gms | German Medical Science

Objectives: Phenotyping of sensory nerve fibers innervating bone, bone marrow and periosteum has demonstrated the presence of signaling molecules including the sensory neuropeptide alpha-calcitonin gene-related peptide (α-CGRP) indicating that the sensory nervous system is part of the endo- and paracrine control of bone metabolism. Previous studies have described a distortion of bone microarchitecture for α-CGRP-deficient mice. Recently, we observed changes in cell survival and activity of osteoblasts and osteoclasts isolated from wildtype (WT) mice when stimulated with α-CGRP. In this study, we investigated and compared cell metabolism of osteoblasts and osteoclasts isolated from α-CGRP-deficient mice and WT controls.

Methods: Isolation/differentiation of bone marrow macrophages (BMM; for 5 days) to osteoclasts and osteoblast-like cells (for 7/14/21 days) from 8-12 weeks old female α-CGRP-/- and WT control (both C57Bl/6J) mice according to established protocols. We analyzed cell migration of osteoblast-like cells out of femoral bone chips (cristal violet staining); proliferation (BrdU incorporation), viability (WST-1 reagent), caspase 3/7-activity (apoptosis rate) and gene expression of calcitonin receptor-like receptor (calcrl; qPCR) in osteoblasts and osteoclasts. Alkaline phosphatase (ALP) activity reflects osteoblast bone formation activity and cathepsin K activity reflects osteoclast resorption activity. Endogenous α-CGRP production was measured by ELISA.

Results and conclusion: We counted reduced numbers of BMM from α-CGRP-/- mice after initial seeding compared to WT with no differences in viability. Migration of osteoblast-like cells from α-CGRP-/- mice out of bone chips was comparable to WT at all time points of outgrowth. Osteoblasts and osteoclasts from WT mice endogenously synthesize and secrete α-CGRP. Gene expression of calcrl was similar in BMM of WT and α-CGRP-/- mice but was lower in α-CGRP-/- osteoblasts after 14 days in osteogenic medium. Proliferation was comparable in BMM from WT and α-CGRP-/- mice. Survival of BMM/osteoclast cultures from α-CGRP-/- mice was by trend higher but cathepsin K activity was significantly lower compared to WT BMM/osteoclast cultures. We did not observe differences in proliferation, survival or activity of osteoblast-like cells from WT and α-CGRP-/- mice throughout osteogenic differentiation (days 7, 14, 21).

We assume that bone resorption rate is slightly reduced in α-CGRP-/- mice as we found decreased BMM numbers and reduced cathepsin K activity of BMM/osteoclast cultures from α-CGRP-/- mice, however, this may be compensated by increased cell survival. In contrast, bone formation rate seems to be unchanged as α-CGRP-/- osteoblast metabolism is not altered. Therefore, we hypothesize that additional conditions as ageing and physical activity - present in vivo - might contribute to the inferior bone properties described for aged α-CGRP-/- mice.