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Cell Biology International (2012) 36, 909–915 (Printed in Great Britain)
Effect of calcitonin gene-related peptide on osteoblast differentiation in an osteoblast and endothelial cell co-culture system
Yang Bo*1, Li Yan†1, Zhang Gang*, Lei Tao‡ and Tan Yinghui*2
*Department of Oral and Maxillofacial Surgery, Xingqiao Hospital, Third Military Medical University, Chongqing, People's Republic of China, †Department of Oral and Maxillofacial Surgery, Chengdu Army General Hospital, Chengdu, People's Republic of China, and ‡Department of Hepatobiliary Surgery, 324th Army Hospital, Chongqing, People's Republic of China

We have investigated the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on the differentiation of OB (osteoblasts) in co-culture with HUVEC (human umbilical vein endothelial cells). Primary human MOB (mandibular OB) and OB-like cells (MG-63) were either cultured directly or indirectly co-cultured with HUVEC at a 1:1 ratio. Expression of OC (osteocalcin) was measured by ELISA, and expression of ALP (alkaline phosphatase) and collagen mRNA was measured by quantitative fluorescent PCR. For mineralization nodus, OB were stained with Alizarin Red-S. When co-cultured with HUVEC, expression of OC and ALP mRNA were increased in MG-63 (P<0.01), and the expression of OC, ALP and collagen mRNA were increased in MOB (P<0.01 or 0.05). When treated with CGRP, OC and ALP mRNA and mineralization nodus numbers were increased in the MG-63 co-culture system (P<0.01 or 0.05); OC, ALP and collagen mRNA, and mineralization nodus numbers were increased in the MOB co-culture system (P<0.01 or 0.05). The effect of CGRP regulation on the differentiation of OB is not only direct but also indirect, via its effect on HUVEC and stimulation of OB.

Key words: calcitonin gene-related peptide, co-culture, endothelial cell, osteoblast

Abbreviations: ALP, alkaline phosphatase, BMP, bone morphogenetic protein, CGRP, calcitonin gene-related peptide, DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, DMEM, Dulbecco's modified Eagle's medium, EC, endothelial cells, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, HBMSC, human bone marrow stromal cells, HUVEC, human umbilical vein endothelial cells, MOB, mandibular OB, OB, osteoblast, OC, osteocalcin, RQ%, relative quantity %, VEC, vascular endothelial cells, VEGF, vascular endothelial growth factor

1These authors contributed equally to this work.

2To whom correspondence should be addressed (email

Received 1 December 2011/16 March 2012; accepted 28 May 2012

Published as Cell Biology International Immediate Publication 28 May 2012, doi:10.1042/CBI20110562

© 2012 The Author(s)
The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence ( which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.

ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)