Architecture of connective tissue regenerated by enamel matrix derivative around hydroxyapatite implanted into tooth extraction sockets in the rat maxilla.

We investigated the architecture of periodontal ligament regenerated by an enamel matrix derivative (EMD, Emdogain®) coating on the surface of hydroxyapatite (EMD-HA). Immediately after extraction of the maxillary first molar in rats, HA alone or EMD-HA was implanted into the socket. At 5 days, and 2 and 4 weeks after implantation, the specimens were examined by light and transmission electron microscopy, and immunohistochemistry for periostin and matrix metalloproteinase (MMP)-13. Histological observations revealed a large number of fibroblasts and well-developed blood capillaries in the fibrous connective tissue surrounding EMD-HA at 5 days. Ultrastructural analysis showed a distinct difference in the architecture of the fibrous connective tissue. As compared with the poorly constructed architecture of HA, EMD-HA had an orderly alignment of fibroblasts and bundled collagen fibers, with some fibroblasts in the cytoplasm showing collagen fiber phagocytosis. Periostin immunoreactivity was observed in the fibrous connective tissue around EMD-HA at each time point, but was not seen in HA at 5 days and 2 weeks. MMP-13 immunoreactivity was intensely localized in fibroblasts at 5 days and 2 weeks in EMD-HA. The present results indicate that EMD may greatly contribute to a well-developed architecture accompanied by orderly alignment of fibroblasts and bundled collagen fibers, through accelerated induction of periostin, maintenance of fibrillogenesis, and degradation of collagen fibers by extracellular proteinase and phagocytosis.

Metabolic mode peculiar to Meckel's cartilage: immunohistochemical comparisons of hypoxia-inducible factor-1alpha and glucose transporters in developing endochondral bones in mice.

The middle portion of Meckel's cartilage resembles endochondral bone formation accompanied by chondrocyte hypertrophy and death, cartilaginous matrix calcification, and chondroclastic resorption. We examined Meckel's cartilage specimens from mice mandibles taken on embryonic days 14-16 (E14-E16) using immunohistochemistry for hypoxia-inducible factor-1alpha (HIF-1alpha), glucose transporter 1 (GLUT1), glucose transporter 3 (GLUT3), and glucose transporter 5 (GLUT5), and using enzyme histochemistry for glucose-6-phosphate isomerase (GPI), lactate dehydrogenase (LDH), and cytochrome oxidase (COX), along with the periodic acid-Schiff (PAS) reaction, and compared the results with those of endochondral bones from E16 hind limbs. Periodic acid-Schiff-positive glycogen, HIF-1alpha, and GLUT immunoreactivity, and GPI, LDH, and COX activities were observed in Meckel's cartilage in E14 and E15 mandibles. In E16 mandibles, hypertrophic chondrocytes showed a transitory loss of HIF-1alpha immunoreactivity and consumed glycogen, while those closest to the resorption front showed intense immunoreactivity for HIF-1, GLUT3, and GLUT5. Hypertrophic chondrocytes of metatarsals possessed HIF-1alpha immunoreactivity in the nuclei and diminished COX activity, whereas developing tibias showed weak HIF-1alpha immunoreactivity even in hypoxic regions characterized by little or no COX activity. These findings suggest that HIF-1alpha becomes stabilized independently of the concentration of oxygen, and largely contributes to the development and resorption of Meckel's cartilage, probably through shifting the predominant metabolic mode from aerobic to anaerobic glycolysis.

Matrix mineralization as a trigger for osteocyte maturation.

The morphology of the osteocyte changes during the cell's lifetime. Shortly after becoming buried in the matrix, an osteocyte is plump with a rich rough endoplasmic reticulum and a well-developed Golgi complex. This "immature" osteocyte reduces its number of organelles to become a "mature" osteocyte when it comes to reside deeper in the bone matrix. We hypothesized that mineralization of the surrounding matrix is the trigger for osteocyte maturation. To verify this, we prevented mineralization of newly formed matrix by administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and then examined the morphological changes in the osteocytes in rats. In the HEBP group, matrix mineralization was disturbed, but matrix formation was not affected. The osteocytes found in the unmineralized matrix were immature. Mature osteocytes were seen in the corresponding mineralized matrix in the control group. The immature osteocytes in the unmineralized matrix failed to show immunoreactivity with anti-sclerostin antibody, whereas mature osteocytes in the mineralized matrix showed immunoreactivity in both control and HEBP groups. These findings suggest that mineralization of the matrix surrounding the osteocyte is the trigger for cytodifferentiation from a plump immature form to a mature osteocyte. The osteocyte appears to start secreting sclerostin only after it matures in the mineralized bone matrix.

