Phytocomponent p-hydroxycinnamic acid stimulates mineralization in osteoblastic MC3T3-E1 cells

Phytocomponent p-hydroxycinnamic acid (HCA) has been shown to have stimulatory effects on bone calcification and inhibitory effects on bone resorption in rat femoral tissues in vitro. Whether HCA has a stimulatory effect on mineralization in osteoblastic cells is unknown. This study was undertaken to determine the effect of HCA on mineralization in osteoblastic MC3T3-E1 cells in vitro. Cells were cultured for 72 h in a minimum essential medium (alpha-MEM) containing 10% fetal bovine serum (FBS), and the cells with subconfluency were changed to a medium containing either vehicle or HCA (10(-7)-10(-5) M) without FBS. Culture with HCA (10(-7)-10(-5) M) did not have a significant effect on cell proliferation and cell death. Deoxyribonucleic acid (DNA) content in osteoblastic cells was significantly increased after culture with HCA (10(-6) or 10(-5) M) for 48 or 72 h. Alkaline phosphatase activity in osteoblastic cells was significantly increased after culture with HCA (10(-7)-10(-5) M) for 24, 48, or 72 h. The results with Alizarin red staining for calcium showed that mineralization was significantly stimulated after culture with HCA (10(-8)-10(-5) M) for 7, 14, or 21 days. This study demonstrates that HCA has stimulatory effects on mineralization in osteoblastic MC3T3-E1 cells.     

Sunflower seed extract enhances the differentiation of osteoblastic MC3T3-E1 cells

Osteoporosis is recognised as one of the major hormonal deficiency diseases, especially in menopausal women and the elderly. The present study investigated whether treatment with sunflower (Helianthus annuus L.) seed extract (SSE) may affect the function of MC3T3-E1 osteogenic cells. In order to determine the growth and differentiation of osteoblast, MTT (3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide) assay, alkaline phosphatase (ALP) activity, collagen synthesis and osteocalcin secretion were performed. Also, the production of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and nitric oxide (NO) in osteoblastic MC3T3-E1 cells was measured. SSE significantly (p<0.05) increased cell growth, ALP activity, collagen content and osteocalcin secretion compared with control. The effect of SSE (50 µg/ml) in increasing cell growth, ALP activity and collagen content was prevented by the presence of 10^?6 M cycloheximide and 10^?6 M ICI182780, suggesting that SSE's effect results from a newly synthesised protein component and might be partly involved in oestrogen action. Treatment with SSE (10 and 50 µg/ml) decreased the 5 µg/ml lipopolysaccharide-induced production of TNF-α, IL-6 and NO in osteoblasts. Our data indicate that the enhancement of osteoblast function by sunflower seed may result in the prevention for osteoporosis and inflammatory bone diseases.     

Effect of centrifugal force on cellular activity of osteoblastic MC3T3-E1 cells in vitro

The effects of centrifugal force on growth and differentiation of osteoblastic cells cultured in alpha-MEM containing 1% Fetal bovine serum were investigated by assays of DNA synthesis, alkaline phosphatase activity and osteocalcin-production in osteoblastic MC3T3-E1 cells. Centrifugation of the cells in low concentrations (1%) of fetal bovine serum caused a 1.9-fold increase of [3H]thymidine incorporation on day 3 from the start of centrifugation, and gradually decreased with culture up to day 9. Alkaline phosphatase activity was not affected by centrifugal force until day 5, and increased rapidly after day 7. Stimulation of DNA synthesis by centrifugation was abolished in the presence of H-7, an inhibitor of protein kinase C. These results suggest that centrifugal force stimulates the proliferation of osteoblastic cells through an autocrine secretion of some diffusable growth- promoting activity. Additional centrifugation of the cells also slightly stimulated alkaline phosphatase activity, although this did not directly influence the cell's osteocalcin-production activity.     

