Ontogeny of sensory and autonomic nerves in the developing mouse skeleton.

BACKGROUND: Bone innervation is implicated in bone modeling and remodeling. This study investigates skeletal nerve development in embryonic and newborn mice, focusing on sensory and autonomic nerves and their temporal occurrence. MATERIALS AND METHODS: The ontogeny of innervation and angiogenesis in the hindlimb skeleton of mice was studied from embryonic day (E) 15 to postnatal day (P) 20. Neuronal tissue was immunohistochemically labeled for detection of growth associated protein 43 (GAP-43), protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), and neuropeptide Y (NPY). Vascular endothelium was labeled for platelet endothelium cell adhesion molecule-1 (PECAM-1). Morphology was evaluated with hematoxylin and eosin staining. RESULTS: GAP-43, PGP 9.5, CGRP, and PECAM-1 were all present at E 15, adjacent to areas with high osteogenic and chondrogenic activity. In the primary ossification centers, GAP-43 was found at E 15, PGP 9.5 at E 17, CGRP at E 19, and NPY at P 4. The same time lag in appearance was observed in the secondary ossification centers. The covering capillary network was initially dense, but became mature and sparse from P 12 onwards. CONCLUSION: A functional nerve supply co-localized with a rich capillary network is seen early in the developing mouse skeleton, especially in areas with high osteogenic activity. Sensory innervation occurs prior to partus, while autonomic innervation (revealed by the presence of NPY and TH) is established post partum. The findings indicate a time-related development of nerves with different qualities, according to skeletal development.

Vasoactive intestinal peptide regulates osteoclast activity via specific binding sites on both osteoclasts and osteoblasts.

Clinical and experimental observations, together with immunohistochemical findings, suggest that neuro-osteogenic interactions may occur in the skeleton. In this study, we have examined the effect of vasoactive intestinal peptide (VIP), one of the neuropeptides present in bone, on the activity of the bone-resorbing osteoclast. Effects on bone resorption were assessed by counting the number of pits formed by rat osteoclasts incubated on devitalized slices of bovine cortical bone. Under conditions with an initially sparse density of stromal cells/osteoblasts, VIP caused a rapid cytoplasmic contraction and decreased motility of osteoclasts. This was coupled with a decrease in the number of resorption lacunae and a decrease in the total area resorbed by the osteoclasts in 48-h cultures. Time-course experiments revealed that the inhibitory effects on contraction and motility were transient and that the cells gradually regained their activity, such that, when culture time was prolonged to 120 h, a stimulatory effect by VIP on bone resorption was observed. When osteoclasts were incubated on bone slices, in the presence of an initially large number of stromal cells/osteoblasts, VIP treatment increased the number of resorption pits and total bone area resorbed in 48-h cultures. Using atomic force microscopy, we provide direct evidence that both osteoclasts and stromal cells/osteoblasts bind VIP. Also, VIP was shown to cause a rapid rise of intracellular calcium in osteoclasts and in a proportion (20%) of stromal cells/osteoblasts. Taken together, these data suggest that differentiated osteoclasts are equipped with receptors for VIP that are linked to a transient inhibition of osteoclast activity and, in addition, that stromal cells/osteoblasts have VIP receptors coupled to a delayed stimulation of osteoclastic resorption.

Neuro-hormonal control of bone metabolism: vasoactive intestinal peptide stimulates alkaline phosphatase activity and mRNA expression in mouse calvarial osteoblasts as well as calcium accumulation mineralized bone nodules.

