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Biochemical and Biophysical Research... Apr 2020Bone homeostasis is maintained by concerted actions of bone-forming osteoblasts and bone-resorbing osteoclasts. A wide range of evidence indicates that a proinflammatory...
Bone homeostasis is maintained by concerted actions of bone-forming osteoblasts and bone-resorbing osteoclasts. A wide range of evidence indicates that a proinflammatory cytokine IL-17 promotes osteoclastogenesis. However, the role of IL-17 in osteoblasts is less well-understood. In the current study, the effect of IL-17 on osteogenic differentiation was investigated in mouse calvarial cells. IL-17 stimulated osteoblast differentiation, mineralization, proliferation, motility, and osteoblast-dependent osteoclastogenesis in vitro. The pro-osteogenic role of IL-17 was dependent on Act1 and the generation of reactive oxygen species. In a critical size calvarial defect model, IL-17 significantly augmented bone regeneration. Importantly, IL-17 also remarkably increased bone remodeling and restored osteoclastogenesis in zoledronate-treated mice. Furthermore, IL-17 conspicuously stimulated the formation of lamellar bones. These data not only provide a clue to understand the role of IL-17 in bone metabolism but also suggest possible applications in bone augmentation therapies.
Topics: Animals; Bone Regeneration; Bone Remodeling; Cells, Cultured; Interleukin-17; Male; Mice; Mice, Inbred C57BL; Osteoblasts; Osteogenesis
PubMed: 32067737
DOI: 10.1016/j.bbrc.2020.02.054 -
Journal of Endodontics Mar 2018Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and CPP-ACP with fluoride (CPP-ACFP) have been shown to provide bioavailable ions to promote mineralization....
INTRODUCTION
Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and CPP-ACP with fluoride (CPP-ACFP) have been shown to provide bioavailable ions to promote mineralization. Hence, the aim of this study was to evaluate the materials' biocompatibility and osteogenic/calcification potential for endodontic applications.
METHODS
Human and mouse osteoblast-like and fibroblast-like cell lines were incubated with 0.05%-3.0% w/v CPP-ACP and CPP-ACFP, and toxicity, proliferation, alkaline phosphatase, interleukin (IL)-1α, and IL-6 production, collagen type I, osteocalcin, and osteopontin production, and mineralization/calcification were determined.
RESULTS
CPP-ACP and CPP-ACFP were non-toxic and had no significant effect on proliferation or production of the inflammatory cytokine IL-1α. Alkaline phosphatase activity of the osteoblast-like cells was significantly increased (P < .05) by CPP-ACP and CPP-ACFP, as was the production of the osteotropic cytokine IL-6, the formation of calcium mineral deposits, and the secretion of mineralization-related proteins (collagen type I and osteocalcin).
CONCLUSIONS
CPP-ACP and CPP-ACFP are biocompatible and have the potential to induce osteoblastic differentiation and mineralization. Potential applications include apexification, perforation repair, vital pulp therapy, and regenerative endodontic procedures.
Topics: Animals; Apatites; Biocompatible Materials; Calcification, Physiologic; Caseins; Cells, Cultured; Humans; Mice; Osteoblasts; Osteogenesis
PubMed: 29275851
DOI: 10.1016/j.joen.2017.11.005 -
Journal of Cellular Physiology Apr 2015Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is a potent transcription factor that represses osteoblast differentiation and bone formation....
Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is a potent transcription factor that represses osteoblast differentiation and bone formation. Previously, we observed that stimuli for osteoblast differentiation, such as bone morphogenetic protein 2 (BMP2), inhibits COUP-TFII expression. This study was undertaken to identify BMP2-regulated and COUP-TFII-targeting microRNAs (miRNAs), and to explore their regulatory roles in osteoblast differentiation. Based on in silico analysis, 12 miRNAs were selected and their expression in BMP2-treated MC3T3-E1 cells was examined. BMP2 induced miR-302a expression in dose- and time-dependent manners with the decrease in COUP-TFII expression. Runx2, a BMP2-downstream transcription factor, specifically regulated miR-302a expression and its promoter activity. A computer-based prediction algorithm led to the identification of two miR-302a binding sites on the 3'-untranslational region of COUP-TFII mRNA (S1: 620-626 bp, S2: 1,016-1,022 bp), and a luciferase assay showed that miR-302a directly targeted S1 and S2. Transfection of miR-302a precursor significantly enhanced expression of osteogenic marker genes with decreasing COUP-TFII mRNA and protein level, alkaline phosphatase activity and matrix mineralization. On the other hand, inhibition of miR-302a significantly attenuated BMP2-induced osteoblast specific gene expression, alkaline phosphatase activity, and matrix mineralization with increasing COUP-TFII mRNA and protein level. These results indicate that miR-302a is induced by osteogenic stimuli and promotes osteoblast differentiation by targeting COUP-TFII. MiR-302a could be a positive regulator for osteoblast differentiation.