Suppressive effect of genistein on rat bone osteoclasts: involvement of protein kinase inhibition and protein tyrosine phosphatase activation

The suppressive effect of genistein on osteoclast-like multinucleated cells from rat femoral tissues was investigated. The bone cells isolated from rat femoral tissues were cultured for 48 h in an alpha-minimal essential medium (5% fetal bovine serum) containing either vehicle or genistein (10(-7)-10(-5) M). Osteoclasts were estimated by staining for tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts. The presence of genistein caused a significant decrease in the number of osteoclasts. Such a decrease was also seen in the presence of calcium choride (10(-5) M). Magnesium chloride (10(-5)-10(-3) M), a blocker of Ca2+ channels, had no effect on the number of osteoclasts. The effect of genistein (10(-5) M) or calcium (10(-3) M) in decreasing osteoclasts was significantly prevented by the presence of magnesium (10-3 M). Vanadate (10(-6)-10(-4) M), an inhibitor of protein tyrosine phosphatase activity, did not have an effect on the number of osteoclasts. The genistein's effect was not altered by vanadate. When isolated osteoclasts were cultured for 24 h in the presence of genistein (10(-7)-10(-5) M), protein kinase activity in the 5500 g supernatant of homogenate of the cells was significantly decreased, while protein tyrosine phosphatase activity was significantly elevated. Such an effect was also seen by the addition of genistein (10(-7)-10(-5) in the enzyme reaction mixture in vitro. The present study suggests that the suppressive effect of genistein on rat bone osteoclasts is partly involved in the inhibition of protein kinase and the activation of protein tyrosine phosphatase in osteoclasts.     

Enhancing effect of poly(L-lactide) on the differentiation of mouse osteoblast-like MC3T3-E1 cells

Poly(L-lactide) (PLLA) has bioabsorbability and biocompatibility, and it is used as biodegradable screws, pins and plates for internal bone fixation. The purpose of this study was to clarify the effects of low molecular weight (Mw) PLLA on the proliferation and differentiation of mouse osteoblast-like MC3T3-E1 cells. MC3T3-E1 cells were cultured with the concentration of 5-50 microg/ml of PLLA with weight average Mw of 5000 (PLLA-5k) and 10,000 (PLLA-10k) for 2 weeks using the micromass culture. Both PLLAs did not affect the proliferation of MC3T3-E1 cells. However, the calcifications of MC3T3-E1 cells were stimulated with increasing the concentration of the PLLAs. Then PLLA-5k increased the calcification of MC3T3-E1 cells more than PLLA-10k. Additionally, both PLLAs increased the alkaline phosphatase (ALP) activity and calcium content of MC3T3-E1 cells up to the similar level to the calcification. These results indicated that low Mw PLLA enhanced the differentiation of MC3T3-E1 cells with no effect on the proliferation. Moreover, it was suggested that the increase of the ALP activity was a key step to stimulate the calcification of MC3T3-E1 cells. The osteoconductivity of implanted PLLA would be based on the enhancing effect of low Mw PLLA on the differentiation of the osteoblasts.     

Hormonal regulation of regucalcin mRNA expression in osteoblastic MC3T3-E1 cells

The effect of various hormones on regucalcin mRNA expression in osteoblastic MC3T3-E1 cells in vitro was investigated. Cells with subconfluency were cultured for 24 or 48 h in a medium containing either vehicle or various hormones without fetal bovine serum. Regucalcin mRNA expression was significantly increased after culture with parathyroid hormone (synthetic human PTH; 10(-7) M), insulin-like growth factor-I (IGF-I; 10(-8) M), or 17beta-estradiol (10(-10) or 10(-9) M) for 48 h. Culture with 1,25-dihydroxyvitamin D3 (10(-7) M) for 48 h caused a significant decrease in regucalcin mRNA expression. Regucalcin mRNA expression was significantly decreased after culture with tumor necrosis factor-alpha (1 or 10 ng/ml of medium) for 24 or 48 h. The effect of PTH or IGF-I in increasing regucalcin mRNA expression was not seen in the presence of staurosporine (10(-8) M), an inhibitor of protein kinase C, or PD98059 (10(-7) M), an inhibitor of mitosis-activated protein kinase (MAP kinase), respectively, suggesting that regucalcin mRNA expression is enhanced through intracellular signaling factors. This study demonstrated that regucalcin mRNA expression in osteoblastic MC3T3-E1 cells is regulated by various hormones.     

