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Time lapse imaging techniques for comparison of mineralization dynamics in primary murine osteoblasts and the late osteoblast/early osteocyte-like cell line MLO-A5.
Cells Tissues Organs. 2009; 189(1-4):6-11.CT

Abstract

Mineralization of bone matrix and osteocyte differentiation occur simultaneously and appear interrelated both spatially and temporally. Although these are dynamic events, their study has been limited to using static imaging approaches, either alone or in combination with chemical and biochemical analysis and/or genetic manipulation. Here we describe the application of live cell imaging techniques to study mineralization dynamics in primary osteoblast cultures compared to a late osteoblast/early osteocyte-like cell line, MLO-A5. Mineral deposition was monitored using alizarin red as a vital stain for calcium. To monitor differentiation into an osteocyte-like phenotype, the calvarial cells were isolated from transgenic mice expressing green fluorescent protein (GFP) driven by an 8-kb dentin matrix protein-1 (Dmp1) promoter that gives osteocyte-selective expression. Time lapse imaging showed that there was a lag phase of 15-20 h after beta-glycerophosphate addition, followed by mineral deposition that was rapid in primary osteoblast cultures but more gradual in MLO-A5 cultures. In primary osteoblast cultures, mineral was deposited exclusively in association with clusters of cells expressing Dmp1-GFP, suggesting that they were already differentiating into osteocyte-like cells. In MLO-A5 cells, the first indication of mineralization was the appearance of punctate areas of alizarin red fluorescence of 4-7 mum in diameter, followed by mineral deposition throughout the culture in association with collagen fibrils. A high amount of cell motility was observed within mineralizing nodules and in mineralizing MLO-A5 cultures. These studies provide a novel approach for analyzing mineralization kinetics that will enable us to dissect in a time-specific manner the essential players in the mineralization process.

Authors+Show Affiliations

School of Dentistry, Department of Oral Biology, University of Missouri at Kansas City, 650 E. 25th Street, Kansas City, MO 64108, USA. dallass@umkc.eduNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

18728354

Citation

Dallas, Sarah L., et al. "Time Lapse Imaging Techniques for Comparison of Mineralization Dynamics in Primary Murine Osteoblasts and the Late Osteoblast/early Osteocyte-like Cell Line MLO-A5." Cells, Tissues, Organs, vol. 189, no. 1-4, 2009, pp. 6-11.
Dallas SL, Veno PA, Rosser JL, et al. Time lapse imaging techniques for comparison of mineralization dynamics in primary murine osteoblasts and the late osteoblast/early osteocyte-like cell line MLO-A5. Cells Tissues Organs (Print). 2009;189(1-4):6-11.
Dallas, S. L., Veno, P. A., Rosser, J. L., Barragan-Adjemian, C., Rowe, D. W., Kalajzic, I., & Bonewald, L. F. (2009). Time lapse imaging techniques for comparison of mineralization dynamics in primary murine osteoblasts and the late osteoblast/early osteocyte-like cell line MLO-A5. Cells, Tissues, Organs, 189(1-4), 6-11. https://doi.org/10.1159/000151745
Dallas SL, et al. Time Lapse Imaging Techniques for Comparison of Mineralization Dynamics in Primary Murine Osteoblasts and the Late Osteoblast/early Osteocyte-like Cell Line MLO-A5. Cells Tissues Organs (Print). 2009;189(1-4):6-11. PubMed PMID: 18728354.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Time lapse imaging techniques for comparison of mineralization dynamics in primary murine osteoblasts and the late osteoblast/early osteocyte-like cell line MLO-A5. AU - Dallas,Sarah L, AU - Veno,Patricia A, AU - Rosser,Jennifer L, AU - Barragan-Adjemian,Cielo, AU - Rowe,David W, AU - Kalajzic,Ivo, AU - Bonewald,Lynda F, Y1 - 2008/08/27/ PY - 2008/8/30/pubmed PY - 2009/2/12/medline PY - 2008/8/30/entrez SP - 6 EP - 11 JF - Cells, tissues, organs JO - Cells Tissues Organs (Print) VL - 189 IS - 1-4 N2 - Mineralization of bone matrix and osteocyte differentiation occur simultaneously and appear interrelated both spatially and temporally. Although these are dynamic events, their study has been limited to using static imaging approaches, either alone or in combination with chemical and biochemical analysis and/or genetic manipulation. Here we describe the application of live cell imaging techniques to study mineralization dynamics in primary osteoblast cultures compared to a late osteoblast/early osteocyte-like cell line, MLO-A5. Mineral deposition was monitored using alizarin red as a vital stain for calcium. To monitor differentiation into an osteocyte-like phenotype, the calvarial cells were isolated from transgenic mice expressing green fluorescent protein (GFP) driven by an 8-kb dentin matrix protein-1 (Dmp1) promoter that gives osteocyte-selective expression. Time lapse imaging showed that there was a lag phase of 15-20 h after beta-glycerophosphate addition, followed by mineral deposition that was rapid in primary osteoblast cultures but more gradual in MLO-A5 cultures. In primary osteoblast cultures, mineral was deposited exclusively in association with clusters of cells expressing Dmp1-GFP, suggesting that they were already differentiating into osteocyte-like cells. In MLO-A5 cells, the first indication of mineralization was the appearance of punctate areas of alizarin red fluorescence of 4-7 mum in diameter, followed by mineral deposition throughout the culture in association with collagen fibrils. A high amount of cell motility was observed within mineralizing nodules and in mineralizing MLO-A5 cultures. These studies provide a novel approach for analyzing mineralization kinetics that will enable us to dissect in a time-specific manner the essential players in the mineralization process. SN - 1422-6421 UR - https://www.unboundmedicine.com/medline/citation/18728354/Time_lapse_imaging_techniques_for_comparison_of_mineralization_dynamics_in_primary_murine_osteoblasts_and_the_late_osteoblast/early_osteocyte_like_cell_line_MLO_A5_ L2 - https://www.karger.com?DOI=10.1159/000151745 DB - PRIME DP - Unbound Medicine ER -