone regeneration by promoting angiogenesis and osteogenesis: Potential role for tissue engineering bone

Bone regeneration is a coordinated process involving the connection between blood vessels and bone cells. Glycoprotein non-metastatic melanoma protein B (GPNMB) is known to be vital in bone formation. However, the effect of GPNMB on bone regeneration and the underlying molecular mechanism are still undefined. Fibroblast growth factor receptor (FGFR)-mediating signaling is pivotal in bone formation and angiogenesis. Therefore, we assessed GPNMB function as a communicating molecule between osteoblasts and angiogenesis, and the possible correlation with FGFR-1 signaling. Recombinant GPNMB dose-dependently increased the differentiation of human bone marrow stromal cells (hBMSCs) into osteoblasts, as well as the mRNA levels of osteoblasts marker alkaline phosphatase (ALP) and osteocalcin (OCN). Furthermore, these increases depended on the activation of FGFR-1 signaling, as pretreatment with FGFR-1 siRNA or its inhibitor SU5402 dramatically dampened GPNMB-induced osteogenesis. Additionally, GPNMB triggered dose-dependently the proliferation and migration of human umbilical vein endothelial cells (hUVECs), FGFR-1 phosphorylation, as well as capillary tube and vessels formation in vitro and in vivo. Blocking FGFR-1 signaling dampened GPNMB-induced angiogenic activity. Following construction of a rodent cranial defect model, scaffolds delivering GPNMB resulted in an evident increase in blood vessels and new bone formation; however, combined delivery of GPNMB and SU5402 abated these increase in defect sites. Taken together, these results suggest that GPNMB stimulates bone regeneration by inducing osteogenesis and angiogenesis via regulating FGFR-1 signaling. Consequently, our findings will clarify a new explanation about how GPNMB induces bone repair, and provide a potential target for bone regeneration therapeutics and bone engineering.

Exogenous polyamines promote osteogenic differentiation by reciprocally regulating osteogenic and adipogenic gene expression

Polyamines are naturally occurring organic polycations that are ubiquitous in all organisms, and are essential for cell proliferation and differentiation. Although polyamines are involved in various cellular processes, their roles in stem cell differentiation are relatively unexplored. In this study, we found that exogenous polyamines, putrescine, spermidine, and spermine, promoted osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) without inducing cell death or apoptosis. Alkaline phosphatase (ALP) activity and the mRNA level of osteogenic genes, including Runx2, ALP, osteopontin, and osteocalcin, were up-regulated by exogenous polyamines. When hBMSCs were cultured at high cell density favoring adipocyte formation, exogenous polyamines resulted in down-regulation of adipogenic genes such as PPARγ, aP2, and adipsin. Extracellular matrix mineralization, a marker for osteoblast maturation, was enhanced in the presence of exogenous polyamines, while lipid accumulation, an indication of adipogenic differentiation, was attenuated. Exogenous polyamines increased the mRNA expression of polyamine-modulated factor 1 (PMF-1) and its downstream effector, spermidine/spermine N(1)-acetyltransferase (SSAT), while that of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was suppressed. These results lead to possible connections between polyamine metabolism and osteogenic differentiation pathways. To summarize, this study provides evidence for the involvement of polyamines in osteogenic differentiation of hBMSCs, and is the first to demonstrate that osteogenic and adipogenic differentiation are reciprocally regulated by exogenous polyamines.

Bacterial Infection Increases Periodontal Bone Loss in Diabetic Rats through Enhanced Apoptosis

Periodontal disease is the most common osteolytic disease in humans and is significantly increased by diabetes mellitus. We tested the hypothesis that bacterial infection induces bone loss in diabetic animals through a mechanism that involves enhanced apoptosis. Type II diabetic rats were inoculated with Aggregatibacter actinomycetemcomitans and treated with a caspase-3 inhibitor, ZDEVD-FMK, or vehicle alone. Apoptotic cells were measured with TUNEL; osteoblasts and bone area were measured in H&E sections. New bone formation was assessed by labeling with fluorescent dyes and by osteocalcin mRNA levels. Osteoclast number, eroded bone surface, and new bone formation were measured by tartrate-resistant acid phosphatase staining. Immunohistochemistry was performed with an antibody against tumor necrosis factor-α. Bacterial infection doubled the number of tumor necrosis factor-α-expressing cells and increased apoptotic cells adjacent to bone 10-fold (P < 0.05). Treatment with caspase inhibitor blocked apoptosis, increased the number of osteoclasts, and eroded bone surface (P < 0.05); yet, inhibition of apoptosis resulted in significantly greater net bone area because of an increase in new bone formation, osteoblast numbers, and an increase in bone coupling. Thus, bacterial infection in diabetic rats stimulates periodontitis, in part through enhanced apoptosis of osteoblastic cells that reduces osseous coupling through a caspase-3-dependent mechanism.