Based upon the immunohistochemical demonstration of neuropeptides in the skeleton, including vasoactive intestinal peptide (VIP), we have addressed the question of whether neuropeptides may exert regulatory roles on bone tissue metabolism or not. In the present communication, we have investigated if VIP can affect anabolic processes in osteoblasts. Osteoblasts were isolated from neonatal mouse calvariae by time sequential enzyme-digestion and subsequently cultured for 2-28 days in the presence of VIP and other modulators of cyclic AMP formation. VIP (10(-6) M) stimulated ALP activity and calcium content. The cyclic AMP phosphodiesterase inhibitors ZK 62 711 (10(-4) M) and isobutyl-methylxanthine (10(-4) M) stimulated ALP activity and synergistically potentiated the effect of VIP. Neither VIP, nor isobutyl-methylxanthine or ZK 62 711, in the absence or presence of VIP, affected cell number. The stimulatory effect of VIP on ALP activity, in the presence of ZK 62 711, was dependent on time and concentration of VIP. The stimulatory effects of VIP and ZK 62 711 on ALP activity was seen also in cells stained for ALP. VIP (10(-6) M), in the presence of ZK 62 711 (10(-6) M), significantly enhanced mRNA for tissue non-specific ALP. VIP (10(-6) M), in the presence of ZK 62 711, stimulated cyclic AMP production. Forskolin and choleratoxin stimulated ALP activity and cyclic AMP formation in a concentration-dependent manner, without affecting cell number. VIP (10(-6) M) and ZK 62 711 (10(-5) M) stimulated, and their combination synergistically enhanced, calcium content in bone noduli. These data show that VIP, without affecting cell proliferation, can stimulate osteoblastic ALP biosynthesis and bone noduli formation by a mechanism mediated by cyclic AMP. Our observations suggest a possibility that anabolic processes in bone are under neurohormonal control.

Vasorelaxation in isolated bone arteries. Vasoactive intestinal peptide, substance P, calcitonin gene-related peptide, and bradykinin studied in pigs.

We assessed the effects of vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP), and bradykinin in arteries (diameter approximately 230 microns) isolated from cancellous bone from pigs. Arterial segments (2 mm long) were mounted on a myograph for measurement of isometric force development. After submaximal precontraction with norepinephrine, VIP (10(-10)-10(-7) M), CGRP (10(-11)-10(-7) M), SP (10(-6) M), and bradykinin (10(-11)-10(-6) M) were added. 44 arterial segments (23 pigs) were investigated. VIP-, CGRP-, and bradykinin induced a concentration-dependent vasorelaxation, while SP mediated a transient relaxation. After mechanical removal of the endothelium, the effects of SP and bradykinin were completely abolished, while the relaxation to CGRP was still pronounced. This indicates that the effects of SP and bradykinin are mediated by the endothelium, while CGRP mainly mediates relaxation by a direct effect on vascular smooth muscle cells. The relaxations to CGRP and bradykinin were still significant after inhibition of nitric oxide synthase with 10(-4) M N omega-nitro-L-arginine (L-NNA) and inhibition of prostaglandin synthesis with 10(-5) M indomethacin, indicating the existence of an alternative vasorelaxing pathway. Our findings support the theory of a vasoregulatory role of neuropeptides in bone.

Neuropeptides in heterotopic bone induced by bone matrix in immunosuppressed rats.

The effects of cyclosporin A on the occurrence of neuroendocrine peptides in bone induced by demineralized allogeneic and xenogeneic bone matrix were studied in rats. Cyclosporin A enhanced bone induction in demineralized allogeneic bone matrix implants by 40% to 50% at 4 weeks, whereas there was no difference to the control group at 8 weeks. In demineralized xenogeneic bone matrix implants there was virtually no cartilage or bone formation at 4 weeks, but some bone and cartilage formation was seen at 8 weeks. In both cyclosporin A treated groups the net bone formation in demineralized xenogeneic bone matrix implants was increased four to five times at 4 weeks. Cyclosporin A treatment did not alter the temporal occurrence or distribution of neuropeptide containing nerve fibers in the bone induced by allogeneic bone matrix. Fibers containing substance P, calcitonin gene related peptide, neuropeptide Y, vasoactive intestinal peptide, and tyrosine hydroxylase were detected in the ossicles of cyclosporin A treated and control rats. In the xenogeneic bone matrix of the control group, no immunoreactive nerve fibers could be detected at 4 weeks, but at 8 weeks all five neuropeptides were detected. However, after cyclosporin A treatment immunoreactive nerve fibers could be seen at 4 weeks in the demineralized xenogeneic bone matrix implants. Thus, immunologic properties of the inductive matrix affect the yield of mineralized bone and the degree of innervation. Cyclosporin A decreases the immune response and enhances the formation of bone and the number of transmitter identified nerves in demineralized xenogeneic bone matrix induced ossicles.

The development of autonomic innervation in bone and joints of the rat.

The development of autonomic nerves in the hindlimb skeleton, was studied in rats from gestational day (G) 15 to postnatal day (P) 24 by immunoreactivity to neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP). Control labelling with antisera to neurofilaments, protein gene-product 9.5 (PGP 9.5), and nerve terminals, synaptophysin (SYN), showed nerve fibres at G15 and nerve terminals at G19 in the perichondrial tissue. From P4, nerve fibres and terminals were observed within the bone organ. Noradrenergic sympathetic nerves, containing NPY, were first discerned at birth, G21, in the perichondrial tissue and within the bone organ at P4. Autonomic cholinergic nerve fibres, indicated by immunoreactivity to VIP, exhibited a similar temporal and regional occurrence. The diaphyseal parts were first supplied with autonomic nerves at P4. The nerve fibres extended into the metaphyses at P6-8 and finally into the epiphyses at P10, concomitant with the first signs of mineralization. Vascular as well as non-vascular nerve fibres were seen. The study shows that developing bone organ is supplied with autonomic nerves from birth, and the the growth of nerves parallels the mineralisation process. Previous studies have demonstrated that NPY potently inhibits parathyroid hormone (PTH) induced effects on osteoblastic bone cells and that VIP is a strong inductor of bone resorption. NPY and VIP also exert vasoregulatory effects. The combined findings suggest an autonomic influence on bone development.

Interleukin-1 immunoreactive nerves in heterotopic bone induced by DBM.

The occurrence of interleukin-1-positive nerves was investigated by immunohistochemistry in developing heterotopic bone, induced by demineralized allogeneic bone matrix (DBM) in the rat. Interleukin-1 immunoreactivity was observed 1 week after implantation and remained until the end of the experiment at 12 weeks. Immunoreactive material was first identified in mononuclear cells at day 7. Interleukin-1 immunoreactive nerve fibers were first observed in the fibrous tissue at 2 weeks after implantation. A maximum density of fibers was reached at 8 weeks. Abundant immunofluorescent fibers were observed in the marrow tissue of the ossicles, and also in the surrounding fibrous tissue. A substantial number were vascular, but in the bone marrow most of the nerve fibers appeared as irregularly arranged, non-vascular terminals with ramifications and varicosities, intermingled between the marrow cells. No fibers could be detected in the proper bone tissue. The distribution of interleukin-1-positive nerves in the ossicles strongly resembled that previously observed in rat long bones. Moreover, the shape and distribution of the fibers exhibited a striking similarity to that of noradrenergic fibers identified previously both in ossicles and normal rat long bones. The late occurrence and predominant distribution in marrow tissue would seem to imply that neuronal interleukin-1 does not participate in the early differentiation of bone cells. The most important finding seems to be the presence of interleukin-1-positive nerve terminals in blood vessel walls and amidst marrow cells.

Neuropeptide Y- and vasoactive intestinal polypeptide-like immunoreactivity in adjuvant arthritis: effects of capsaicin treatment.

The occurrence of the neuropeptides vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) in ankle joints and dorsal root ganglia (L2-L6) was analyzed in normal and arthritic Lewis rats. In addition the effect of capsaicin pretreatment was investigated. The study included 92 rats consisting of 4 groups, 23 rats in each; normal rats, normal rats given capsaicin, arthritic rats and arthritic rats pretreated with capsaicin. The localization of the neuropeptides was assessed by immunohistochemistry and the tissue concentrations were determined by radioimmunoassay (RIA). In the arthritic rats, there was a slight increase in NPY immunoreactive nerve fibres in the ankle joint synovium and bone marrow, as compared to normal rats. Notably, there was an intense fluorescence and significant increase (p < 0.01, 41%) in the number of NPY-positive megakaryocytes in the tibial bone marrow of arthritic rats. RIA showed that the concentration of NPY-like immunoreactivity (LI) was increased by 50% in the ankle joint. Pretreatment with capsaicin did not affect the increased level of NPY-LI in the ankle joint of arthritic rats. The concentration of NPY-LI in the dorsal root ganglia was not altered in arthritic rats, nor was it affected by the capsaicin treatment. No NPY immunoreactive cells could be detected in the dorsal root ganglia. The number of VIP immunoreactive nerve fibres observed in ankle joints of arthritic and normal rats did not differ. However, RIA measurements showed an 11% increase in the VIP concentration in arthritic rats, which was unaffected by capsaicin treatment. In dorsal root ganglia, RIA disclosed a 21% increase in VIP-LI, although no VIP-positive cells could be detected. Capsaicin treatment did not affect the increased concentration of VIP-LI in the dorsal root ganglia.

