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Zeitschrift Fur Naturforschung. C,... Jul 2019Osteoporosis is one of the most common bone diseases, occurring due to an imbalance between bone formation and bone resorption. The aim of this study was to investigate...
Osteoporosis is one of the most common bone diseases, occurring due to an imbalance between bone formation and bone resorption. The aim of this study was to investigate the effects of Ishige sinicola, a brown alga, on osteoblast differentiation through the activation of the bone morphogenetic protein 2 (BMP-2)/runt-related transcription factor 2 (Runx2) signalling pathway in MC3T3-E1 cells. A cell proliferation assay, alkaline phosphatase (ALP) activity assay, alizarin red staining, and expression analysis of osteoblastic genes were carried out to assess MC3T3-E1 cell proliferation and osteoblastic differentiation. We found that I. sinicola extract (ISE) increased cell proliferation in a dose-dependent manner. Ishige sinicola extract markedly promoted ALP activity and mineralization. Alizarin red S staining demonstrated that ISE treatment tended to increase extracellular matrix calcium accumulation. Moreover, ISE up-regulated the osteoprotegerin/receptor activator of nuclear factor κB ligand ratio. Ishige sinicola extract also increased the protein expression levels of type 1 collagen, ALP, osteocalcin, osterix, BMP-2, and Runx2. Therefore, ISE showed potential in stimulating osteoblastic bone formation, and it might be useful for the prevention and treatment of osteoporosis.
Topics: Animals; Bone Morphogenetic Protein 2; Calcium; Cell Differentiation; Cell Line; Cell Proliferation; Collagen Type I; Core Binding Factor Alpha 1 Subunit; Mice; NF-kappa B; Osteoblasts; Osteocalcin; Phaeophyceae; Plant Extracts; Signal Transduction; Sp7 Transcription Factor
PubMed: 31085751
DOI: 10.1515/znc-2018-0044 -
Molecular and Cellular Endocrinology Dec 2018The osteoporosis-resistant nature of skull bones implies inherent differences exist between their cellular responses and those of other osteoporosis-susceptible skeletal...
The osteoporosis-resistant nature of skull bones implies inherent differences exist between their cellular responses and those of other osteoporosis-susceptible skeletal sites. Phenotypic differences in calvarial and femoral osteoblastic responses to induction of osteogenesis, mechanical loading, estrogen, growth factor and cytokine stimulation were investigated. Primary rat calvarial and femoral adult male osteoblasts were cultured and osteoblastic mineralisation and maturation determined using Alizarin Red staining and expression of osteogenic marker genes assessed. Expression of known mechanically-responsive genes was compared between sites following loading of scaffold-seeded cells in a bioreactor. Cell proliferation and differentiation following growth factor and estrogen stimulation were also compared. Finally expression of estrogen receptors and associated genes during osteogenic differentiation were investigated. Calvarial osteoblasts exhibited delayed maturation (45d. vs 21d.) and produced less mineralised matrix than femoral osteoblasts when osteogenically induced. PDGF-BB and FGF2 both caused a selective increase in proliferation and decrease in osteoblastic differentiation of femoral osteoblasts. Mechanical stimulation resulted in the induction of the expression of Ccl2 and Anx2a selectively in femoral osteoblasts, but remained unchanged in calvarial cells. Estrogen receptor beta expression was selectively upregulated 2-fold in calvarial osteoblasts. Most interestingly, the estrogen responsive transcriptional repressor RERG was constitutively expressed at 1000-fold greater levels in calvarial compared with femoral osteoblasts. RERG expression in calvarial osteoblasts was down regulated during osteogenic induction whereas upregulation occurred in femoral osteoblasts. Bone cells of the skull are inherently different to those of the femur, and respond differentially to a range of stimuli. These site-specific differences may have important relevance in the development of strategies to tackle metabolic bone disorders.
Topics: Alkaline Phosphatase; Animals; Cell Proliferation; Co-Repressor Proteins; Estrogens; Femur; Gene Expression Regulation; Intercellular Signaling Peptides and Proteins; Male; Osteoblasts; Osteogenesis; Phenotype; Rats, Wistar; Receptors, Estrogen; Signal Transduction; Skull; Stress, Mechanical
PubMed: 29928929
DOI: 10.1016/j.mce.2018.06.011 -
Biological Trace Element Research Aug 2017Bone remodeling is a vital physiological process of healthy bone tissue in humans. Imbalances in this vital process lead to pathological conditions, including...