Effect of hydroxyapatite sol on cell proliferation and alkaline phosphatase activity of osteoblastic MC3T3-E1 cells

The effect of hydroxyapatite sol (HAm) which is composed of unheating hydroxyapatite microcrystal particle on cell proliferation and alkaline phophatase activity in osteoblastic MC3T3-E1 cells, was examined in the presence or absence of fetal calfe serum (FCS) in the medium. In the absence of FCS, HAm inhibited the cell proliferation of MC3T3-E1 cells in a dose-dependent manner, but not in the presence of FCS. Inhibitory effect by HAm was observed only in directly contact with the cells. Similar inhibition effects by HAm were also observed on ALP activity of the cells. However, inhibitory effect by HAm on ALP extracted from the cells was not observed. After HAm treatment, ALP activity in the medium was increased. HAm pretreated with FCS did not inhibit the ALP activity of the cells at all. These results suggest that serum may play an important role in the biocompatibility of HAm and osteoblastic cells.     

miR-132-3p过表达对MC3T3-E1细胞增殖分化的影响

文题释义： miRNA：是一类小的非编码RNA,长度约为22个核苷酸,其主要通过结合靶标mRNA的3'UTR区诱导靶标mRNA降解或抑制其翻译,从而在转录及转录后水平调控相关基因的表达。miRNA在细胞增殖、分化及凋亡等多种生物学活动过程中起着非常重要的调控作用,并且发现它是调控骨组织细胞增殖和分化过程的重要因子之一。 MC3T3-E1细胞：是新生C57BL/6小鼠颅顶骨中分离培养所建立的一株成骨细胞株,它能够展现骨组织中成骨细胞的各个发育阶段和各种生物学特性。作为研究成骨细胞增殖和分化的理想模型,被广泛应用于国内外各种骨组织工程学研究。 背景：机械牵引力能够影响MC3T3-E1细胞的增殖分化过程,并引起细胞内miR-132-3p的差异表达。然而,牵引力是否通过调控miR-132-3p的表达来影响成骨细胞增殖分化仍需进一步研究。 目的：明确12%牵引力作用下MC3T3-E1细胞中成骨分化标志因子及miR-132-3p表达变化,并进一步探讨miR-132-3p对细胞增殖分化的影响。 方法：MC3T3-E1细胞分别加载0%,12%牵张应力,检测应力加载后碱性磷酸酶活性、骨钙蛋白及miR-132-3p mRNA的表达水平;细胞内瞬时转染miR-132-3p模拟物及其阴性对照,qRT-PCR检测转染后碱性磷酸酶、骨钙蛋白、Runt标志转录因子2 mRNA的表达,CCK-8法检测miR-132-3p对细胞增殖能力的影响。 结果与结论：①12%牵张应力作用下,MC3T3-E1细胞中碱性磷酸酶活性、骨钙蛋白mRNA表达水平下调(P < 0.01),miR-132-3p表达水平显著升高(P < 0.05);②细胞内转染miR-132-3p后,miR-132-3p模拟物组成骨分化标志因子碱性磷酸酶、骨钙蛋白、Runt标志转录因子2 mRNA表达水平显著降低(P < 0.05);③相比于阴性对照组,miR-132-3p 模拟物转染24,48,72 h后细胞增殖能力明显降低(P < 0.001),且在转染48 h后降低最明显;④结果说明12%周期性循环牵张应力能够通过过表达miR-132-3p负向调节MC3T3-E1细胞的增殖和成骨分化。 ORCID: 0000-0003-0696-3329(孙芬) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Stimulatory effect of menaquinone-7 on bone formation in elderly female rat femoral tissues in vitro: prevention of bone deterioration with aging