Topics: Animals; Bone Morphogenetic Protein 2; COUP Transcription Factor II; Cell Differentiation; Mice; MicroRNAs; Osteoblasts; RNA, Messenger; Transcription, Genetic
PubMed: 25215426
DOI: 10.1002/jcp.24822 -
Biochemical Genetics Oct 2021MiR-133b is considered to be lowly expressed in osteoporosis patients. This study aimed to probe the role and in-depth mechanism of miR-133b in modulating osteoblast...
MiR-133b is considered to be lowly expressed in osteoporosis patients. This study aimed to probe the role and in-depth mechanism of miR-133b in modulating osteoblast biological behavior and differentiation. The differential expressions of miR-133b and GNB4 in patients with osteoporosis and healthy control were analyzed based on the GEO database. Osteoblastic differentiation of hFOB 1.19 cells was induced in the culture medium containing 10 mM β-glycerophosphate, 50 nm dexamethasone, and 100 μg/ml ascorbic acid. The level of GNB4 was detected using quantitative real-time PCR (qRT-PCR) and Western blot. Cell viability and apoptosis were measured by Cell Counting Kit-8 (CCK-8) and flow cytometry assays, respectively. Western blot was also utilized to measure the levels of osteoblast-related proteins, including ALP, Runx2, Osterix, and OPN. GNB4 was identified and confirmed as a downstream target gene of miR-133b. The expression of miR-133b was declined while the expression of GNB4 was increased in osteoporosis patients. Importantly, up-regulation of miR-133b caused the increase of cell viability and the decrease of apoptosis, which could be blocked by overexpression of GNB4. Also, up-regulation of miR-133b promoted osteoblasts differentiation, as shown by the increase in the expression of ALP, Runx2, Osterix, and OPN. Similarly, this promoting impact resulted from miR-133b overexpression can be reversed via up-regulation of GNB4. These findings revealed that miR-133b can promote the viability and differentiation of osteoblasts by targeting GNB4, hoping to lay a feasible theoretical foundation for the clinical treatment of osteoporosis.
Topics: Apoptosis; Case-Control Studies; Cell Differentiation; Cell Proliferation; Cells, Cultured; GTP-Binding Protein beta Subunits; Humans; MicroRNAs; Osteoblasts; Osteogenesis; Osteoporosis
PubMed: 33687637
DOI: 10.1007/s10528-021-10048-9 -
Biochemical and Biophysical Research... Jun 2020Berberine is an essential phytochemical for the treatment of various diseases; however, when used to treat osteoporosis, it has minor effect as compared with that of the...
Berberine is an essential phytochemical for the treatment of various diseases; however, when used to treat osteoporosis, it has minor effect as compared with that of the currently available drugs. This study aimed to find a new compound that would have a better anti-osteoporotic effect than that of berberine. Based on structure and activity relationship study, we identified compound 2c, a berberine derivative, to be the most potent compound to affect osteoblast differentiation. Compound 2c was more effective than berberine and exhibited no toxicity within its effective concentration. Compound 2c increased, in a dose-dependent manner, ALP activity during osteoblast differentiation and enhanced the mRNA expression of osteogenic factors including ALP, Runx2, and Osterix. Furthermore, compound 2c increased the transcriptional activity induced by BMP4 on the ALP and BSP promoter. Taken together, compound 2c shows promise as a therapeutic agent for osteoporosis by promoting osteoblast differentiation.
Topics: Animals; Berberine; Cell Differentiation; Cell Line; Gene Expression Regulation; Mice; Osteoblasts; Osteogenesis
PubMed: 32446353
DOI: 10.1016/j.bbrc.2020.03.145 -
Bioorganic & Medicinal Chemistry Jul 2018We recently developed an amphiphilic peptide, E1Y9 (Ac-E-YEYKYEYKY-NH), that self-assembles into nanofibers and forms a hydrogel in the presence of Ca ion. Four...