Increased levels of substance P and calcitonin gene-related peptide in rat adjuvant arthritis. A combined immunohistochemical and radioimmunoassay analysis.

OBJECTIVE: To analyze the occurrence of substance P (SP) and calcitonin gene-related peptide (CGRP) in ankle joints and corresponding dorsal root ganglia (L2-L6) of rats with adjuvant arthritis. METHODS: Arthritis was induced by inoculation with heat-killed mycobacteria. The morphologic distribution of SP and CGRP was assessed by immunohistochemical analysis. Tissue concentrations of the neuropeptides were determined by radioimmunoassay. RESULTS: Neuronal CGRP-like immunoreactivity was clearly increased in the synovium and the dorsal root ganglia, whereas the increase in SP-positive structures was less pronounced. The tissue concentrations of SP and CGRP were significantly increased both in ankle joints and in dorsal root ganglia. CONCLUSION: Levels of sensory neuropeptides are increased under conditions of joint inflammation.

Capsaicin effects on substance P and CGRP in rat adjuvant arthritis.

The effects of capsaicin on the sensory neuropeptides substance P and calcitonin gene-related peptide were analyzed in the ankle joints and dorsal root ganglia (L2-L6) of adult female Lewis rats. The study included 23 normal rats and 23 arthritic rats, all injected subcutaneously with capsaicin (total dose 200 mg/kg bw). Another two groups of animals from a previous study, i.e., 23 normal rats and 23 arthritic rats not given capsaicin served as controls. Adjuvant arthritis was induced by inoculation with heat-killed mycobacteria. The morphological distribution of sensory neuropeptides was assessed by immunohistochemistry and the tissue concentrations were determined by radioimmunoassay. In normal rats, capsaicin significantly reduced the concentrations of substance P and calcitonin gene-related peptide in ankle joints (54 and 36%, respectively) as well as dorsal root ganglia (40 and 54%, respectively). In arthritic rats those pretreated with capsaicin had significantly lower concentrations of substance P and calcitonin gene-related peptide in dorsal root ganglia (19 and 42%, respectively) compared to the arthritic controls. In the ankle joints, however, only the SP concentration was reduced (42%). Notably, this was accompanied by a 40% reduction in inflammatory response as assessed by comparing the ankle joint weights of the experimental groups. In general, there was a good correlation between the neuropeptide concentrations in ipsilateral ankle joints and the corresponding dorsal root ganglia as assessed in individual rats. The present study of adjuvant induced arthritis shows that capsaicin administration reduces the otherwise up-regulated levels of sensory neuropeptides in dorsal root ganglia and ankle joints. However, capsaicin at the dose given can only mitigate, not completely prevent the development of joint inflammation. Nonetheless, the findings suggest that antineuronal therapy targeted against specific neurotransmitters may prove useful in inflammatory joint disease.

Vasoconstrictive action of neuropeptide Y in bone. The porcine tibia perfused in vivo.

The hemodynamic effects of neuropeptide Y (NPY) and norepinephrine (NE) in bone were studied by infusion into the nutrient artery of an in vivo and in situ perfused tibia in 19 pigs. NPY and NE caused elevation of the perfusion pressure and decline in intraosseous pressure, which was evidence of intraosseous vasoconstriction. The study suggests that NPY, along with NE, acts as a sympathetic neurotransmitter in the control of vascular tone in bone.

Interleukin-1 immunoreactive nerve fibres in rat joint synovium.

The occurrence of interleukin-1 immunoreactive nerves in the synovial membrane of rat knee joints was investigated by immunohistochemistry. Synovial tissue sections from 11 rats consistently showed interleukin-1 positive nerve fibres. The majority of the fibres appeared in blood vessel walls. However, varicose interleukin-1 positive fibres were also seen to terminate amidst synoviocytes. The overall distribution resembled that of autonomic fibres previously identified in synovium. Further investigation by double staining disclosed the co-existence of interleukin-1 and neuropeptide Y in the synovial nerve fibres. It has been suggested that the nervous system is implicated in the pathogenesis of arthritis. Considering the role of the cytokine interleukin-1 in various immunogenic and inflammatory conditions, it may prove that neuronal interleukin-1 in the synovial membrane represents a pathway for mediating such effects in joint tissue.

