Induction Of Mineralization Research Articles

Differentiation potential of osteoblast from cultured C2C12 cells on zirconia disk

We examined the adaptability of zirconia as a fixture for implants. A mouse myoblast cell line (C2C12) was seeded on Ce-TZP and titanium disks, and on poly-L-lysine-coated glass slides. Proliferation potency was determined by cell counting and mineral induction by BMP2 was studied. The osteoblastic differentiation potency was determined by alkaline phosphatase (ALP) and alizarin red (ARS) staining. ALP activity and calcium concentrations were colorimetrically measured. The number of cells on all materials was approximately equal. ALP and ARS staining showed densely-stained images, demonstrating the induction of C2C12 cells to express the osteoblastic phenotype. RT-PCR showed that mRNA expressions of type I collagen, osteocalcin, osterix and ALP were up-regulated. With regard to ALP activity and calcium concentration of C2C12 cells, no significant differences were observed between Ce-TZP and titanium disks. We conclude that Ce-TZP has the biological activity comparable to titanium and has the utility as fixture of dental implants.

Cellular Toxicity and Apoptosis Studies in Osteocarcinoma Cells, a Comparison of 177Lu-EDTMP and Lu-EDTMP

The most promising bone pain palliative agent such as 177Lu-EDTMP emerges as a newer radiopharmaceutical for cancer management. Thus, it was of interest to study the cell uptake of this agent in osteocarcinoma cell line and investigate the underlying mechanism of cellular toxicity. The cell binding studies of 177Lu-EDTMP were carried out in osteocarcinoma cells (MG63) after induction of bone mineralization. Cellular toxicity studies were carried out with varying amounts of 177Lu-EDTMP and compared with equivalent amount of cold Lu-EDTMP. Cell viability was assessed by trypan blue, LDH and MTT assay. Different studies such as DNA fragmentation and Western blotting for apoptosis related proteins were carried out to elucidate the mechanism of cell death. Maximum cell binding of 177Lu-EDTMP, observed with mineralized MG63 cells was 19 ± 0.122%. Nearly 12% cell death was observed in MG63 cells treated with 37 MBq of 177Lu-EDTMP as compared to controls. Apoptosis studies were carried out by ELISA to estimate DNA fragmentation and it was found that DNA enrichment factor was 1.8, compared to the corresponding control. Down regulation of anti-apoptotic protein, bcl-2 and cleavage of PARP protein was evident by Western blot results. These studies indicate that the 177Lu-EDTMP binds to mineralized bone cells and induces apoptotic cell death in MG63 cells.

1α,25‐dihydroxyvitamin D3 stimulates activin A production to fine‐tune osteoblast‐induced mineralization

In healthy bones, mineralization has to be tightly controlled to avoid pathological phenotypes. In this study, we investigated interactions between 1α,25(OH)2 D3 (1,25D3) and activin A in the regulation of osteoblast induced mineralization. In human osteoblast cultures, we demonstrated that besides stimulation of mineralization, 1,25D3 also induced activin A, a strong inhibitor of mineralization. Simultaneously, follistatin (FST), the natural antagonist of activin A, was down-regulated by1,25D3. This resulted in an increase in activin A activity during 1,25D3 treatment. We also showed that in 1,25D3-treated osteoblasts, mineralization can be further increased when activin A activity was abrogated by adding exogenous FST. This observation implies that, besides stimulation of mineralization, 1,25D3 also controls activin A-mediated inhibition of mineralization. Besides activin A, 1,25D3 also induces osteocalcin (BGLAP), another inhibitor of mineralization. Warfarin, which has been shown to inactivate osteocalcin, increased 1,25D3-induced mineralization. Interaction between these two systems became evident from the synergistic increase in BGLAP expression upon blocking activin activity in 1,25D3-treated cultures. In conclusion, we demonstrate that 1,25D3 stimulation of mineralization by human osteoblasts is suppressed by concomitant induction of inhibitors of mineralization. Mineralization induction by 1,25D3 may actually be controlled via interplay with activin A and osteocalcin. Finally, this complex regulation of mineralization substantiates the significance of tight control of mineralization to prevent excessive mineralization and consequently reduction in bone quality and strength.

Localization and quantitative co-localization of enamelin with amelogenin.

