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Effects of Apatite Cement Containing Atelocollagen on Attachment to and Proliferation and Differentiation of MC3T3-E1 Osteoblastic Cells.

ABSTRACT: To improve the osteoconductivity of apatite cement (AC) for reconstruction of bone defects after oral maxillofacial surgery, we previously fabricated AC containing atelocollagen (AC(ate)). In the present study, we examined the initial attachment, proliferation and differentiation of mouse osteoblastic cells (MC3T3-E1 cells) on the surface of conventional AC (c-AC), AC(ate) and a plastic cell dish. The number of osteoblastic cells showing initial attachment to AC(ate) was greater than those attached to c-AC and similar to the number attached to the plastic cell wells. We also found that osteoblastic cells were well spread and increased their number on AC(ate) in comparison with c-AC and the wells without specimens, while the amount of procollagen type I carboxy-terminal peptide (PIPC) produced in osteoblastic cells after three days on AC(ate) was greater as compared to the others. There was no significant difference in regard to alkaline phosphatase (ALP) activity and osteocalcin production by osteoblastic cells among the three surface types after three and six days. However, after 12 days, ALP activity and the produced osteocalcin were greater with AC(ate). In conclusion, AC(ate) may be a useful material with high osteoconductivity for reconstruction of bone defects after oral maxillofacial surgery.

SUBMITTER: Takechi M

PROVIDER: S-EPMC5502976 | biostudies-other | 2016 Apr

REPOSITORIES: biostudies-other

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Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation.

Project description:AIM: To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects. METHODS: MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 was measured using real-time PCR. Western blot analysis was used to detect the expression of AMPK and eNOS proteins. RESULTS: Bezafibrate increased the viability and proliferation of MC3T3-E1 cells in a dose- and time-dependent manner. Bezafibrate (100 ?mol/L) significantly enhanced osteoblastic mineralization and expression of the differentiation markers ALP, collagen I and osteocalcin. Bezafibrate (100 ?mol/L) increased phosphorylation of AMPK and eNOS, which led to an increase of NO production by 4.08-fold, and upregulating BMP-2 and Runx-2 mRNA expression. These effects could be blocked by AMPK inhibitor compound C (5 ?mol/L), or the PPAR? inhibitor GSK0660 (0.5 ?mol/L), but not by the PPAR? inhibitor MK886 (10 ?mol/L). Furthermore, GSK0660, compound C, or N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mmol/L) could reverse the stimulatory effects of bezafibrate (100 ?mol/L) on osteoblast proliferation and differentiation, whereas MK886 only inhibited bezafibrate-induced osteoblast proliferation. CONCLUSION: Bezafibrate stimulates proliferation and differentiation of MC3T3-E1 cells, mainly via a PPAR?-dependent mechanism. The drug might be beneficial for osteoporosis by promoting bone formation.

| S-EPMC4002510 | biostudies-literature

Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells.

Project description:BackgroundAdiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells.ResultsAdiponectin receptor type 1 (AdipoR1) mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase) was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR), in the cells. AdipoR1 small interfering RNA (siRNA) transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01-0.5 mM) in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01-1.0 mug/ml) also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively.ConclusionTaken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions.

| S-EPMC2214728 | biostudies-literature

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Promoting Effect of Pinostrobin on the Proliferation, Differentiation, and Mineralization of Murine Pre-osteoblastic MC3T3-E1 Cells.

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| S-EPMC6151515 | biostudies-other

LGR5 enhances the osteoblastic differentiation of MC3T3-E1 cells through the Wnt/β-catenin pathway.

Project description:Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a Wnt-associated gene that contributes to cell proliferation and self-renewal in various organs. LGR5 is expressed in Ewing sarcoma, and LGR5-overexpressing mesenchymal stem cells promote fracture healing. However, the effects of LGR5 on osteoblastic differentiation remain unclear. The aim of the present study was to explore the function of LGR5 in osteoblastic differentiation. LGR5 was overexpressed or knocked down in the MC3T3-E1 pre-osteoblastic cell line via lentiviral transfection and its function in osteoblastic differentiation was investigated. The mRNA expression levels of the osteoblast differentiation markers alkaline phosphatase (ALP), osteocalcin and collagen type I a1 were determined, and ALP and Alizarin red staining were performed. In addition, the effects of LGR5 modulation on β-catenin and the expression of target genes in the Wnt pathway were investigated. The results revealed that the overexpression of LGR5 promoted osteoblastic differentiation. This was associated with enhancement of the stability of β-catenin and its levels in the cell nucleus, which enabled it to activate Wnt signaling. By contrast, the inhibition of LGR5 decreased the osteogenic capacity of MC3T3-E1 cells. These results indicate that LGR5 is a positive regulator of osteoblastic differentiation, whose effects are mediated through the Wnt/β-catenin signaling pathway. This suggests suggesting that the regulation of LGR5/Wnt/β-catenin signaling has potential as a therapy for osteoporosis.

| S-EPMC8237272 | biostudies-literature

Astragalin Promotes Osteoblastic Differentiation in MC3T3-E1 Cells and Bone Formation <i>in vivo</i>.