In situ localization of gelatinolytic activity during development and resorption of Meckel's cartilage in mice.

Degradation of Meckel's cartilage in the middle portion is accompanied by hypertrophy and death of chondrocytes, calcification of the cartilaginous matrix, and chondroclastic resorption. We hypothesize that the gelatinolytic activity of matrix metalloproteinases (MMPs) largely contributes to the degradation of extracellular matrix (ECM) in the process. The activity in Meckel's cartilage of mouse mandibular arches at embryonic days 14-16 (E14-E16) was examined by a combination of in situ zymography (ISZ), using quenched fluorescent dye-labeled gelatin as a substrate, with CTT (a selective inhibitor of MMP-2 and -9) or with EDTA (a general MMP inhibitor). On E14 and E15, ISZ showed fluorescence in the perichondrium, in the intercellular septa between chondrocytes, and in the nucleus of chondrocytes. CTT attenuated fluorescence, and EDTA eliminated it. On E16, calcified cartilaginous matrix showed intense fluorescence, and dot-like fluorescence was observed in as-yet uncalcified intercellular septa, even after CTT treatment. EDTA inhibited fluorescence, but unexpectedly intense fluorescence was found in the cytoplasm of hypertrophic chondrocytes facing the resorption front. MMP-2, -9, and -13 immunoreactivity was detected in the perichondrium and chondrocytes of Meckel's cartilage. These findings suggest that MMPs and other proteinases capable of degrading gelatin play an integral role in the development, calcification, and resorption of Meckel's cartilage through ECM reconstitution.

A histotopographic study of the perineal body in elderly women: the surgical applicability of novel histological findings.

Female perineal structures located around the perineal body were histologically examined using semiserial sections obtained from 15 elderly female cadavers. The smooth muscle content of the perineal body was greater in multiparous women. The connective tissue of the perineal body extended inferolaterally and provided a fibromuscular mass that was 10-30 mm long mediolaterally and 3-15 mm long superoinferiorly. The lateral extension (LEX) of the perineal body occupies a space that is surrounded by the vestibular bulb, internal anal sphincter, and levator ani slings. The LEX did not directly connect to the ischiopubic bony rami but did connect indirectly via the vestibular bulb and ischiocavernosus. Thus, the LEX appears to play a critical role for maintaining the topographical relationship between the vagina and the rectum. The surgical approximation of bilateral LEX instead of levator ani may be of key importance when doing a perineorrhaphy. As pudendal nerve branches run along the inferior margin of the LEX, a mediolateral episiotomy may not be the best option.

Effects of local and whole body irradiation on appearance of osteoclasts during wound healing of tooth extraction sockets in rats.

We examined effects of local and whole body irradiation before tooth extraction on appearance and differentiation of osteoclasts in the alveolar bone of rat maxillary first molars. Wistar rats weighting 100 g were divided into three groups: non-irradiation group, local irradiation group, and whole body irradiation group. In the local irradiation group, a field made with lead blocks was placed over the maxillary left first molar tooth. In the whole body irradiation group, the animals were irradiated in cages. Both groups were irradiated at 8 Gy. The number of osteoclasts around the interradicular alveolar bone showed chronological changes common to non-irradiated and irradiated animals. Several osteoclasts appeared one day after tooth extraction, and the maximal peak was observed 3 days after extraction. Local irradiation had no difference from non-irradiated controls. In animals receiving whole body irradiation, tooth extraction one day after irradiation caused smaller number of osteoclasts than that 7 day after irradiation during the experimental period. Whole body-irradiated rats had small osteoclasts with only a few nuclei and narrow resorption lacunae, indicating deficiency of radioresistant osteoclast precursor cells. Injection of intact bone marrow cells to whole body-irradiated animals immediately after tooth extraction recovered to some content the number of osteoclasts. These findings suggest that bone resorption in the wound healing of alveolar socket requires radioresistant, postmitotic osteoclast precursor cells from hematopoietic organs, but not from local sources around the alveolar socket, at the initial phase of wound healing.