Bone remodeling is a vital physiological process of healthy bone tissue in humans. Imbalances in this vital process lead to pathological conditions, including periodontal diseases. In this study, we characterized the effects of micromolar levels of NaF on the proliferation and osteogenic differentiation of MC3T3-E1 osteoblastic cells. NaF significantly enhanced the proliferation, alkaline phosphatase (ALP) activity, and mineralization of MC3T3-E1 cells. Quantitative real-time PCR analysis revealed that the expression of mRNAs encoding runt-related transcription factor 2 (Runx2), Osterix, Osteopontin and Osteocalcin was up-regulated in NaF-treated MC3T3-E1 cells compared with untreated controls. Western blot analysis demonstrated that Runx2 and Osterix were inhibited by Runx2 siRNA but were re-activated by treatment with NaF. Furthermore, in vivo evidence indicated that NaF protects against Porphyromonas gingivalis-induced periodontal inflammation and alveolar bone loss in a P. gingivalis-challenged experimental periodontitis animal model. These data suggest that NaF promotes the osteoblastic differentiation of MC3T3-E1 cells through the Runx2/Osterix pathway and may be effective for the treatment of bone-related disorders.
Topics: Animals; Antigens, Differentiation; Cell Differentiation; Cell Line; Core Binding Factor Alpha 1 Subunit; Mice; Osteoblasts; Signal Transduction; Sodium Fluoride
PubMed: 28083749
DOI: 10.1007/s12011-017-0930-5 -
Cells, Tissues, Organs 2009The progressive ankylosis gene (ank) is a transmembrane protein that transports intracellular pyrophosphate to the extracellular milieu. Human mutations of ank lead to...
The progressive ankylosis gene (ank) is a transmembrane protein that transports intracellular pyrophosphate to the extracellular milieu. Human mutations of ank lead to craniometaphyseal dysplasia, a disease which is characterized by the overgrowth of craniofacial bones and osteopenia in long bones, suggesting that ANK plays a regulatory role in osteoblast differentiation. To determine the role of ANK in osteoblast differentiation, we suppressed ANK expression in the osteoblastic MC3T3-E1 cell line using siRNA and determined the expression of osteoblastic marker genes and the transcription factors osterix and runx2. In addition, we determined the osteoblastic differentiation of bone marrow stromal cells isolated from the bone marrow of ank/ank mice, which express a truncated, nonfunctional ANK protein, or wild-type littermates. Suppression of ANK expression in MC3T3-E1 cells led to a decrease in bone marker gene expression, including alkaline phosphatase, bone sialoprotein, osteocalcin and type I collagen. In addition, osterix gene expression was decreased in ANK expression-suppressed MC3T3 cells, whereas runx2 expression was increased. Bone marrow stromal cells isolated from ank/ank mice cultured in the presence of ascorbate-2-phosphate for up to 35 days showed markedly reduced mineralization compared to the mineralization of bone marrow stromal cells isolated from wild-type littermates. In conclusion, these findings suggest that ANK is a positive regulator of differentiation events towards a mature osteoblastic phenotype.
Topics: Alkaline Phosphatase; Animals; Bone Marrow Cells; Cell Differentiation; Cell Line; Formates; Membrane Proteins; Mice; Osteoblasts; Phosphate Transport Proteins; Stromal Cells
PubMed: 18728347
DOI: 10.1159/000151725 -
Developmental Biology Apr 2008Our laboratory and others have shown that overexpression of Dlx5 stimulates osteoblast differentiation. Dlx5(-/-)/Dlx6(-/-) mice have more severe craniofacial and limb...
Our laboratory and others have shown that overexpression of Dlx5 stimulates osteoblast differentiation. Dlx5(-/-)/Dlx6(-/-) mice have more severe craniofacial and limb defects than Dlx5(-/-), some of which are potentially due to defects in osteoblast maturation. We wished to investigate the degree to which other Dlx genes compensate for the lack of Dlx5, thus allowing normal development of the majority of skeletal elements in Dlx5(-/-) mice. Dlx gene expression in cells from different stages of the osteoblast lineage isolated by FACS sorting showed that Dlx2, Dlx5 and Dlx6 are expressed most strongly in less mature osteoblasts, whereas Dlx3 is very highly expressed in differentiated osteoblasts and osteocytes. In situ hybridization and Northern blot analysis demonstrated the presence of endogenous Dlx3 mRNA within osteoblasts and osteocytes. Dlx3 strongly upregulates osteoblastic markers with a potency comparable to Dlx5. Cloned chick or mouse Dlx6 showed stimulatory effects on osteoblast differentiation. Our results suggest that Dlx2 and Dlx6 have the potential to stimulate osteoblastic differentiation and may compensate for the absence of Dlx5 to produce relatively normal osteoblastic differentiation in Dlx5 knockout mice, while Dlx3 may play a distinct role in late stage osteoblast differentiation and osteocyte function.
Topics: Animals; Animals, Newborn; Bone Marrow Cells; Cell Differentiation; Cloning, Molecular; Gene Expression Regulation; Homeodomain Proteins; Mice; Mice, Inbred Strains; Mice, Transgenic; Osteoblasts; Osteocytes; Polymerase Chain Reaction; RNA, Messenger; Stromal Cells; Transcription Factors
PubMed: 18280462
DOI: 10.1016/j.ydbio.2008.01.001 -
Indian Journal of Biochemistry &... Feb 2013R-spondin (Rspo)s proteins are a new group of Wnt/beta-catenin signaling agonists. These signaling molecules are known to be involved in the developmental stages of...
R-spondin (Rspo)s proteins are a new group of Wnt/beta-catenin signaling agonists. These signaling molecules are known to be involved in the developmental stages of skeletal system. Recent studies in various murine osteoblast models have proposed that Rspo 1 may interact with Wnt signaling pathway to induce differentiation in osteoblasts. Though findings in murine osteoblasts implicate a synergestic role of Rspo 1 with Wnt signaling, still no study has addressed the similar role in more clinically applicable osteoblast models i.e., human cell lines or primary cells. Therefore, in the present study, we investigated the possible role of Rspo 1 during differentiation process of human in vitro osteoblast cell models like primary osteoblasts or human osteoprogenitor cell line hFOB 1.19 along with murine preosteoblast cell line MC3T3 E-1. Our results showed increase in Rspo 1 at transcript level during differentiating phase of human primary osteoblasts and human FOB 1.19 cells. We also found that Rspo 1 (100 ng/mL) acts additively with Wnt3a to activate Wnt signaling, as confirmed by luciferase activity after transfection of TOPFLASH construct to hFOB 1.19 cells. Similar additive role of Rspo 1 and Wnt3a was apparent in alkaline phosphatase (ALP) activity analysis of human primary cells. Moreover, a reduction in ALP activity was observed with knock-down of Rspo 1 by transfected shRNA in hFOB 1.19 cells. These results suggested the possibility of autocrine regulation by Rspo 1 on the osteogenic activities in human in vitro osteoblast models. Furthermore, these results were corroborated in MC3T3-E1, murine osteoblast cell model. Osteoblastic differentiation was induced by transfection of Rspo 1 which was confirmed by increased ALP staining and qRT-PCR analysis of osteogenic markers, such as Runx2 and osteocalcin. In conclusion, present study highlights the role of Rspo 1 in bone remodeling where it activates Wnt signaling to induce differentiation, as shown in human as well murine in vitro osteoblast cell models.
Topics: Animals; Cell Differentiation; Cell Line; Humans; Mice; Osteoblasts; Osteogenesis; Thrombospondins; Wnt Proteins; Wnt Signaling Pathway
PubMed: 23617070
DOI: No ID Found -
Journal of Bone and Mineral Metabolism Sep 2012This study investigated the effects of different frequencies of low intensity ultrasound on osteoblast migration using an in vitro scratch-wound healing assay. Mouse...
This study investigated the effects of different frequencies of low intensity ultrasound on osteoblast migration using an in vitro scratch-wound healing assay. Mouse calvarial-derived MC3T3-E1 osteoblasts in culture were exposed to continuous 45 kHz ultrasound (25 mW/cm(2)) or pulsed 1 MHz ultrasound (250 mW/cm(2)) for 30 min followed by 2 days' culture. Ultrasound treatment with either kHz or MHz output similarly and significantly increased cell numbers after 2 days in culture compared with untreated control cultures. In the scratch-wound healing assay the presence of the cell proliferation inhibitor mitomycin C (MMC) did not influence scratch-wound closure in control cultures indicating that cell migration was responsible for the in vitro wound healing. Application of ultrasound significantly stimulated wound closure. MMC did not affect kHz-stimulated in vitro wound healing; however, MMC reduced in part the scratch-wound closure rate in MHz-treated cultures suggesting that enhanced cell proliferation as well as migration was involved in the healing promoted by MHz ultrasound. In conclusion, both continuous kHz and pulsed MHz ultrasound promoted osteoblastic migration; however, subtle differences were apparent in the manner the different ultrasound regimens enhanced in vitro scratch-wound healing.