Project description:Astragalin (AG) is a biologically active flavonoid compound that can be extracted from a number of medicinal plants. However, the effects of AG on osteoblastic differentiation in mouse MC3T3-E1 cells and on bone formation in vivo have not been studied fully. In this study, we found that the activities of alkaline phosphatase (ALP) and mineralized nodules in MC3T3-E1 cells were both significantly increased after treatment with AG (5, 10, and 20 μM). Meanwhile, the mRNA and protein levels of osteoblastic marker genes in MC3T3-E1 cells after AG treatment were markedly increased compared with a control group. In addition, the levels of BMP-2, p-Smad1/5/9, and Runx2 were significantly elevated in AG-treated MC3T3-E1 cells. Moreover, we found that the protein levels of Erk1/2, p-Erk1/2, p38, p-p38, and p-JNK were also significantly increased in AG-treated MC3T3-E1 cells compared to those in the control group. Finally, in vivo experiments demonstrated that AG significantly promoted bone formation in an ovariectomized (OVX)-induced osteoporotic mouse model. This was evidenced by significant increases in the values of osteoblast-related parameters (BFR/BS, MAR, Ob.S/BS, and Ob.N/B.Pm) and bone histomorphometric parameters (BMD, BV/TV, Tb.Th, and Tb.N.) in OVX mice after AG treatment (5, 10, and 20 mg/kg). Collectively, these results demonstrated that AG may promote osteoblastic differentiation in MC3T3-E1 cells via the activation of the BMP and MAPK pathways and promote bone formation in vivo. These novel findings indicated that AG may be a useful bone anabolic agent for the prevention and treatment of osteoporosis.

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| S-EPMC6801562 | biostudies-literature

Co-culture with periodontal ligament stem cells enhanced osteoblastic differentiation of MC3T3-E1 cells and osteoclastic differentiation of RAW264.7 cells.

Project description:Periodontal ligament stem cells (PDLSCs) are characterized by having multipotential differentiation and immunoregulatory properties, which are the main mechanisms of PDLSCs-mediated periodontal regeneration. Periodontal or bone regeneration requires coordination of osteoblast and osteoclast, however, very little is known about the interactions between PDLSCs and osteoblast-like cells or osteoclast precursors. In this study, the indirect co-culture approach was introduced to preliminarily elucidate the effects of PDLSCs on differentiation of osteoblast-like cells and osteoclast precursors in vitro.Human PDLSCs were obtained from premolars extracted and their stemness was identified in terms of their colony-forming ability, proliferative capacity, cell surface epitopes and multi-lineage differentiation potentials. A noncontact co-culture system of PDLSCs and preosteoblastic cell line MC3T3-E1 or osteoclast precursor cell line RAW264.7 was established, and osteoblastic differentiation of MC3T3-E1 and osteoclastic differentiation of RAW264.7 were evaluated.PDLSCs exhibited features of mesenchymal stem cells. Further investigation through indirect co-culture system showed that PDLSCs enhanced ALP activity, expressions of ALP, Runx2, BSP, OPN mRNA and BSP, OPN proteins and mineralization matrix deposition in MC3T3-E1. Meanwhile, they improved maturation of osteoclasts and expressions of TRAP, CSTK, TRAF6 mRNA and TRAP, TRAF6 proteins in RAW264.7.PDLSCs stimulates osteoblastic differentiation of osteoblast precursors and osteoclastic differentiation of osteoclast precursors, at least partially, in a paracrine fasion.

| S-EPMC4713569 | biostudies-literature

ELP2 negatively regulates osteoblastic differentiation impaired by tumor necrosis factor α in MC3T3-E1 cells through STAT3 activation.

Project description:Tumor necrosis factor-α (TNF-α) is a pluripotent signaling molecule. The biological effect of TNF-α includes slowing down osteogenic differentiation, which can lead to bone dysplasia in long-term inflammatory microenvironments. Signal transducer and activator of transcription 3 (STAT3)-interacting protein 1 (StIP1, also known as elongator complex protein 2, ELP2) play a role in inhibiting TNF-α-induced osteoblast differentiation. In the present study, we investigated whether and how ELP2 activation mediates the effects of TNF-α on osteoblastic differentiation. Using in vitro cell cultures of preosteoblastic MC3T3-E1 cells, we found that TNF-α inhibited osteoblastic differentiation accompanied by an increase in ELP2 expression and STAT3 activation. Forced ELP2 expression inhibited osteogenic differentiation of MC3T3-E1 cells, with a decrease in the expression of osteoblast marker genes, alkaline phosphatase activity, and matrix mineralization capacity. In contrast, ELP2 silencing ameliorated osteogenic differentiation in MC3T3-E1 cells, even after TNF-α stimulation. The TNF-α-induced inhibitory effect on osteoblastic differentiation was therefore mediated by ELP2, which was associated with Janus kinase 2 (JAK2)/STAT3 activation. These results suggest that ELP2 is upregulated at the differentiation of MC3T3-E1 cells into osteoblasts and inhibits osteogenic differentiation in response to TNF-α through STAT3 activation.

| S-EPMC6618314 | biostudies-literature

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Project description:The primary pathological change in postmenopausal osteoporosis (PM-OP) is bone collagen loss caused by estrogen depletion. Osteoblasts synthesize type I collagen, which composes the organic matrix of bone. Although isopsoralen stimulates osteoblastic cell proliferation and differentiation, transforming growth factor (TGF)-β1 is an important cell signaling factor for stimulating collagen synthesis. To explore the association between isopsoralen and the synthesis of collagen in vitro, the molecular and biological association between isopsoralen and TGF-β signaling was examined. (CAGA) 12-luciferase-reporter gene was used to measure TGF-β1 signaling activity. Type I collagen was detected by semiquantitative reverse transcription polymerase chain reaction, and mothers against decapentaplegic homolog 7 (Smad7) protein expression levels were analyzed by western blotting. The expression of collagen in MC3T3-E1 cells stimulated with isopsoralen was significantly upregulated compared with the control groups (P<0.05). Conversely, isopsoralen significantly decreased Smad7 protein expression compared with the control groups (P<0.05). Moreover, it was observed that isopsoralen activates the TGF-β1 signaling pathway and ultimately promotes collagen synthesis through inhibition of Smad7 protein expression. Therefore, isopsoralen is a potential target for the treatment of PM-OP.

| S-EPMC5526118 | biostudies-other

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