Contributions of matrix metalloproteinases toward Meckel's cartilage resorption in mice: immunohistochemical studies, including comparisons with developing endochondral bones.

The middle portion of Meckel's cartilage (one of four portions that disappear with unique fate) degrades via hypertrophy and the cell death of chondrocytes and via the resorption of cartilage by chondroclasts. We have examined the immunolocalization of matrix metalloproteinase-2 (MMP-2), MMP-9, MMP-13, and MMP-14 (members of the MMP activation cascade) and galectin-3 (an endogenous substrate for MMP-9 and an anti-apoptotic factor) during resorption of Meckel's cartilage in embryonic mice and have compared the results with those of developing endochondral bones in hind limbs. MMP immunoreactivity, except for MMP-2, is present in nearly all chondrocytes in the middle portion of Meckel's cartilage. On embryonic day 15 (E15), faint MMP-2-immunoreactive and intense MMP-13-immunoreactive signals occur in the periosteal bone matrix deposited by periosteal osteoblasts on the lateral surface, whereas MMP-9 and MMP-14 are immunolocalized in the peripheral chondrocytes of Meckel's cartilage. The activation cascade of MMPs by face-to-face cross-talk between cells may thus contribute to the initiation of Meckel's cartilage degradation. On E16, immunopositive signaling for MMP-13 is detectable in the ruffled border of chondroclasts at the resorption front, whereas immunostaining for galectin-3 is present at all stages of chondrocyte differentiation, especially in hypertrophic chondrocytes adjacent to chondroclasts. Galectin-3-positive hypertrophic chondrocytes may therefore coordinate the resorption of calcified cartilage through cell-to-cell contact with chondroclasts. In metatarsal specimens from E16, MMPs are detected in osteoblasts, young osteocytes, and the bone matrix of the periosteal envelope, whereas galectin-3 immunoreactivity is intense in young periosteal osteocytes. In addition, intense MMP-9 and MMP-14 immunostaining has been preferentially found in pre-hypertrophic chondrocytes, although galectin-3 immunoreactivity markedly decreases in hypertrophic chondrocytes. These results indicate that the degradation of Meckel's cartilage involves an activation cascade of MMPs that differs from that in endochondral bone formation.

Operating behind Denonvilliers' fascia for reliable preservation of urogenital autonomic nerves in total mesorectal excision: a histologic study using cadaveric specimens, including a surgical experiment using fresh cadaveric models.

PURPOSE: Little is known about which urogenital nerves are liable to be injured along surgical planes in front of or behind Denonvilliers' fascia. METHODS AND RESULTS: Using semiserial histology for five fixed male pelves, we demonstrated that: 1) left/right communicating branches of bilateral pelvic plexuses run immediately in front of Denonvilliers' fascia; and 2) a lateral continuation of Denonvilliers' fascia separates the urogenital neurovascular bundle from the mesorectum. Notably, the mesorectum contains no or few extramural ganglion cells. At the level of the seminal vesicles, incision in front of Denonvilliers' fascia seems likely to injure superior parts of the pelvic plexus and the left/right communication. Moreover, at the prostate level, this incision misleads the surgical plane into the neurovascular bundle. Fresh cadaveric dissections of five unfixed male pelves confirmed that the surgical plane in front of Denonvilliers' fascia continues to a fascial space for the pelvic plexus containing ganglion cell clusters lateral and/or inferior to the seminal vesicles. CONCLUSIONS: To preserve all autonomic nerves for urogenital function, optimal total mesorectal excision for rectal cancer requires dissection behind Denonvilliers' fascia.

Radiation-induced apoptosis is independent of caspase-8 but dependent on cytochrome c and the caspase-9 cascade in human leukemia HL60 cells.