Extraction and quantitation of neuropeptides in bone by radioimmunoassay.

The feasibility of extracting and quantifying neuropeptides in bone by radioimmunoassay was investigated in a study including 60 diaphyseal rat femora. Substance P, calcitonin gene-related peptide, neuropeptide Y and vasoactive intestinal polypeptide, previously identified in bone by immunohistochemistry, were extracted from separate homogenates of bone, periosteum and bone marrow in a solution of 4% EDTA and 2 M acetic acid. Measurable amounts of all four neuropeptides in bone, periosteum and bone marrow were obtained by radioimmunoassay in a reproducible manner. The neuropeptide immunoreactivities were characterized by reverse-phase high performance liquid chromatography. Among the four neuropeptides analyzed, neuropeptide Y consistently exhibited the highest concentrations in the different tissues. Overall, cortical bone showed the lowest neuropeptide concentrations. The concentration of vasoactive intestinal polypeptide was higher in periosteum than in bone marrow, whereas that of calcitonin gene-related peptide was uniform in these tissues. The distributional differences observed in bone tissue may be explained by a variety of physiological roles attributed to neuropeptides such as regulation of nociception, vasoactivity, immune function and local bone metabolism. The described methodology offers a new means of investigating a neuropeptidergic involvement in various disorders of the skeleton.

Extraction of neuropeptides from joint tissue for quantitation by radioimmunoassay. A study in the rat.

The feasibility of extracting neuropeptides from rat knee joints for quantitation by radioimmunoassay was tested. The investigation, based on 25 adult Lewis rats, focused on substance P, calcitonin gene-related peptide, neuropeptide Y, and vasoactive intestinal polypeptide. The relative recovery of the peptides in different extraction media was assessed Both knee joints including the articulating epiphysis were dissected and cut into small pieces. The series was divided into five subgroups, 10 joints in each, for extraction in five different media: 1) 1 M acetic acid in 4% EDTA, 2) 2 M acetic acid in 4% EDTA, 3) neutral water in 4% EDTA, 4) 2 M acetic acid in 4% EDTA and 95% alcohol, and 5) 2 M acetic acid without EDTA. Measureable concentrations of the four neuropeptides were reproducibly assessed by RIA. Although all extraction media provided measurable concentrations, 2 M acetic acid in 4% EDTA was found to give the highest overall yield of the four neuropeptides analyzed. Reverse-phase HPLC confirmed that the immunoreactivities assessed by RIA corresponded to the four neuropeptides of interest. Experimental and clinical evidence suggest a neurogenic involvement in the pathophysiology of inflammatory joint disease, e.g., rheumatoid arthritis. The extraction procedure described offers a means of determining neuropeptide concentrations in joint tissue under normal and pathologic conditions by RIA.

Sensory and autonomic innervation of the facet joint in the rat lumbar spine.

The presence of sensory and autonomic nerves in the synovial membrane of the lumbar facet joint in rats was investigated by immunohistochemistry. Substance P and calcitonin gene-related peptide immunoreactivities, representing sensory nerves, were observed as varicose fibers in the synoviocyte layer. The fibers were predominantly nonvascular. The autonomic innervation was identified by the presence of neuropeptide Y- and tyrosine hydroxylase-positive fibers. Most of these fibers were found adjacent to or within blood vessel walls. Immunoreactivity to vasoactive intestinal polypeptide was seen in varicose nerve terminals in the synoviocyte layer, mostly unrelated to blood vessels. There is accumulating evidence of an involvement of both the sensory and sympathetic nervous systems in inflammatory joint disease. The neuropeptides now identified in lumbar facet joints may prove to play a significant role in the pathogenesis of low-back pain.

Neuropeptide Y, tyrosine hydroxylase and vasoactive intestinal polypeptide-immunoreactive nerve fibers in the vertebral bodies, discs, dura mater, and spinal ligaments of the rat lumbar spine.

