Recent evidence suggests that bone metabolism may be influenced by the innervation of skeletal tissues. Innervation of skeletal tissues might directly influence bone volume by the release or secretion of osteogenic growth factors in the form of neuropeptides. These neuropeptides could act locally on osteoblasts to increase bone formation and/or mineralization. Since calcitonin gene-related peptide (CGRP) and Substance P (SP) are the most abundant neuropeptides present in sensory nerves in bone, the current studies were intended to test the hypothesis that these two neuropeptides may have direct effects on osteoblast growth, differentiation, and mineralization. Replicate cultures of murine calvarial osteoblasts were grown with and without CGRP and/or SP, across a range of physiologic to pharmacologic doses. Cell growth was assessed by changes in cell layer protein. Differentiation was assessed by changes in cellular alkaline phosphatase levels, and mineralization was measured by alizarin red staining. Although we did not observe effects of CGRP or SP on cell growth, we did observe effects on differentiation—a 48 hour exposure to CGRP induced a dose-dependent increase in alkaline phosphatase levels in the calvarial osteoblasts (p<0.001). Alkaline phosphatase activity was also increased in a dose-dependent manner following treatment with SP (r =0.979 , p<0.001). The murine cell line MC3T3-E1 was used to determine the effect of CGRP and SP on osteoblastic mineralization. After 7–21 days of continuous treatment with CGRP and SP in mineralization media, cells were fixed and mineralization was assessed by staining with alizarin red. The results of those studies showed that both CGRP and SP increased the rate and the extent of mineralization (p<0.001) and the effects of both neuropeptides together were greater than either alone (p<0.001). The effects of CGRP, SP, and the combination of peptides on osteoblast gene expression, as assessed by RT-PCR, have further revealed that exposure to CGRP and/or SP increased the mRNA levels for the Cox 2 protein, suggesting a possible role for prostaglandins as determinants of the osteogenic action(s) of CGRP and SP. Together, these findings are consistent with the hypothesis that neuropeptides released from neurons in bone may be involved in the local regulation of bone volume, by effects on osteoblast growth, differentiation, and mineralization.
School of Medicine
Linkhart, Thomas A.
Wright, Kenneth R.
Doctor of Philosophy (PhD)
Year Degree Awarded
Date (Title Page)
Library of Congress/MESH Subject Headings
Osteoblasts; Cell Differentiation; Nerve Growth Factors; Neuropeptides; Receptors - Neurokinin-1; Interleukins; Growth Inhibitors; Bone Morphogenetic Proteins; Calcification - Physiologic;
Subject - Local
Calcitonin Gene-related Peptide; Substance P; Osteoblast Growth and Differentiation; Cellular Alkaline Phosphatase Levels; Osteoblast Mineralization
Loma Linda University Libraries
This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.
Moran, Colleen M., "Effects of Neurotrophic Factors on Osteoblast Growth and Differentiation" (2011). Loma Linda University Electronic Theses, Dissertations & Projects. Paper 306.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives