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The Effect of Exogenous Zinc Concentration on the Responsiveness of MC3T3-E1 Pre-Osteoblasts to Surface Microtopography: Part I (Migration).

ABSTRACT: Initial cell-surface interactions are guided by the material properties of substrate topography. To examine if these interactions are also modulated by the presence of zinc, we seeded murine pre-osteoblasts (MC3T3-E1, subclone 4) on micropatterned polydimethylsiloxane (PDMS) containing wide (20 µm width, 30 µm pitch, 2 µm height) or narrow (2 µm width, 10 µm pitch, 2 µm height) ridges, with flat PDMS and tissue culture polystyrene (TC) as controls. Zinc concentration was adjusted to mimic deficient (0.23 µM), serum-level (3.6 µM), and zinc-rich (50 µM) conditions. Significant differences were observed in regard to cell morphology, motility, and contact guidance. We found that cells exhibited distinct anisotropic migration on the wide PDMS patterns under either zinc-deprived (0.23 µM) or serum-level zinc conditions (3.6 µM). However, this effect was absent in a zinc-rich environment (50 µM). These results suggest that the contact guidance of pre-osteoblasts may be partly influenced by trace metals in the microenvironment of the extracellular matrix.

SUBMITTER: Dorst K

PROVIDER: S-EPMC5452741 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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The Effect of Exogenous Zinc Concentration on the Responsiveness of MC3T3-E1 Pre-Osteoblasts to Surface Microtopography: Part I (Migration).

Dorst Kathryn K Rammelkamp Derek D Hadjiargyrou Michael M Gersappe Dilip D Meng Yizhi Y

Materials (Basel, Switzerland) 20131127 12

Initial cell-surface interactions are guided by the material properties of substrate topography. To examine if these interactions are also modulated by the presence of zinc, we seeded murine pre-osteoblasts (MC3T3-E1, subclone 4) on micropatterned polydimethylsiloxane (PDMS) containing wide (20 µm width, 30 µm pitch, 2 µm height) or narrow (2 µm width, 10 µm pitch, 2 µm height) ridges, with flat PDMS and tissue culture polystyrene (TC) as controls. Zinc concentration was adjusted to mimic defici ...[more]

PMID: 28788406

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Project description:BackgroundWe have previously reported that repeated treatment of human periodontal ligament cells and murine pre-osteoblast MC3T3-E1 cells with transforming growth factor-beta 1 (TGF-β1) inhibited their osteoblastic differentiation because of decreased insulin-like growth factor-1 (IGF-1) secretion. We also found that IGF-1/PI3K signaling plays an important role in osteoblast differentiation induced by TGF-β1 treatment; however, the downstream signaling controlling this remains unknown. The aim of this current study is to investigate whether Akt activation is required for osteoblast differentiation.Methodology/principal findingsMC3T3-E1 cells were cultured in osteoblast differentiation medium (OBM) with or without 0.1 ng/mL TGF-β1. OBM containing TGF-β1 was changed every 12 h to provide repeated TGF-β1 administration. MC3T3-E1 cells were infected with retroviral vectors expressing constitutively active (CA) or dominant-negative (DN)-Akt. Alkaline phosphatase (ALP) activity and osteoblastic marker mRNA levels were substantially decreased by repeated TGF-β1 treatment compared with a single TGF-β1 treatment. However, expression of CA-Akt restored ALP activity following TGF-β1 treatment. Surprisingly, ALP activity increased following multiple TGF-β1 treatments as the number of administrations of TGF-β1 increased. Activation of Akt significantly enhanced expression of osteocalcin, but TGF-β1 treatment inhibited this. Mineralization of MC3T3-E1 cells was markedly enhanced by CA-Akt expression under all medium conditions. Exogenous IGF-1 restored the down-regulation of osteoblast-related gene expression by repeated TGF-β1 administration. However, in cells expressing DN-Akt, these levels remained inhibited regardless of IGF-1 treatment. These findings indicate that Akt activation is required for the early phase of osteoblast differentiation of MC3T3-E1 cells induced by TGF-β1. However, Akt activation is insufficient to reverse the inhibitory effects of TGF-β1 in the late stages of osteoblast differentiation.ConclusionsTGF-β1 could be an inducer or an inhibitor of osteoblastic differentiation of MC3T3-E1 cells depending on the state of Akt phosphorylation. Our results indicate that Akt is the molecular switch for TGF-β1-induced osteoblastic differentiation of MC3T3-E1 cells.

Project description:BackgroundIn mouse, it was discovered that resveratrol (Res) enhanced osteoporosis (OP) by boosting osteogenesis. Besides, Res can also have an impact on MC3T3-E1 cells, which are crucial for the control of osteogenesis and thus increase osteogenesis. Although some articles have discovered that Res enhanced autophagy to promote the value-added differentiation of MC3T3, it is unclear exactly how this affects the process of osteogenesis in mouse. Therefore, we will show that Res encourages MC3T3-E1 proliferation and differentiation in mouse pre-osteoblasts and further investigate the autophagy-related mechanism for this impact.Methods(1) MC3T3-E1 cells were separated into blank control group and various concentrations (0.01, 0.1, 1, 10, 100µmol/L) of group in order to determine the ideal Res concentration. In the Res group, Cell Counting Kit-8 (CCK-8) was used to measure the proliferation activity of pre-osteoblasts in mice in each group after resveratrol intervention. Alkaline Phosphatase (ALP) and alizarin red staining were used to gauge the degree of osteogenic differentiation, and RT-qPCR was used to measure the expression levels of Runx2 and OCN in the osteogenic differentiation ability of the cells. (2) In the experiment, four groups were set up: the control group, 3MA group, Res group, and Res + 3MA group. To examine cell mineralization, ALP and alizarin red staining were utilized. RT-qPCR and Western blot detection of cell autophagy activity levels and osteogenic differentiation capacity in each group following intervention.Results(1) Resveratrol might increase the number of mice pre-osteoblast, with the impact being most pronounced at 10µmol/L (P < 0.05). The nodules developed substantially more often than in the blank control group, and Runx2 and OCN expressions significantly increased (P < 0.05). (2) In contrast to the Res group, after 3MA purine blocked autophagy, the Res + 3MA group's alkaline phosphatase staining and the development of mineralized nodules were reduced. Runx2, OCN, LC3II / LC3I expression decreased, p62 expression increased (P < 0.05).ConclusionThe present study partially or indirectly demonstrated that Res may, through increased autophagy, induce osteogenic differentiation of MC3T3-E1 cells.

Dataset Information

The Effect of Exogenous Zinc Concentration on the Responsiveness of MC3T3-E1 Pre-Osteoblasts to Surface Microtopography: Part I (Migration).

Publications

The Effect of Exogenous Zinc Concentration on the Responsiveness of MC3T3-E1 Pre-Osteoblasts to Surface Microtopography: Part I (Migration).

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