Menaquinone-7 (MK-7) is vitamin K2 which is a series of vitamins with multiisoprene units at the 3-position of the naphthoquinone. MK-7 has been shown to prevent bone loss in ovariectomized rats, an animal model for osteoporosis. This study was undertaken to determine whether MK-7 has a stimulatory effect on bone components of elderly female rats in vitro. The femoral-diaphyseal and -metaphyseal tissues obtained from young (4 weeks old) or elderly (50 weeks old) female rats were cultured for 48 h in a Dullbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. Calcium content, alkaline phosphatase activity and deoxyribonucleic acid (DNA) in the diaphyseal and metaphyseal tissues obtained from elderly rats were significantly decreased as compared with those of young rats, indicating that aging causes a deterioration of bone formation. The presence of MK-7 (10(-6) or 10(-5) M) caused a significant increase in biochemical components in the femoral-diaphyseal and -metaphyseal tissues obtained from elderly rat in vitro. The anabolic effect of MK-7 (10(-6) or 10(-5) M) on the femoral calcium content was significantly enhanced in the presence of phytoestrogen genistein (10(-6) or 10(-5) M), suggesting that the mode of action of MK-7 differ from that of genistein. The effect of MK-7 (10(-5) M) in increasing calcium content, alkaline phosphatase activity and DNA content in the diaphyseal and metaphyseal tissues was completely abolished in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis in vitro. These findings demonstrate that MK-7 has a stimulatory effect on bone formation in the femoral tissues of elderly female rats in vitro. MK-7 may have a preventive role for bone deterioration with aging.     

Pinacidil stimulates osteoblast function in osteoblastic MC3T3-E1 cells

Abstract

This study examined the effect of pinacidil, a nonselective adenosine triphosphate–sensitive potassium channel opener, on the function of osteoblastic MC3T3-E1 cells. Pinacidil caused a significant elevation of collagen synthesis, alkaline phosphatase activity, osteocalcin synthesis and mineralization in the cells (p?<?0.05). Pinacidil significantly decreased the production of osteoclast differentiation inducing factors such as TNF-α, IL-6 and receptor activator of nuclear factor-κB ligand in the presence of antimycin A, which inhibits mitochondrial electron transport. Moreover, pinacidil prevented antimycin A-induced reactive oxygen species and nitrotyrosine production. These results demonstrate that pinacidil may have positive effects on skeletal structure.     

Role of Sandhika: A Polyherbal Formulation on MC3T3-E1 Osteoblast-like Cells

Sandhika is a polyherbal formulation, (water soluble fraction of Commiphora mukul, Boswellia serrata, Semecarpus anacardium and Strychnos nux vomica), which has been in clinical use in India for last 20 years. Its modified formulation BHUx has shown specific inhibition of cyclooxygenase (COX)-2 and lipoxygenase (LOX)-15 and has prevented diet-induced atherosclerosis in rabbits. In order to explore the possibility of the use of Sandhika for the management of osteoporosis, we have examined its influence on MC3T3-E1 osteoblast-like cells in presence of lipopolysaccharide (1 μg/ml) in terms of calcium nodule formation and alkaline phosphatase activity. MC3T3-E1 osteoblast-like cells (80% confluence in 6-well plates) were treated with water extract of Sandhika, for 10 days, in the concentration range of 0.5 to 16 mg/ml final concentration, in presence of LPS. Media was changed on every third day and culture supernatant was collected after every change to assess the alkaline phosphatase activity and on the tenth day, cells were washed and stained with “Alizarin S” for visualization of calcium nodules by using Meta Morph software (Universal Imaging, Downingtown, PA). The results showed significant enhancement in calcium nodule formation in the dose dependent manner up to 2 mg/ml, followed by gradual decrease at higher concentrations. This change was accompanied with the increase in the alkaline phosphatase activity in these plates, indicating a potential anabolic effect of this polyherbal formulation on osteoblast-like cells under inflammatory conditions induced by LPS.     