Enamelin and amelogenin are vital proteins in enamel formation. The cooperative function of these two proteins controls crystal nucleation and morphology in vitro. We quantitatively analyzed the co-localization between enamelin and amelogenin by confocal microscopy and using two antibodies, one raised against a sequence in the porcine 32 kDa enamelin region and the other raised against full-length recombinant mouse amelogenin. We further investigated the interaction of the porcine 32 kDa enamelin and recombinant amelogenin using immuno-gold labeling. This study reports the quantitative co-localization results for postnatal days 1-8 mandibular mouse molars. We show that amelogenin and enamelin are secreted into the extracellular matrix on the cuspal slopes of the molars at day 1 and that secretion continues to at least day 8. Quantitative co-localization analysis (QCA) was performed in several different configurations using large (45 μm height, 33 μm width) and small (7 μm diameter) regions of interest to elucidate any patterns. Co-localization patterns in day 8 samples revealed that enamelin and amelogenin co-localize near the secretory face of the ameloblasts and appear to be secreted approximately in a 1:1 ratio. The degree of co-localization decreases as the enamel matures, both along the secretory face of ameloblasts and throughout the entire thickness of the enamel. Immuno-reactivity against enamelin is concentrated along the secretory face of ameloblasts, supporting the theory that this protein together with amelogenin is intimately involved in mineral induction at the beginning of enamel formation.

Epigenetic Marks Define the Lineage and Differentiation Potential of Two Distinct Neural Crest-Derived Intermediate Odontogenic Progenitor Populations

Epigenetic mechanisms, such as histone modifications, play an active role in the differentiation and lineage commitment of mesenchymal stem cells. In the present study, epigenetic states and differentiation profiles of two odontogenic neural crest-derived intermediate progenitor populations were compared: dental pulp (DP) and dental follicle (DF). ChIP on chip assays revealed substantial H3K27me3-mediated repression of odontoblast lineage genes DSPP and dentin matrix protein 1 (DMP1) in DF cells, but not in DP cells. Mineralization inductive conditions caused steep increases of mineralization and patterning gene expression levels in DP cells when compared to DF cells. In contrast, mineralization induction resulted in a highly dynamic histone modification response in DF cells, while there was only a subdued effect in DP cells. Both DF and DP progenitors featured H3K4me3-active marks on the promoters of early mineralization genes RUNX2, MSX2, and DLX5, while OSX, IBSP, and BGLAP promoters were enriched for H3K9me3 or H3K27me3. Compared to DF cells, DP cells expressed higher levels of three pluripotency-associated genes, OCT4, NANOG, and SOX2. Finally, gene ontology comparison of bivalent marks unique for DP and DF cells highlighted cell-cell attachment genes in DP cells and neurogenesis genes in DF cells. In conclusion, the present study indicates that the DF intermediate odontogenic neural crest lineage is distinguished from its DP counterpart by epigenetic repression of DSPP and DMP1 genes and through dynamic histone enrichment responses to mineralization induction. Findings presented here highlight the crucial role of epigenetic regulatory mechanisms in the terminal differentiation of odontogenic neural crest lineages.

The Survival Role of Peroxisome Proliferator-activated Receptor Gamma Induces Odontoblast Differentiation against Oxidative Stress in Human Dental Pulp Cells

IntroductionPeroxisome proliferator-activated receptor gamma (PPARγ) has well-known anti-inflammatory action in human dental pulp cells (HDPCs). The purpose of this study was to investigate whether the anti-inflammatory action of PPARγ involves in cellular cytoprotection and supports odontoblast differentiation under oxidative stress in HDPCs. MethodsTo simulate long-term oxidative stress, pulp cells were treated with 150 μmol hydrogen peroxide (H2O2) for 12 days. The replication deficiency adenovirus (adenovirus PPARγ) was introduced for PPARγ overexpression in pulp cells. The cellular cytotoxicity and reactive oxygen species formation by H2O2 were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 2′,7′-dichlorodihydrofluorescein diacetate with fluorescence-activated cell sorting assay. To determine the roles of PPARγ, several molecules of odontogenic/osteogenic and signal pathway were analyzed by reverse-transcription polymerase chain reaction and Western hybridization. Dentin mineralization was determined by alizarin red stain and alkaline phosphatase activity assay. ResultsPulp cells treated with long-term H2O2 showed high reactive oxygen species formation, low cell viability, down-expression of antioxidant molecules (Cu/Zn and Mn superoxide dismutase), and odontogenic/osteogenic markers (eg, dentin sialophosphoprotein, dentin matrix protein-1, osteopontin, bone sialoprotein, Runx-2, and bone morphogenetic protein 2 and 7). In addition, pulp cells with oxidative stress underwent the activation of ERK1/2, activator protein-1, and nuclear factor-κB translocation to the nucleus. However, the PPARγ-overexpressed cells gave opposite results although under oxidative stress. Furthermore, PPARγ and its agonist rosiglitazone exhibited an induction of dentin mineralization under oxidative stress. ConclusionsPPARγ in pulp cells increases cell viability, odontoblastic differentiation, and dentin mineralization under oxidative stress. These results offer new insights into the potential antioxidative activity of PPARγ and its agonist for therapeutic agents for pulp vitality in HDPCs.