Topics: Animals; Cell Line; Cell Movement; Cell Proliferation; Cell Survival; Mice; Mitomycin; Osteoblasts; Ultrasonics; Ultrasonography; Wound Healing
PubMed: 22752127
DOI: 10.1007/s00774-012-0368-y -
ASGSB Bulletin : Publication of the... Oct 1995One physiologic consequence of extended periods of weightlessness is the rapid loss of bone mass associated with skeletal unloading. Conversely, mechanical loading has... (Review)
Review
One physiologic consequence of extended periods of weightlessness is the rapid loss of bone mass associated with skeletal unloading. Conversely, mechanical loading has been shown to increase bone formation and stimulate osteoblastic function. The mechanisms underlying mechanotransduction, or how the osteoblast senses and converts biophysical stimuli into cellular responses has yet to be determined. For non-innervated mechanosensitive cells like the osteoblast, mechanotransduction can be divided into four distinct phases: 1) mechanocoupling, or the characteristics of the mechanical force applied to the osteoblast, 2) biochemical coupling, or the mechanism through which mechanical strain is transduced into a cellular biochemical signal, 3) transmission of signal from sensor to effector cell and 4) the effector cell response. This review examines the characteristics of the mechanical strain encountered by osteoblasts, possible biochemical coupling mechanisms, and how the osteoblast responds to mechanical strain. Differences in osteoblastic responses to mechanical strain are discussed in relation to the types of strain encountered and the possible transduction pathways involved.
Topics: Animals; Biomechanical Phenomena; Bone Demineralization, Pathologic; Bone and Bones; Integrins; Ion Channel Gating; Osteoblasts; Rats; Signal Transduction; Space Flight; Stress, Mechanical; Weightlessness
PubMed: 11538550
DOI: No ID Found -
Phytotherapy Research : PTR Apr 2010Natural compounds with bone-forming (or anabolic) activity have been recently focused on in bone research. The present study investigated the effect of undecylenic acid...
Natural compounds with bone-forming (or anabolic) activity have been recently focused on in bone research. The present study investigated the effect of undecylenic acid (UA) on osteoblast differentiation in mouse osteoblastic MC3T3-E1 subclone 4 cells and primary mouse calvarial cells. Low concentrations of UA (up to 5 microM) exhibited no cytotoxicity and significantly increased the expression and activity of alkaline phosphatase (early differentiation marker of osteoblast) and calcium deposition with the induction of expression of the osteocalcin gene in both cells. Interestingly, at low concentration of UA, the induction of NF-kappaB p65 translocation into nucleus and the up-regulation of AP-1 and NFATc1 transcript levels were also observed, suggesting that the stimulatory effect of UA on osteoblast differentiation could be mediated through the activation of transcription factors. Additionally, although the patterns of UA-induced activation of MAP kinases (JNK and p38) were not completely consistent with the increase of both ALP activity and calcium deposition by UA, MAP kinases might be partially involved in the biological function of UA during the early and late stages of osteoblast differentiation.
Topics: Animals; Cell Differentiation; Cell Line; Mice; Mitogen-Activated Protein Kinases; Osteoblasts; Transcription Factors; Undecylenic Acids
PubMed: 19777559
DOI: 10.1002/ptr.2984 -
Endocrinology Oct 1986PTH stimulates osteoclastic bone resorption in vivo and in organ culture. We have previously found that if osteoclasts are disaggregated from bone and incubated on bone...
PTH stimulates osteoclastic bone resorption in vivo and in organ culture. We have previously found that if osteoclasts are disaggregated from bone and incubated on bone slices, PTH does not increase bone resorption, but does so if osteoblastic cells are added to the cultures. This suggests that PTH acts primarily on osteoblasts, which are induced by the presence of the hormone to stimulate osteoclastic bone resorption. In the present paper we describe investigations into the mechanism by which osteoblastic cells stimulate osteoclasts. We found that increased resorption could not be accounted for by changes in the bone substrate. Osteoblast-like cells (UMR106) incubated with PTH did, however, release a factor into the culture supernatant that stimulated osteoclastic bone resorption. This factor was stable for at least 7 days when stored at 4 C and survived freeze-thawing, but was inactivated by heating to 65 C for 30 min. Activity was lost entirely after dialysis using a Spectrapor membrane with a mol wt cut-off (MWCO) of 2000. The small size of the molecule was confirmed after ultrafiltration across Amicon filters YM2 and YC05. There was no loss of activity across YM2 (MWCO, 1000), but, in contrast, there was no stimulation in the conditioned medium after ultrafiltration across YC05 (MWCO, 500).
Topics: Animals; Bone Resorption; Cattle; Cell Line; Culture Media; Culture Techniques; Dialysis; Female; Hot Temperature; Osteoblasts; Osteoclasts; Osteosarcoma; Parathyroid Hormone; Rats; Rats, Inbred Strains; Ultrafiltration
PubMed: 3463505
DOI: 10.1210/endo-119-4-1654