Osteostatin improves the osteogenic activity of fibroblast growth factor-2 immobilized in Si-doped hydroxyapatite in osteoblastic cells

Si-doped hydroxyapatite (Si-HA) is a suitable ceramic for the controlled release of agents to improve bone repair. We recently showed that parathyroid hormone-related protein (PTHrP) (107-111) (osteostatin) has remarkable osteogenic features in various in vitro and in vivo systems. Fibroblast growth factor (FGF)-2 modulates osteoblastic function and induces angiogenesis, and can promote osteoblast adhesion and proliferation after immobilization on Si-HA. In the present study we examined whether osteostatin might improve the biological efficacy of FGF-2-coated Si-HA in osteoblastic MC3T3-E1 cells in vitro. We found that Si-HA/FGF-2 in the presence or absence of osteostatin (100 nM) similarly increased cell growth (by about 50%). However, addition of the latter peptide to Si-HA/FGF-2 significantly enhanced gene expression of Runx2, osteocalcin, vascular endothelial growth factor (VEGF) and the VEGF receptors 1 and 2, without significantly affecting that of FGF receptors in these cells. Moreover, secreted VEGF in the MC3T3-E1 cell conditioned medium, which induced the proliferation of pig endothelial-like cells, was also enhanced by these combined factors. The synergistic action of osteostatin and Si-HA/FGF-2 on the VEGF system was abrogated by a mitogen-activated protein kinase inhibitor (U0126) and by the calcium antagonist verapamil. This action was related to an enhancement of alkaline phosphatase activity and matrix mineralization in MC3T3-E1 cells, and also in primary human osteoblastic cells. These in vitro data show that osteostatin increases the osteogenic efficacy of a Si-HA/FGF-2 biomaterial by a mechanism involving mitogen-activated protein kinases and intracellular Ca(2+). These findings provide an attractive strategy for bone tissue engineering.     

Grooves affect primary bone marrow but not osteoblastic MC3T3-E1 cell cultures

To elucidate the influence of microtextures on bone cell performance, primary adult rat bone marrow cells (RBMC) and osteoblastic MC3T3-E1 cells were cultured on tissue culture pretreated plates to which grooves at different density were applied. RBMC cells were found to be significantly affected by grooves in the substratum in contrast to osteoblastic MC3T3-E1 cells, taking culture morphology, total cell number, cell mass, and cell activity (MTT-dehydrogenase), parameter for differentiation of osteoblast progenitor cells into (pre-)osteoblasts (alkalinephosphatase activity, ALP) and tartrate-resistant acid phosphatase (TRAP) activity as indices. TRAP is located in lysosomes and secretory granules mainly although not solely in osteoclasts. By applying grooves to and/or by chemical treatment of unpretreated pure polysterene plates it could be concluded that the effects on RBMC cells were evoked not only by the presence of grooves but also by the surface chemistry of the grooved and ungrooved surface areas.     

Effects of Bordetella bronchiseptica dermonecrotic toxin on the structure and function of osteoblastic clone MC3T3-e1 cells.

The effects of Bordetella bronchiseptica dermonecrotic toxin on the structure and function of a clonal osteoblastic cell line, MC3T3-E1, were investigated. The toxin induced a morphological change in the cells from a spindle shape to a spherical form with many blebs. The toxin-treated cells were viable and grew to form confluent cell layers composed of irregularly shaped cells and multinuclear cells. The toxin inhibited elevation of alkaline phosphatase activity in the cells in a dose-dependent manner at concentrations from 10 pg to 10 ng/ml. The accumulation of type I collagen in the cells was also reduced by the toxin. Since high alkaline phosphatase activity and accumulation of collagen are closely linked to differentiation of the cells into osteoblasts, it is considered likely that B. bronchiseptica dermonecrotic toxin impairs the ability of the cells to differentiate.