Coupling effect of CGRP in microcirculation and bone regeneration

Objectives: Microcirculation is closely related to osteogenesis. The neuropeptide, calcitonin gene-related peptide (CGRP), can up-regulate proliferation and activities of osteoblasts and human umbilical vein endothelial cells. CGRP-gene knockout mice (CGRP-/-) represent a valid model to explore the coupling effect of CGRP on microcirculation and bone regeneration.
Methods: CGRP was loaded in alginate microbeads and the release kinetics of CGRP was assayed by ELISA kit. The microbeads were mixed with collagen I gel before implantation in 4mm cranial defects of mice. A total of 54 male mice were used across six groups, including (a) seeded with collagen I gel and CGRP-loaded microbeads in CGRP-/- (n=9), (b) seeded with collagen I gel without microbeads in CGRP-/- (n=9), (c) empty defects in CGRP-/- (n=9), and the same groups in wild type mice. The mice were sacrificed at 2, 4, and 8 weeks. Histologic changes were analyzed by HE, Masson and immunohistochemical staining. Micro-CT was used to measure new bone formation. The expression of Runx2, OCN, VEGF and CD31 were measured by RT-PCR. All the data were statistically analyzed (ANOVA).
Results: New bone volume in CGRP-/- groups, without CGRP-loaded microbeads, is less than that in the wild type group (P<0.05). Addition of CGRP increased bone formation significantly in CGRP-/- group (P<0.05), almost equivalent to that in wild type groups. Bone formation in CGRP-/- group with collagen I alone increased slightly compared with the empty group (P>0.05). The density of blood vessels increased and the vessel lumen dilated after CGRP application in CGRP-/- groups. CGRP up regulated expression of Runx and VEGF at 2w and OCN at 4w and 8w.
Conclusions: CGRP possibly possesses coupling effect on microcirculation and bone regeneration. This study may complement the present theory of effect and mechanism of CGRP.