Mineralization induction effects of osteopontin, bone sialoprotein, and dentin phosphoprotein on a biomimetic collagen substrate

Native bone tissue is composed of a matrix of collagen, noncollagenous proteins, and calcium phosphate minerals, which are primarily hydroxyapatite. The SIBLING (small integrin-binding ligand, N-linked glycoprotein) family of proteins is the primary noncollagenous protein group found in mineralized tissues. In this work, the mineralization induction capabilities of three of the SIBLING members, bone sialoprotein (BSP), osteopontin (OPN), and the calcium-binding subdomain of dentin sialophosphoprotein, dentin phosphoprotein (DPP), are directly compared on a biomimetic collagen substrate. A self-assembled, loosely aligned collagen fibril substrate was prepared, and then (125) I-radiolabeled adsorption isotherms were developed for BSP, OPN, and DPP. The results showed that BSP exhibited the highest binding capacity for collagen at lower concentrations, followed by DPP and OPN. However, at the highest concentrations, all three proteins had similar adsorption levels. The adsorption isotherms were then used to identify conditions that resulted in identical amounts of adsorbed protein. These substrates were prepared and placed in simulated body fluid for 5, 10, and 24 h at 37°C. The resulting mineral morphology was assessed by atomic force microscopy, and the composition was determined using photochemical assays. Mineralization was seen in the presence of all the proteins. However, DPP was seen to be the only protein that formed individual mineral nodules similar to those seen in developing bone. This suggests that DPP plays a significant role in the biomineralization process and that the incorporation of DPP into tissue engineering constructs may facilitate the induction of biomimetic mineral formation.

NHERF1 regulation of PTH-dependent bimodal Pi transport in osteoblasts

Control of systemic inorganic phosphate (Pi) levels is crucial for osteoid mineralization. Parathyroid hormone (PTH) mediates actions on phosphate homeostasis mostly by regulating the activity of the type 2 sodium–phosphate cotransporter (Npt2), and this action requires the PDZ protein NHERF1. Osteoblasts express Npt2 and in response to PTH enhance osteogenesis by increasing mineralized matrix. The regulation of Pi transport in osteoblasts is poorly understood. To address this gap we characterized PTH-dependent Pi transport and the role of NHERF1 in primary mouse calvarial osteoblasts. Under proliferating conditions osteoblasts express Npt2a, Npt2b, PTH receptor, and NHERF1. Npt2a mRNA expression was lower in calvarial osteoblasts from NHERF1-null mice. Under basal conditions Pi uptake in osteoblasts from wild-type mice was greater than that of knockout mice. PTH inhibited Pi uptake in proliferating osteoblasts from wild-type mice, but not in cells from knockout mice. In vitro induction of mineralization enhanced osteoblast differentiation and increased osterix and osteocalcin expression. Contrary to the results with proliferating osteoblasts, PTH increased Pi uptake and ATP secretion in differentiated osteoblasts from wild-type mice. PTH had no effect on Pi uptake or ATP release in differentiated osteoblasts from knockout mice. NHERF1 regulation of PTH-sensitive Pi uptake in proliferating osteoblasts is mediated by cAMP/PKA and PLC/PKC, while modulation of Pi uptake in differentiated osteoblasts depends only on cAMP/PKA signaling. The results suggest that NHERF1 cooperates with PTH in differentiated osteoblasts to increase matrix mineralization. We conclude that NHERF1 regulates PTH that differentially affects Na-dependent Pi transport at distinct stages of osteoblast proliferation and maturation.

