Establishment of an Osteoid Preosteocyte-like Cell MLO-A5 That Spontaneously Mineralizes in Culture

Kato, Yoichi; Boskey, Adele L.; Spevak, Lyudmila; Dallas, Mark; Hori, Masayuki; Bonewald, Lynda F.

doi:10.1359/jbmr.2001.16.9.1622

Cited by 188 publications

(211 citation statements)

References 39 publications

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“…Cell layers were collected in 50 mM ammonium bicarbonate (pH 8.0), lyophilized, and analyzed as potassium bromide (KBr) pellets on a Thermo-Nicolet Spectrometer 4700 (Madison, WI). The data was collected under nitrogen purge, and the spectral baseline was corrected and analyzed using GRAMS/386 software (Galactic Industries, Salem, NH, USA) as previously described [31]. The mineral content was calculated based on the spectrally derived mineral-to-matrix ratio (the integrated areas of the phosphate absorbance (900-1200 cm −1 ) and protein amide I band (1585-1720cm −1 )).…”

Section: Fourier Transform Infrared (Ftir) Analysismentioning

confidence: 99%

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

Salasznyk

Klees

Williams

et al. 2007

Experimental Cell Research

263

225

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The intracellular signaling events controlling human mesenchymal stem cell (hMSC) differentiation into osteoblasts are not entirely understood. We recently demonstrated that contact with extracellular matrix (ECM) proteins is sufficient to induce osteogenic differentiation of hMSC through an ERKdependent pathway. We hypothesized that FAK signaling pathways provide a link between activation of ERK 1/2 by ECM, and stimulate subsequent phosphorylation of the Runx2/Cbfa-1 transcription factor that controls osteogenic gene expression. We plated hMSC on purified collagen I (COLL-I) and vitronectin (VN) in the presence or absence of FAK-specific siRNA, and assayed for phosphorylation of Runx2/Cbfa-1 as well as expression of established osteogenic differentiation markers (bone sialoprotein-2, osteocalcin, alkaline phosphatase, calcium deposition, and spectroscopically determined mineral:matrix ratio). We found that siRNA treatment reduced FAK mRNA levels by >40% and decreased ECM-mediated phosphorylation of FAK Y397 and ERK 1/2. Serine phosphorylation of Runx2/Cbfa-1 was significantly reduced after 8 days in treated cells. Finally, FAK inhibition blocked osterix transcriptional activity and the osteogenic differentiation of hMSC, as assessed by lowered expression of osteogenic genes (RT-PCR), decreased alkaline phosphatase activity, greatly reduced calcium deposition, and a lower mineral:matrix ratio after 28 days in culture. These results suggest that FAK signaling plays an important role in regulating ECMinduced osteogenic differentiation of hMSC.

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Section: Fourier Transform Infrared (Ftir) Analysismentioning

confidence: 99%

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

Salasznyk

Klees

Williams

et al. 2007

Experimental Cell Research

263

225

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“…The majority of these studies have utilized an osteoblastic cell line in culture media that typically contain ionic calcium levels between 1.8 mM [Minimum Essential Medium (MEM), aMEM, and Dulbecco's Modified Eagle Medium (DMEM)] and 2.6 mM [Biggers, Gwatkin and Judah b (BGJ/b) Medium, Fitton Jackson Modification], ionic phosphate levels of 0.9 mM (DMEM) to 1.6 mM (BGJ/b), and 10 mM b-glycerophosphate [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. The role of the bglycerophosphate is generally accepted to be as a source of phosphate for mineral growth, but it may affect mineral deposition in other ways as well [15].…”

mentioning

confidence: 99%

Mineralization of Decalcified Bone Occurs Under Cell Culture Conditions and Requires Bovine Serum But Not Cells

