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Comparison of the action of transient and continuous PTH on primary osteoblast cultures expressing differentiation stage-specific GFP.
J Bone Miner Res. 2005 Jan; 20(1):5-14.JB

Abstract

Primary calvarial osteoblast cultures derived from type I collagen promoter-GFP reporter transgenic mice were used to examine progression of the osteoblast lineage. This system was validated by assessing the effect of PTH on osteoblast growth in real time. The anabolic effect of PTH seemed to be the result of enhanced osteoblast differentiation rather than expansion of a progenitor population.

INTRODUCTION

Activation of green fluorescent protein (GFP) marker genes driven by Col1a1 promoter fragments has been associated with the level of osteoblast differentiation. GFP-marked cultures provide an approach to continuously monitor the level of osteoblast differentiation in real time without the termination of cultures.

MATERIALS AND METHODS

Neonatal calvarial cells transgenic for pOBCol2.3GFP and pOBCol3.6GFP were used to establish calvarial osteoblast cultures. Parathyroid hormone (PTH) was added either continuous (days 1-21) or transient (days 1-7) to examine its diverse effect on osteoblast differentiation in cultures for 21 days. Three fluorescent markers were used: (1) pOBCol3.6GFP, which is activated in preosteoblastic cells; (2) pOBCol2.3GFP, which is restricted to differentiated osteoblasts; and (3) xylenol orange (XO), which stains the mineralized nodules. Progression of osteoblast differentiation indicated by fluorescent markers was documented throughout the entire period of culture. Recorded fluorescent images were analyzed in the patterns of expression and quantitated in the area of expression.

RESULTS

Continuous PTH blocked osteoblast differentiation, which was evident by the attenuation of pOBCol3.6GFP and an absence of pOBCol2.3GFP. In contrast, transient PTH inhibited the initial osteoblast differentiation but ultimately resulted in a culture with more mineralized nodules and enhanced osteoblast differentiation expressing strong levels of pOBCol3.6GFP and pOBCol2.3GFP. Quantitative analysis showed that transient PTH first decreased then later increased areas of GFP expression and XO staining, which correlated with results of Northern blot and alkaline phosphatase activity. Transient PTH caused a decrease in DNA content during the treatment and after the removal of PTH.

CONCLUSION

GFP-marked cultures combined with fluorescent image analysis have the advantage to assess the effect of PTH on osteoblast differentiation in real time. Results suggest that the anabolic effect of transient PTH is caused by an enhancement in osteoblast differentiation rather than an increase in the population of progenitor cells.

Authors+Show Affiliations

Department of Pediatric Dentistry, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030-1610, USA. ywang@nso.uchc.eduNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15619664

Citation

Wang, Yu-Hsiung, et al. "Comparison of the Action of Transient and Continuous PTH On Primary Osteoblast Cultures Expressing Differentiation Stage-specific GFP." Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research, vol. 20, no. 1, 2005, pp. 5-14.
Wang YH, Liu Y, Buhl K, et al. Comparison of the action of transient and continuous PTH on primary osteoblast cultures expressing differentiation stage-specific GFP. J Bone Miner Res. 2005;20(1):5-14.
Wang, Y. H., Liu, Y., Buhl, K., & Rowe, D. W. (2005). Comparison of the action of transient and continuous PTH on primary osteoblast cultures expressing differentiation stage-specific GFP. Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research, 20(1), 5-14.
Wang YH, et al. Comparison of the Action of Transient and Continuous PTH On Primary Osteoblast Cultures Expressing Differentiation Stage-specific GFP. J Bone Miner Res. 2005;20(1):5-14. PubMed PMID: 15619664.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Comparison of the action of transient and continuous PTH on primary osteoblast cultures expressing differentiation stage-specific GFP. AU - Wang,Yu-Hsiung, AU - Liu,Yaling, AU - Buhl,Kathy, AU - Rowe,David W, Y1 - 2004/10/25/ PY - 2004/06/10/received PY - 2004/08/03/revised PY - 2004/08/20/accepted PY - 2004/12/28/pubmed PY - 2005/5/26/medline PY - 2004/12/28/entrez SP - 5 EP - 14 JF - Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research JO - J. Bone Miner. Res. VL - 20 IS - 1 N2 - UNLABELLED: Primary calvarial osteoblast cultures derived from type I collagen promoter-GFP reporter transgenic mice were used to examine progression of the osteoblast lineage. This system was validated by assessing the effect of PTH on osteoblast growth in real time. The anabolic effect of PTH seemed to be the result of enhanced osteoblast differentiation rather than expansion of a progenitor population. INTRODUCTION: Activation of green fluorescent protein (GFP) marker genes driven by Col1a1 promoter fragments has been associated with the level of osteoblast differentiation. GFP-marked cultures provide an approach to continuously monitor the level of osteoblast differentiation in real time without the termination of cultures. MATERIALS AND METHODS: Neonatal calvarial cells transgenic for pOBCol2.3GFP and pOBCol3.6GFP were used to establish calvarial osteoblast cultures. Parathyroid hormone (PTH) was added either continuous (days 1-21) or transient (days 1-7) to examine its diverse effect on osteoblast differentiation in cultures for 21 days. Three fluorescent markers were used: (1) pOBCol3.6GFP, which is activated in preosteoblastic cells; (2) pOBCol2.3GFP, which is restricted to differentiated osteoblasts; and (3) xylenol orange (XO), which stains the mineralized nodules. Progression of osteoblast differentiation indicated by fluorescent markers was documented throughout the entire period of culture. Recorded fluorescent images were analyzed in the patterns of expression and quantitated in the area of expression. RESULTS: Continuous PTH blocked osteoblast differentiation, which was evident by the attenuation of pOBCol3.6GFP and an absence of pOBCol2.3GFP. In contrast, transient PTH inhibited the initial osteoblast differentiation but ultimately resulted in a culture with more mineralized nodules and enhanced osteoblast differentiation expressing strong levels of pOBCol3.6GFP and pOBCol2.3GFP. Quantitative analysis showed that transient PTH first decreased then later increased areas of GFP expression and XO staining, which correlated with results of Northern blot and alkaline phosphatase activity. Transient PTH caused a decrease in DNA content during the treatment and after the removal of PTH. CONCLUSION: GFP-marked cultures combined with fluorescent image analysis have the advantage to assess the effect of PTH on osteoblast differentiation in real time. Results suggest that the anabolic effect of transient PTH is caused by an enhancement in osteoblast differentiation rather than an increase in the population of progenitor cells. SN - 0884-0431 UR - https://www.unboundmedicine.com/medline/citation/15619664/Comparison_of_the_action_of_transient_and_continuous_PTH_on_primary_osteoblast_cultures_expressing_differentiation_stage_specific_GFP_ L2 - https://doi.org/10.1359/JBMR.041016 DB - PRIME DP - Unbound Medicine ER -