We recently developed an amphiphilic peptide, E1Y9 (Ac-E-YEYKYEYKY-NH), that self-assembles into nanofibers and forms a hydrogel in the presence of Ca ion. Four E1Y9-derivatives (E1Y9-ALK, E1Y9-DGR, E1Y9-PRG and E1Y9-RGD) were designed as conjugates of E1Y9 with bioactive peptide sequences named as ALK (ALKRQGRTLYGF), DGR (DGRDSVAYG), PRG (PRGDSGYRGDS) and RGD (RGDS), respectively, and stimulated osteoblast cells growth as well as differentiation. In this study, E1Y9/E1Y9-derivative mixed hydrogels were constructed to serve as scaffolds for osteoblastic differentiation of MC3T3-E1 cells. E1Y9 and E1Y9-derivatives co-assembled into networked nanofibers and formed hydrogels in response to Ca ion. The pre-osteoblast cell line MC3T3-E1 was cultured and differentiated on mixed hydrogels. An E1Y9/E1Y9-ALK mixed hydrogel exhibited the highest cell proliferation and differentiation activity among the peptide hydrogels. The peptide sequence ALK promoted expression of RUNX2 and osteopontin, a key transcription factor and bone tissue matrix protein, respectively, during the differentiation stage. During the later stage, localization of RUNX2 and osteopontin was regulated in the cytosol and extracellularly, respectively, indicating that the E1Y9/E1Y9-ALK mixed hydrogel controlled the differentiation of MC3T3-E1 cells. Thus, the E1Y9/E1Y9-ALK mixed hydrogel developed in this study showed potential for the culture and regulation of differentiation of osteoblast cells for bone regeneration.
Topics: Amino Acid Sequence; Animals; Calcium; Cell Adhesion; Cell Differentiation; Cell Line; Cell Proliferation; Core Binding Factor Alpha 1 Subunit; Hydrogels; Mice; Osteoblasts; Osteopontin; Peptides
PubMed: 29699909
DOI: 10.1016/j.bmc.2018.04.039 -
Current Osteoporosis Reports Feb 2016Neural pathways are now a well-appreciated factor in the regulatory milieu controlling the maintenance of bone mass. A number of neural pathways from the brain to bone... (Review)
Review
Neural pathways are now a well-appreciated factor in the regulatory milieu controlling the maintenance of bone mass. A number of neural pathways from the brain to bone have been identified. These pathways often involve elements of the energy homeostatic apparatus, indicating links between the regulation of bone metabolism and energy balance. Neuropeptide Y is one such factor that co-regulates these two processes. Initial studies outlined the skeletal actions of NPY from within the brain and the interactions with energy homeostatic processes. However, in recent years, an appreciation for the actions of NPY within bone cells has expanded. Cells of the osteoblastic lineage express both NPY ligand and a cognate receptor NPY, Y1R. Murine studies have demonstrated that both ligand and receptor actively control bone mass and osteoblast activity and interact with mechanical signals to integrate with the local loading environment. Local NPY signalling regulates osteoprogenitor production and differentiation, to cover the entire osteoblastic lineage. In addition, several recent studies have demonstrated extra-skeletal actions of osteoblastic NPY signalling, to regulate energy expenditure and with it adiposity, and in a separate study, to control release of a factor-controlling beta cell mass and insulin production/release and with it glucose tolerance. Thus, osteoblastic neuropeptide production and signalling illustrates the rapidly widening sphere of influence of skeletal tissue, and suggests a far more complex and interconnected physiology then is currently appreciated.
Topics: Animals; Bone Density; Bone and Bones; Cell Proliferation; Energy Metabolism; Homeostasis; Hypothalamus; Mice; Neuropeptide Y; Osteoblasts; Osteocytes; Receptors, Neuropeptide Y
PubMed: 26872458
DOI: 10.1007/s11914-016-0300-9 -
Molecular Medicine Reports Dec 2016Mammalian stanniocalcin 2 (STC2) is a glycoprotein hormone with multiple functions. The present study determined the importance of STC2 in osteoblast differentiation. It...
Mammalian stanniocalcin 2 (STC2) is a glycoprotein hormone with multiple functions. The present study determined the importance of STC2 in osteoblast differentiation. It was revealed that the expression of STC2 was increased during the differentiation of MC3T3-E1 cells to osteoblasts and that knockdown of STC2 reduced osteoblast differentiation and mineralization, whereas STC2 overexpression increased differentiation and mineralization. Knockdown of STC2 downregulated the expression of osteoblast-associated genes, including runt‑related transcription factor 2, collagen type I α 1 chain, osterix and osteocalcin. Overexpression of STC2 upregulated the expression of these osteoblastic genes. In addition, overexpression of STC2 enhanced the phosphorylation of extracellular signal‑regulated kinase 1/2 (ERK1/2), whereas inhibition of ERK phosphorylation reduced osteoblast differentiation of MC3T3‑E1 cells overexpressing STC2. These findings indicated that STC2 may promote osteoblast differentiation and mineralization by regulating ERK activation.