Effects of calcium salts of acidic monomers on mineral induction of phosphoprotein immobilized to agarose beads

The aim of this study is to evaluate the mineralizing potential of acidic monomers and their calcium salts for mineralization, using an in vitro mineral induction model. Phosvitin (PV) was used as a model phosphoprotein in this study. PV was immobilized on agarose beads with divinyl sulfone. Five aliquots of agarose-immobilized PV, acidic monomers, and their calcium salts were incubated in mineralizing solution at various concentrations. The PV beads and acidic monomers were incubated at 37°C. Samples were taken at several time points during the incubation. Then, the agarose beads were analyzed for bound calcium by atomic absorption spectrometry. The mineral formed on the agarose beads was identified as an apatite by microarea X-ray diffraction. Additionally, the specimens were observed using scanning electron microscopy (SEM). Mineral induction time decreased with increasing solution saturation. 4-METCa salt [calcium salt of 4-methacryloxyethyl trimellitate (CMET)] significantly reduced the mineral induction time. Using these data, the interfacial tension for mineral induction of PV and CMET was determined to be 90.1 and 92.7 ergs/cm(2), respectively. The mineral induced in each specimen after incubation for 24 h was identified by its X-ray diffraction pattern as apatite. SEM observation showed that lath-shaped crystals were formed on the surfaces of the CMET. We conclude that CMET could play a role in dentin remineralization.

Abstract 300: Leptin Promotes the Osteoblast Differentiation and Mineralization of Primary Cultures of Vascular Smooth Muscle Cells by Regulating GSK-3β

Obesity is an independent risk factor for cardiovascular disease (CVD) although the precise molecular mechanisms that link obesity to CVD are not understood. Recent studies suggest that factors secreted by adipose tissue may play an adverse role in cardiovascular health. We have previously demonstrated that the adipocytokine, leptin, promotes vascular calcification in apolipoprotein E - deficient mice and that this increase in calcification is associated with an increase in the expression of several osteoblast-specific markers within the vessel wall. In an effort to understand the mechanism by which leptin exerts these effects, we cultured primary bovine aortic smooth muscle cells (BASMCs) in the presence of 0 to 2 μg/ml leptin for up to 12 days. Osteogenic differentiation of BASMCs was determined by an increase in the expression of osteoblast-specific markers, and the induction of both alkaline phosphatase activity and mineralization. Consistent with previous studies, we found that treatment of BASMCs with leptin induced osteoblast differentiation in a dose-dependent manner. To investigate the underlying mechanism, we examined changes in the expression levels of key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway. We found that exposure to leptin induced the Erk 1/2-dependent inactivation of GSK-3β, through Ser9 phosphorylation, and a subsequent increase in the nuclear accumulation of β-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3β (Ad-GSK-3β S9A) resulted in a > 2-fold increase in GSK-3β activity, a decrease in the expression of the osteoblast-specific marker, osteopontin, and a significant decrease in leptin-induced alkaline phosphatase activity. Together, our results provide a possible mechanism by which elevated leptin concentrations, associated with obesity, promote osteoblast differentiation, and vascular calcification in vivo.

Astragaloside II induces osteogenic activities of osteoblasts through the bone morphogenetic protein-2/MAPK and Smad1/5/8 pathways

Radix Astragalus has been identified to exert beneficial effects in preventing postmenopausal bone loss. However, the active ingredients and mechanism of action remain unknown. In this study, we examined the effect of Astragaloside II (AST II), which is a monomer of Astragalus saponin, on the viability, proliferation, differentiation and maturation of rat primary osteoblasts, as well as its relevant molecular mechanism. We found that AST II exhibits a significant induction of proliferation, differentiation and mineralization in primary osteoblasts. AST II stimulates osteoblast differentiation at various stages, from early to late stage of differentiated osteoblasts. Furthermore, induction of differentiation by AST II is associated with increased expression of bone morphogenetic protein-2 (BMP-2), activation of Smad1/5/8, ERK1/2 and p38, and increased expression of core-binding factor 1 (Cbfa1)/Runx2. BMP antagonist (Noggin) blocks the effect of AST II on cell differentiation, and Smad1/5/8, p38, Cbfa1 expression, but only partly decreases ERK1/2 activation. This indicates that BMP-2 is essential in AST II-mediated osteoblast differentiation and Smad1/5/8, p38, Cbfa1 activation, and is partly involved in ERK1/2 activation. In conclusion, although in vivo studies are required in the future, as a phyto-saponin of Radix Astragalus, AST II may become a novel candidate that is beneficial for stimulating the osteoblastic activity resulting in bone formation, which has not been recognized and reported previously.