Hamlin

Price

2004

Calcif Tissue Int

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Abstract. The purpose of this study was to develop an in vitro model system for bone matrix mineralization in the absence of cells. For this model, we utilized EDTAdecalcified new-born rat tibias with the cartilaginous ends intact, allowing us to visually determine the specificity of mineralization within the bone. Our results show that supplementation of DMEM culture medium with 10mM b-glycerophosphate and 15% fetal bovine serum (FBS) results in non-physiological mineral percipitation in the tibia because of the generation of supraphysiological (5mM) levels of inorganic phosphate in the medium. The same medium supplemented only with inorganic phosphate to a final concentration of 2mM failed to mineralize a decalcified tibia matrix. However, additional supplementation of this medium with as little as 5% FBS resulted in mineralization of those regions of the type I collagen where mineral was found prior to decalcification, with no evidence for mineralization in the cartilage at the bone ends or in the periosteum. Analysis of the mineral by Fourier-transform infrared spectroscopy and powder X-ray diffraction shows that tibias that have been decalcified and then remineralized contain an apatitic mineral that is strikingly similar to the mineral in normal bone. Tendon, a type I collagen matrix not normally mineralized in vivo , also mineralizes when incubated in DMEM containing 2mM Pi and as little as 1.5% FBS, but not when incubated in DMEM without serum. These data indicate that serum contains a nucleator of type I collagen matrix mineralization, and that mineralization of type I collagen under cell culture conditions requires serum but not living cells.

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“…Therefore, we have used the MLO‐Y4 cell line which serve as a good osteocyte model as they respond to mechanical stimulation by releasing prostaglandin‐E 2 ,46 ATP,47 and nitric oxide,48 integral to osteocytes’ orchestration of adaptive bone remodeling. We also used MLO‐A5 cells with their higher expression of ALP and osteocalcin represent cells undergoing osteoblast–osteocyte transition which have the ability to mineralize the osteoid matrix they are embedded in 27. Used together, we believe that these models are a good representation of osteocyte behavior with which to test our hypothesis, although we appreciate that some of the key regulators of bone homeostasis such as SOST/sclerostin and FGF23 are not expressed by these cell lines 49, 50…”

Section: Discussionmentioning

confidence: 99%

Osteocyte physiology and response to fluid shear stress are impaired following exposure to cobalt and chromium: Implications for bone health following joint replacement

et al. 2016

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The effects of metal ion exposure on osteocytes, the most abundant cell type in bone and responsible for coordinating bone remodeling, remain unclear. However, several studies have previously shown that exposure to cobalt (Co2+) and chromium (Cr3+), at concentrations equivalent to those found clinically, affect osteoblast and osteoclast survival and function. In this study, we tested the hypothesis that metal ions would similarly impair the normal physiology of osteocytes. The survival, dendritic morphology, and response to fluid shear stress of the mature osteocyte‐like cell‐line MLO‐Y4 following exposure to clinically relevant concentrations and combinations of Co and Cr ions were measured in 2D‐culture. Exposure of MLO‐Y4 cells to metal ions reduced cell number, increased dendrites per cell and increased dendrite length. We found that combinations of metal ions had a greater effect than the individual ions alone, and that Co2+ had a predominate effect on changes to cell numbers and dendrites. Combined metal ion exposure blunted the responses of the MLO‐Y4 cells to fluid shear stress, including reducing the intracellular calcium responses and modulation of genes for the osteocyte markers Cx43 and Gp38, and the signaling molecules RANKL and Dkk‐1. Finally, we demonstrated that in the late osteoblasts/early osteocytes cell line MLO‐A5 that Co2+ exposure had no effect on mineralization, but Cr3+ treatment inhibited mineralization in a dose‐dependent manner, without affecting cell viability. Taken together, these data indicate that metal exposure can directly affect osteocyte physiology, with potential implications for bone health including osseointegration of cementless components, and periprosthetic bone remodeling. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1716–1723, 2017.

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Establishment of an Osteoid Preosteocyte-like Cell MLO-A5 That Spontaneously Mineralizes in Culture

Cited by 188 publications

References 39 publications

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

Mineralization of Decalcified Bone Occurs Under Cell Culture Conditions and Requires Bovine Serum But Not Cells

Osteocyte physiology and response to fluid shear stress are impaired following exposure to cobalt and chromium: Implications for bone health following joint replacement

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