Aortic valvular interstitial cells apoptosis and calcification are mediated by TNF-related apoptosis-inducing ligand

Background/ObjectivesCalcific aortic valvular disease (CAVD) is an actively regulated process characterized by the activation of specific osteogenic signaling pathways and apoptosis. We evaluated the involvement in CAVD of the TNF-related apoptosis-inducing ligand (TRAIL), an apoptotic molecule which induces apoptosis by interacting with the death receptor (DR)-4 and DR5, and whose activity is modulated by the decoy receptor (DcR)-1 and DcR2. MethodsSections of calcific and normal aortic valves, obtained at surgery time, were subjected to immunohistochemistry and confocal microscopy for TRAIL immunostaining. Valvular interstitial cells (VICs) isolated from calcific (C-VICs) and normal (N-VICs) aortic valves were investigated for the gene and protein expression of TRAIL receptors. Cell viability was assayed by MTT. Von Kossa staining was performed to verify C-VIC ability to produce mineralized nodules. TRAIL serum levels were detected by ELISA. ResultsHigher levels of TRAIL were detected in calcific aortic valves and in sera from the same patients respect to controls. C-VICs express significantly higher mRNA and protein levels of DR4, DR5, DcR1, DcR2 and Runx2 compared to N-VICs. C-VICs and N-VICs, cultured in osteogenic medium, express significantly higher mRNA levels of DR4, Runx2 and Osteocalcin compared to baseline. C-VICs and N-VICs were sensitive to TRAIL-apoptotic effect at baseline and after osteogenic differentiation, as demonstrated by MTT assay and caspase-3 activation. TRAIL enhanced mineralized matrix nodule synthesis by C-VICs cultured in osteogenic medium. ConclusionsTRAIL is characteristically present within calcific aortic valves, and mediates the calcification of aortic valve interstitial cells in culture through mechanism involving apoptosis.

Response of immortalized murine cementoblast cells to hypoxia in vitro

ObjectivesThe aim of the study was to investigate the impact of hypoxia on proliferation, apoptosis and mineralization of cementoblast-like cells (OCCM-30) in vitro. MethodsThe effects of different periods of hypoxia (2% O2) on proliferation, apoptosis, cementoblastic potential and root cementum resorption capability of OCCM-30 were evaluated, by using MTT, flow cytometry, alkaline phosphatase (ALP) activity assay, reverse transcription-polymerase chain reaction measurement, enzyme-linked immunosorbent assay and mineralization nodule formation assay. ResultsOCCM-30 viability was significantly inhibited by hypoxia while the apoptosis ratio was enhanced in a time-dependent manner; hypoxia inducible factor-1α and vascular endothelial growth factor mRNA were induced by hypoxia in different manners; temporary hypoxia (<24h) stimulated cementoblastic function of OCCM-30, while long-term hypoxia inhibited it, manifested by decreased mRNA level or release of ALP, osteocalcin, bone sialoprotein, osteopontin and osteoprotegerin. In addition, hypoxia affected mineralized nodule formation of OCCM-30 in a time-dependent fashion; moreover, root cementum resorption function was also induced by hypoxia, manifested by increased receptor activator of nuclear factor kappa B ligand mRNA and protein expression. ConclusionTemporary exposure of OCCM-30 to hypoxia inhibited proliferation, promoted apoptosis and mineralization, while longer duration of hypoxia could inhibit the cementoblast function. The findings may provide theoretical basis for developing novel therapeutics to prevent root resorption during orthodontic treatment.

Increased reactivity and in vitro cell response of titanium based implant surfaces after anodic oxidation

In the quest for improved bone growth and attachment around dental implants, chemical surface modifications are one possibility for future developments. The biological properties of titanium based materials can be further enhanced with methods like anodic polarization to produce an active rather than a passive titanium oxide surface. Here we investigate the formation of hydroxide groups on sand blasted and acid etched titanium and titanium-zirconium alloy surfaces after anodic polarization in an alkaline solution. X-ray photoelectron spectroscopy shows that the activated surfaces had increased reactivity. Furthermore the activated surfaces show up to threefold increase in OH(-) concentration in comparison to the original surface. The surface parameters Sa, Sku, Sdr and Ssk were more closely correlated to time and current density for titanium than for titanium-zirconium. Studies with MC3T3-E1 osteoblastic cells showed that OH(-) activated surfaces increased mRNA levels of osteocalcin and collagen-I.

Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro

Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca(2+) on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca(2+) on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca(2+) (0, 1, 2, 3, 4, and 5mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca(2+) stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5mM) in the culture, Ca(2+) significantly inhibited the activity of ALP on days 7 and 14 (P<0.01 or P<0.05), significantly suppressed collagen synthesis (P<0.01 or P<0.05), and significantly elevated calcium deposition (P<0.01) and mRNA levels of osteocalcin (P<0.01 or P<0.05) and osteopontin (P<0.01 or P<0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.