Topics: Animals; Calcification, Physiologic; Cell Differentiation; Cell Line; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Gene Knockdown Techniques; Glycoproteins; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Mice; Organ Specificity; Osteoblasts; Phosphorylation
PubMed: 27878259
DOI: 10.3892/mmr.2016.5951 -
International Journal of Molecular... Dec 2022The alizarin red S assay is considered the gold standard for quantification of osteoblast mineralization and is thus widely used among scientists. However, there are...
The alizarin red S assay is considered the gold standard for quantification of osteoblast mineralization and is thus widely used among scientists. However, there are several restrictions to this method, e.g., moderate sensitivity makes it difficult to uncover slight but significant effects of potentially clinically relevant substances. Therefore, an adaptation of the staining method is appropriate and might be obtained by increasing the mineralization ability of osteoblasts. In this study, cell culture experiments with human (SaOs-2) and murine (MC3T3-E1) osteoblasts were performed under the addition of increasing concentrations of calcium chloride (1, 2.5, 5, and 10 mM) or calcitonin (1, 2.5, 5, and 10 nM). After three or four weeks, the mineralization matrix was stained with alizarin red S and the concentration was quantified photometrically. Only calcium chloride was able to significantly increase mineralization, and therefore enhanced the sensitivity of the alizarin red S staining in a dose-dependent manner in both osteoblastic cell lines as well as independent of the cell culture well surface area. This cost- and time-efficient optimization enables a more sensitive analysis of potentially clinically relevant substances in future bone research.
Topics: Animals; Mice; Humans; Cell Differentiation; Calcification, Physiologic; Calcium Chloride; Osteoblasts
PubMed: 36614166
DOI: 10.3390/ijms24010723 -
The Journal of Steroid Biochemistry and... Oct 2014Maintenance of an adequate vitamin D status, as indicated by the level of circulating 25-hydroxyvitamin D (25(OH)D), is associated with higher bone mass and decreased... (Review)
Review
Maintenance of an adequate vitamin D status, as indicated by the level of circulating 25-hydroxyvitamin D (25(OH)D), is associated with higher bone mass and decreased risk of fracture. However, the molecular actions of vitamin D hormone (1,25(OH)2D3) in bone are complex, and include stimulation of osteoclastogenesis via RANK-ligand up-regulation, as well as the inhibition of mineralisation. We hypothesise that these divergent data may be reconciled by autocrine actions of 1,25(OH)2D3 which effect skeletal maintenance, as opposed to endocrine 1,25(OH)2D3 which acts to maintain serum calcium homeostasis. We have previously described local metabolism of 1,25(OH)2D3 within osteoblasts, with effects on gene expression and cell function. The aim of the current study was to investigate potential autocrine actions of 1,25(OH)2D3 within cells that exhibit osteocyte-like properties. Late osteoblastic MLO-A5 cells were cultured in the presence of 25(OH)D for 9 days with gene expression analysed pre- and post-mineralisation. Gene expression analysis revealed maturation within this time frame to an osteocyte-like stage, evidenced by increased Dmp1 and Phex mRNA expression. Expression of Cyp27b1 in 25(OH)D treated MLO-A5 cells was associated with elevated media levels of 1,25(OH)2D3 (p<0.05), induction of Cyp24a1 (p<0.001) and elevated ratios of Opg:Rankl mRNA (p<0.01). Chronic 25(OH)D exposure also increased osteocalcin mRNA in MLO-A5 cells, which contrasted with the dose-dependent inhibition of osteocalcin mRNA observed with acute treatment in MLO-Y4 cells (p<0.01). Treatment of MLO-Y4 cells with 25(OH)D also inhibited Phex mRNA expression (p<0.05), whilst Enpp1 gene expression was induced (p<0.01). Overall, the current study demonstrates that osteocyte-like cells convert physiological levels of 25(OH)D to 1,25(OH)2D3, with changes in gene expression that are consistent with increased osteocyte maturation. Although the physiological role of local metabolism of 1,25(OH)2D3 within osteocytes requires further investigation, the abundance and diverse functions of this cell type within bone underscore its potential importance. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.
Topics: Animals; Calcitriol; Cell Differentiation; Humans; Osteoblasts; Osteocytes; Vitamins
PubMed: 24125735
DOI: 10.1016/j.jsbmb.2013.10.003