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A method for demineralization of bone, preserving the antigenicity of neuroactive peptides, was developed. In all parts of rat long bones, nerves immunoreactive to substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and tyrosine hydroxylase (TH) were detected after immunohistochemical staining. The majority of nerves were vascular, although several non-vascular endings were observed at the growth plate and amidst marrow cells. An abundance of nerves were demonstrated near the epiphyseal plate and in the periosteum, regions of high osteogenic activity. The occurrence of different nerve types was analyzed at different stages of heterotopic osteogenesis, induced by allogeneic bone matrix. Nerve fibres immunoreactive to SP, CGRP, NPY and TH occurred amidst differentiating chondroblastic cells in the second week. They gradually increased in number during the ensuing eight weeks. In an in vitro study of osteoblastic cells (UMR 106-01, ROS 17/2.8, Saos-2, MC3T3-E1) receptors to CGRP, VIP, noradrenaline (NA) and NPY were demonstrated as assessed by analysis of cyclic AMP formation. In UMR cells, NPY inhibited the effects of NA and parathyroid hormone (PTH), which is the first demonstration of a receptor interaction between a local neuropeptide and a systemic calcium regulating hormone. The combined findings indicate a neuroendocrine influence on bone physiology.

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Background: Previous studies have shown that the neuropeptide calcitonin gene-related peptide (CGRP) have an influence on osteoclastic bone resorption and that CGRP and substance P (SP), both wellknown markers for sensory neurons, behave as growth factors.Materials and Methods: The ontogeny of the sensory nerves in the hindlimb skeleton of the rat was studied from gestational day (GD) 15 to neonatal day (ND) 24 by immunohistochemistry. Neurofilaments and nerve terminals were labelled with protein gene-product 9.5 (PGP 9.5) and synaptophycin (SYN), respectively.Results: PGP 9.5 appeared at GD 15 and SYN at GD 19, both in the perichondrial tissue of the long bones. One week later, at ND 4 nerve fibre, immunoreactive to PGP 9.5 and SYN were observed within the bone organ. Sensory nerves, indicated by CGRP and SP, were first discerned at GD 18-19 in the periosteal tissue of the diaphyseal and metaphyseal regions and in the bone organ at ND 4. Approximately at ND 6, vascular as well as non-vascular nerves extended into the metaphyses and at ND 8 into the epiphyses, concomitant with the first signs of mineralization.Conclusions: The study shows that a functional sensory nerve supply of the developing bone organ occurs immediatly prior to partus, apparently parallel with an increasing mineralization. The combined findings may indicate a sensory influence on developmental processes in the skeleton.