Effect of calcitonin gene-related peptide on osteoporotic fracture healing in vitro

Abstract

Objective To explore the effects of calcitonin gene-related peptide (CGRP) on proliferation of vascular endothelial cells (VECs) and osteogenic differentiation of bone mesenchymal stem cells (BMSCs) of osteoporotic rats in vitro, and cell/molecular mechanism of promoting osteoporotic fracture healing. Methods 10 3-month-old SD rats were employed to make osteoporosis model by taking bilateral ovariectomy. Micro-CT analysis was used to determine the success of modeling at 6 months of postoperation. BMSCs and VECs were isolated from osteoporotic rats by blood adherence method. 10-7-10-10 mol/L CGRP were added to the culture medium. CCK-8 method was used to examine the cell proliferation. Effect of CGRP on osteogenic differentiation of BMSCs: Alkaline phosphatase (ALP) activity, and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of ALP, CollagenⅠ, BMP-2, Osteonectin and RunX2 gene. Effect of CGRP on vascularization of VECs: Vascular cell endothelial growth factor (VEGF) protein secretion and gene expression level were tested by ELISA and RT-PCR. Results BMSCs and VECs of osteoporotic rats were isolated successfully.The optical density (OD) values of CCK-8 method increased in a dose-dependent manner, for BMSCs at 7 d, the control group of OD value was 0.58±0.02, the CGRP group 10-7-10-10 mol/L was to 0.80±0.03, 0.79±0.03, 0.74±0.03, 0.69±0.03, difference was significant; for VECs, the control group of OD value was 2.20±0.01, the CGRP group 10-7-10-10 mol/L was to 4.12±0.04, 3.90±0.02, 3.56±0.04, 3.12±0.04, difference was significant. Effect of CGRP on osteogenic differentiation of BMSCs: Alkaline phosphatase (ALP) activity, and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of ALP, CollagenⅠ, BMP-2, Osteonectin and RunX2 gene. Effect of CGRP on vascularization of VECs: Vascular cell endothelial growth factor (VEGF) protein secretion and gene expression level were tested by ELISA and RT-PCR, difference between the control group and CGRP group were all significant. Conclusion 10-7 mol/L, 10-10 mol/L of CGRP can directly promote the proliferation of the BMSCs and VECs of osteoporotic rats, promote the osteogenic differentiation of BMSCs, improve the expression of VEGF gene and protein secretion, promote angiogenesis. Key words: Calcitonin gene-related peptide; Osteoporosis; Fractures, bone