Effects of Wnt/β‐catenin signalling on proliferation and differentiation of apical papilla stem cells

The Wnt signalling pathway has been shown to play an important role in tooth development, however its effects with stem cells from the apical papilla (SCAP) have remained unclear. The purpose of this study was to determine effects of Wnt/β-catenin on proliferation and differentiation of SCAP in vitro. SCAP were obtained, identified and cultured. Cell proliferation, alkaline phosphatase (ALP) activity, mRNA expression of mineralization-related genes and mineralized nodule formation were measured in presence or absence of various concentrations of lithium chloride. MTT assay and flow cytometry demonstrated that Wnt/β-catenin activity could promote proliferation of SCAP. Real-time PCR analysis found that Wnt/β-catenin strongly upregulated expression of dentine sialophosphoprotein, osteocalcin and ALP in SCAP after incubation with mineralization induction medium, while ALP and alizarin red staining indicated that Wnt/β-catenin enhanced ALP activity and formation of mineralized nodules. Our results suggest that canonical Wnt/β-catenin signalling promotes proliferation and odonto/osteogenic differentiation of SCAP.

Carbon Nanotubes in Nanocomposites and Hybrids with Hydroxyapatite for Bone Replacements

Hydroxyapatite (HA), as a bone mineral component, has been an attractive bioceramic for the reconstruction of hard tissues. However, its poor mechanical properties, including low fracture toughness and tensile strength, have been a significant challenge to the application of HA for the replacement of load-bearing and/or large bone defects. Among materials studied to reinforce HA, carbon nanotubes (CNTs: single-walled or multiwalled) have recently gained significant attention because of their unprecedented mechanical properties (high strength and toughness) and physicochemical properties (high surface area, electrical and thermal conductivity, and low weight). Here, we review recent studies of the organization of HA-CNTs at the nanoscale, with a particular emphasis on the functionalization of CNTs and their dispersion within an HA matrix and induction of HA mineralization. The organization of CNTs and HA implemented at the nanoscale can further be developed in the form of coatings, nanocomposites, and hybrid powders to enable potential applications in hard tissue reconstruction.

Bone vs. fat: Embryonic origin of progenitors determines response to androgen in adipocytes and osteoblasts

Although androgen is considered an anabolic hormone, the consequences of androgen receptor (AR) overexpression in skeletally-targeted AR-transgenic lines highlight the detrimental effect of enhanced androgen sensitivity on cortical bone quality. A compartment-specific anabolic response is observed only in male and not in female AR3.6-transgenic (tg) mice, with increased periosteal bone formation and calvarial thickening. To identify anabolic signaling cascades that have the potential to increase bone formation, qPCR array analysis was employed to define expression differences between AR3.6-tg and wild-type (WT) periosteal tissue. Notably, categories that were significantly different between the two genotypes included axonal guidance, CNS development and negative regulation of Wnt signaling with a node centered on stem cell pathways. Further, fine mapping of AR3.6-tg calvaria revealed that anabolic thickening in vivo is not uniform across the calvaria, occurring only in frontal and in not parietal bones. Multipotent fraction 1 progenitor populations from both genotypes were cultured separately as frontal bone neural crest stem-like cells (fNCSC) and parietal bone mesenchymal stem-like cells (pMSC). Both osteoblastic and adipogenic differentiation in these progenitor populations was influenced by embryonic lineage and by genotype. Adipogenesis was enhanced in WT fNCSC compared to pMSC, but transgenic cultures showed strong suppression of lipid accumulation only in fNCSC cells. Osteoblastogenesis was significantly increased in transgenic fNCSC cultures compared to WT, with elevated alkaline phosphatase (ALP) activity and induction of mineralization and nodule formation assessed by alizarin red and von Kossa staining. Osteocalcin (OC) and ALP mRNA levels were also increased in fNCSC cultures from AR3.6-tg vs. WT, but in pMSC cultures ALP mRNA levels, mineralization and nodule formation were decreased in AR3.6-tg cells. Expression differences identified by array in long bone periosteal tissue from AR3.6-tg vs. WT were recapitulated in the fNCSC samples while pMSC profiles reflected cortical expression. These observations reveal the opposing effects of androgen signaling on lineage commitment and osteoblast differentiation that is enhanced in cells derived from a neural crest origin but inhibited in cells derived from a mesodermal origin, consistent with in vivo compartment-specific responses to androgen. Combined, these results highlight the complex action of androgen in the body that is dependent on the embryonic lineage and developmental origin of the cell. Further, these data these data suggest that the periosteum surrounding long bone is derived from neural crest.

GPM6B regulates osteoblast function and induction of mineralization by controlling cytoskeleton and matrix vesicle release

Neuronal membrane glycoprotein gene (GPM6B) encodes a membrane glycoprotein that belongs to the proteolipid protein family. We identified GPM6B as a gene that is strongly upregulated during osteoblast differentiation. To investigate the role of GPM6B in the process of bone formation, we silenced GPM6B expression during osteogenic differentiation of human mesenchymal stem cells (hMSCs). GPM6B silencing in hMSCs resulted in reduced alkaline phosphate (ALP) activity along with reduced mineralization of extracellular matrix (ECM). Microarray expression analysis of GPM6B-depleted osteogenic hMSCs revealed significant changes in genes involved in cytoskeleton organization and biogenesis. Immunocytochemistry results confirm changes in the distribution of actin filaments, as well as the shape and size of focal adhesions on GPM6B silencing. Moreover, we demonstrated that production and release of ALP-positive matrix vesicles (MVs) were reduced. In conclusion, we identified GPM6B as a novel regulator of osteoblast function and bone formation. This finding demonstrates the significance of cytoskeleton organization for MV production and subsequent mineralization.

Expression of Phosphophoryn Is Sufficient for the Induction of Matrix Mineralization by Mammalian Cells

Mineralized tissues such as dentin and bone assemble extracellular matrices uniquely rich in a variety of acidic phosphoproteins. Although these proteins are presumed to play a role in the process of biomineralization, key questions regarding the nature of their contributions remain unanswered. First, it is not known whether highly phosphorylated proteins alone can induce matrix mineralization, or whether this activity requires the involvement of other bone/dentin non-collagenous proteins. Second, it remains to be established whether the protein kinases that phosphorylate these acidic proteins are unique to cells responsible for producing mineralized tissues. To begin to address these questions, we consider the case of phosphophoryn (PP), due to its high content of phosphate, high affinity for Ca(2+), and its potential role in hydroxyapatite nucleation. We have created a model system of biomineralization in a cellular environment by expressing PP in NIH3T3 fibroblasts (which do not produce a mineralized matrix); as a positive control, PP was expressed in MC3T3-E1 osteoblastic cells, which normally mineralize their matrices. We show that expression of PP in NIH3T3 cells is sufficient for the induction of matrix mineralization. In addition, assessment of the phosphorylation status of PP in these cells reveals that the transfected NIH3T3 cells are able to phosphorylate PP. We suggest that the phosphorylation of PP is essential for mineral formation. The principle goal of this study is to enrich the current knowledge of mineralized tissue phosphorylation events by analyzing them in the context of a complete cellular environment.

Effects of Leukemia Inhibitory Factor on Proliferation and Odontoblastic Differentiation of Human Dental Pulp Cells

Introduction The purpose of this study was to determine whether the leukemia inhibitory factor (LIF) is expressed in human dental tissue and exerts its effect on proliferation and odontoblastic differentiation of the dental pulp cells (DPCs). Methods An immunohistochemical assay was used to detect the expression of LIF and leukemia inhibitory factor receptor (LIFR) in the human dental pulp. The proliferation of DPCs was examined by culturing human primary DPCs in the presence of LIF with different doses or the neutralizing antibody to LIF. Western blot was performed to assay the phosphorylation of Janus kinase 2 (Jak2) and signal transducer and activator of transcription 3 (Stat3) in the presence or absence of LIF and/or AG 490, a specific inhibitor of Jak2. The odontoblastic differentiation of DPCs was determined using the alkaline phosphatase (ALP) activity assay, quantification of bone sialoprotein (BSP) and dentin sialophosphoprotein (DSPP) gene expression, and mineralization nodule formation. Results LIF and LIFR were present in the odontoblasts and DPCs. LIF induced proliferation of DPCs, which was inhibited by the LIF neutralizing antibody and AG 490. LIF induced phosphorylation of Jak2 and Stat3 but not in the presence of the AG490. ALP activity of DPCs, in the absence or presence of mineralization induction medium, was inhibited by LIF. Furthermore, the mineralization nodule formation and the expression of BSP and DSPP were inhibited by LIF. This inhibition on differentiation was attenuated by the AG490. Conclusions LIF and LIFR are expressed in the human dental pulp. LIF promotes the proliferation of DPCs, and the odontoblastic differentiation is inhibited via the Jak2-Stat3 signaling pathway.

