gms | German Medical Science

Objectives: The periosteum is a membrane that covers bone surfaces and is richly innervated by both sympathetic nervous system and pain fibers. These nerves release Norepinephrine (NE) and calcitonin gene-related peptide alpha (aCGRP), respectively. NE signals through the beta2-adrenergic receptor (ADRB2) in bone, which regulates the differentiation and function of osteoblasts. Also, aCGRP exerts an osteoanabolic action by promoting osteoblast function. In periosteal cells, however, the precise impact of NE and ADRB2 in relation to aCGRP signaling is unclear. Therefore, this study was designed to assess the impact of NE signaling and its interaction with aCGRP on periosteal cells in vitro.

Methods: Primary periosteal cells and bone marrow-derived osteoblasts were generated from adult WT, ADRB2-/-, and aCGRP-/- mice. Periosteal cells were isolated by enzymatic digestion of dissected whole femur bones, while osteoblasts were obtained from flushed bone marrow. Both cell types were differentiated in osteogenic medium and stimulated with NE, aCGRP, and the aCGRP antagonist BIBN for short- and long-term periods. Evaluation methods used in this study included alizarin red staining to assess matrix mineralization, and qRT-PCR and ELISA to measure gene and protein expression.

Results and conclusion: NE reduced matrix mineralization of bone marrow-derived osteoblasts but did not affect osteogenic differentiation or expression of osteoblast markers in WT periosteal cells. However, NE induced the expression of the pro-angiogenic factors hypoxia-inducible factor-1 alpha (HIF1A), vascular endothelial growth factor A (VEGFA), and aCGRP in periosteal cells in an ADRB2-dependent manner. In periosteal cells deficient in aCGRP, treatment with NE did not induce HIF1A and VEGFA. This lack of induction was also observed when cultures were co-stimulated with the aCGRP antagonist BIBN. Conversely, the stimulatory effect of NE on the expression of HIF1A and VEGFA was synergistically enhanced by co-stimulation of periosteal cells with aCGRP.

The findings suggest that NE/aCGRP signaling regulates the pro-angiogenic properties of periosteal cells, which may be important for adequate vascularization of the fracture callus.