The occurrence of autonomic nerves in the lumbar spine of rats was investigated by immunohistochemical technique. Both peptidergic nerves, represented by immunoreactivity to neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), and noradrenergic nerves as reflected by tyrosine hydroxylase (TH) immunoreactivity, were identified. NPY- and TH-positive nerves were predominantly found in the blood vessels walls. They occurred in the bone and periosteum of the vertebral body, disc, dura mater, and in the spinal ligaments. They were particularly numerous along the growth plate and in the outer layers of the intervertebral discs. VIP-positive fibers were predominantly nonvascular. They occurred in all tissues analyzed, but were most abundant in the vertebral bone marrow and periosteum. The VIP-immunoreactive fibers in the outer fibrous layers of the disc and the spinal ligaments were occasionally observed in blood vessel walls. No immunoreactivity could be detected in the nucleus pulposus. In the dura mater, NPY-, TH- and VIP-positive fibers were found both in the ventral and dorsal portion. In view of the vasoconstrictive properties of both NPY and noradrenaline, it may be assumed that the abundance of NPY- and TH-immunoreactive nerves fibers in blood vessel walls reflects a vasoregulatory activity. The predominance of nonvascular VIP-positive fibers in the vertebral bone marrow and periosteum may represent an involvement in local bone physiology.

Neuroendocrine regulation of cyclic AMP formation in osteoblastic cell lines (UMR-106-01, ROS 17/2.8, MC3T3-E1, and Saos-2) and primary bone cells.

The effect of four different neuropeptides and norepinephrine (NE) on cyclic AMP formation in four different osteoblastic cell lines and in isolated neonatal mouse calvarial bone cells has been examined. In the rat osteosarcoma cell line UMR-106-01, vasoactive intestinal polypeptide (VIP, 0.001-1 microM), calcitonin gene-related peptide (CGRP, 0.3-30 nM), and NE (0.1-300 microM), but not neuropeptide Y (NPY, 0.001-1 microM) or substance P (SP, 0.1-10 microM), caused a dose-dependent stimulation of cyclic AMP formation. The stimulatory effects were synergistically potentiated by forskolin (0.1-3 microM). The effects of NE and VIP were time dependent, with an optimal effect seen at 5 minutes. The amount of cyclic AMP accumulated in cells stimulated with NE and VIP was in the same range. The amplitude of the cyclic AMP response induced by CGRP was smaller than that caused by VIP and NE. In the human osteosarcoma cell line Saos-2, NE (0.1 microM) and VIP (0.3 microM) stimulated cyclic AMP formation, and the effect was synergistically potentiated by forskolin. In the absence of forskolin, no effect of CGRP (30 nM) could be seen in the Saos-2 cells, but in the presence of forskolin (3 microM) a stimulatory effect was observed. SP and NPY did not change basal cyclic AMP levels in Saos-2 cells. In the osteoblastic osteosarcoma cell line of rat, ROS 17/2.8, NE (0.1 microM) caused a significant stimulatory action on cyclic AMP formation that was synergistically potentiated by forskolin (3 microM), VIP, CGRP, and SP did not affect the cellular content of cyclic AMP in ROS 17/2.8.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroendocrine peptides in bone.

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.

The occurrence of neuropeptides at different stages of DBM-induced heterotopic bone formation.

In developing heterotopic bone in the rat, induced by allogeneic bone matrix, we immunohistochemically detected nerves containing substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and tyrosine hydroxylase (TH). After 10 days they were dicernible amidst differentiating chondroblastoid cells in fibrous tissue around and within the implants. Over the next 3 weeks, the nerves increased in number and gradually attained a shape and distribution resembling normal osseal nerves; varicose fibres frequently occurred in periosteum-like fibrous tissue and bone marrow adjacent to newly formed bone. At 8 weeks, NPY-fibres increased, particularly in the marrow and this abundance of NPY fibres remained at 16 weeks. VIP-immunoreactive fibres were only observed in the surrounding periosteum-like fibrous tissue 4-6 weeks after implantation. These observations, in combination with recent findings of receptors to neuropeptides on bone cells, suggest a neurogenic influence on physiological processes in bone tissue.