GPM6B regulates osteoblast differentiation and induction of mineralization by controlling cytoskeleton and matrix vesicle release

GPM6B regulates osteoblast differentiation and induction of mineralization by controlling cytoskeleton and matrix vesicle release

Notch Signaling Pathway Enhances Bone Morphogenetic Protein 2 (BMP2) Responsiveness of Msx2 Gene to Induce Osteogenic Differentiation and Mineralization of Vascular Smooth Muscle Cells

Vascular calcification is regulated in a process similar to bone formation. BMP2 (bone morphogenetic protein 2) is essential for osteoblastic differentiation of mesenchymal progenitor cells and thus has been implicated in the development of vascular calcification. Here we examined whether Notch signaling interacts with BMP2 signaling to regulate osteogenic differentiation and mineralization of vascular smooth muscle cells (SMCs). BMP2 alone scarcely induced the expression of alkaline phosphatase (ALP), an ectoenzyme crucially required for active biomineralization, in human aortic SMCs (HASMCs), despite its strong induction in osteoblast precursor MC3T3-E1 cells. Notably, overexpression of the Notch1 intracellular domain (N1-ICD) markedly enhanced BMP2-mediated induction of ALP activity and mineralization of HASMCs. In HASMCs, expression of Msx2 gene, a well documented BMP2 target gene in osteoblasts, was barely induced by BMP2 alone, and N1-ICD clearly enhanced the BMP2-driven Msx2 gene expression. Deletion and site-directed mutation analysis of Msx2 gene promoter revealed that the RBPJk-binding site was necessary for BMP2 responsiveness. Using the RBPJk-deficient cells and siRNA for RBPJk, we showed that RBPJk was required for BMP2 induction of Msx2 gene expression and ALP activity. Moreover, we showed that Smad1, a transcription factor downstream of BMP2 signaling, interacted with N1-ICD to form a complex within the Msx2 promoter. Immunohistochemistry of human calcifying atherosclerotic plaques revealed colocalized expression of Notch1, BMP2, and Msx2. These results indicate that the Notch intracellular domain·RBPJk complex enhances the BMP2-induced Msx2 gene expression by cooperating with Smad1 and suggest that Notch signaling makes vascular SMC responsive to BMP2 and promotes vascular calcification.

Asperosaponin VI, A Saponin Component from Dipsacus asper Wall, induces Osteoblast Differentiation through Bone Morphogenetic Protein‐2/p38 and Extracellular Signal‐regulated Kinase 1/2 Pathway

Osteoporosis is a reduction in skeletal mass because of the loss of osteoblastic activity or an increase in osteoclastic activity. The survival of osteoblast cells plays a crucial role in the development of osteoporosis. Asperosaponin VI (ASA VI) is a kind of saponin in the medicinal herb Dipsacus asper Wall which has long been used as an antiosteoporosis drug. The assay of cell proliferation, alkaline phosphatase (ALP) activity and measurement of mineralized matrix, showed that ASA VI exhibited a significant induction of proliferation, differentiation and mineralization in MC3T3-E1 and primary osteoblastic cells. Induction of differentiation by ASA VI was associated with increased bone morphogenetic protein-2 (BMP-2), indicating that BMP-2 is essential in ASA VI to mediate osteoblast maturation and differentiation. In addition, ASA VI may induce differentiation by increasing the activity of p38 and ERK1/2. In conclusion, ASA VI may induce osteoblast maturation and differentiation, and then increase bone formation via increasing BMP-2 synthesis, and activating p38 and ERK1/2.