We investigated the role of the caspase activation cascade in apoptosis induced by ionizing radiation or hydrogen peroxide (H(2)O(2)) in human leukemia HL60 cells. Electron paramagnetic resonance (EPR) spectra revealed that hydroxyl and hydrogen radicals were generated in the culture medium after exposure to radiation or H(2)O(2). Initial accumulation of DNA fragments at 2 h after exposure was delayed in irradiated cells compared with H(2)O(2)-treated cells, although formation of abasic sites immediately after exposure was significantly higher in irradiated cells and similar quantities of hydroxyl radicals were produced under both conditions. Activity assay of caspases revealed that caspase-3, -8 and -9 were activated 2 h after exposure to H(2)O(2), whereas in irradiated cells caspase-3 and -9 activation occurred 4 h after exposure but increased caspase-8 activation was not observed. Release of cytochrome c into cytosol was seen at 2 h after radiation and H(2)O(2) treatment. Radiation did not affect proapoptotic proteins (Bax and Bid), whereas H (2)O(2) increased accumulation of Bax in the mitochondrial membrane 2 h to 6 h after treatment, independently of the truncation of Bid by activated caspase-8. Moreover, treatment with the caspase-8 inhibitor Z-IETD-FMK increased cell survival and prevented accumulation of DNA fragments in H(2)O(2)-treated cells, but not in irradiated cells. These results suggest that, unlike the caspase cascade of H(2)O(2)-induced apoptosis, cytochrome c and caspase-9 are important for the intrinsic pathway of radiation-induced apoptosis, independent of caspase-8.

Immunolocalization of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in Meckel's cartilage compared with developing endochondral bones in mice.

We examined the immunolocalization of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in areas of resorption caused by osteoclasts/chondroclasts on embryonic days 14-16 (E14-16) in Meckel's cartilage, and compared the results with those in endochondral bones in mice. Intense RANKL and OPG immunoreactivity was detected in the chondrocytes in Meckel's cartilage. On E15, when the incisor teeth were closest to the middle portion of Meckel's cartilage, tartrate-resistant acid phosphatase (TRAP)-positive cells appeared on the lateral side of the cartilage. Furthermore, the dental follicle showed moderate immunoreactivity for RANKL and OPG, whereas osteoblasts derived from perichondral cells were immunonegative for RANKL and OPG in that area. On E16, cartilage resorption by TRAP-positive cells had progressed at the differential position, and intensely immunoreactive products of RANKL were overlapped on and found to exist next to TRAP-positive cells in the resorption area. In developing metatarsal tissue, OPG immunoreactivity was intense in periosteal osteoblasts, whereas RANKL was only faintly seen in some of the periosteal cells. In epiphyseal chondrocytes of the developing femur, RANKL immunoreactivity was moderate, and OPG scarcely detected. These results indicate a peculiarity of RANKL and OPG immunolocalization in resorption of Meckel's cartilage. Growth of the incisor teeth may be involved in the time- and position-specific resorption of Meckel's cartilage through local regulation of the RANKL/OPG system in dental follicular cells and periosteal osteoblasts, whereas RANKL and OPG in chondrocytes seem to contribute to resorption through regulation of the chondroclast function.

Impaired alveolization in mice deficient in membrane-type matrix metalloproteinase 1 (MT1-MMP).

To clarify the involvement of membrane-type matrix metalloproteinase 1 (MT1-MMP) in lung organogenesis, we studied the lung morphology of 13-day-old MT1-MMP null mice. The lung architecture in MT1-MMP null mice was abnormal, and the airspace compartments were characterized by smooth walls and larger size. Most of the compartment wall consisted of one or two layers of cells and interstitial connective tissue that was thicker than that of normal alveoli. The wall frequently had capillaries on both sides of the interstitial connective tissue. These findings indicate that the lung in MT1-MMP null mice at 13 days of age is comparable to that of neonatal mice, i.e., it represents the stage before alveolization, suggesting that the generation of a large respiratory surface - the final process of lung development - is impaired in MT1-MMP null mice. Moreover, a zymography assay revealed decreased activity of matrix metalloproteinase 2 (MMP-2) in MT1-MMP null mice, suggesting that activation of pro-MMP-2 by MT1-MMP is critical in this process.

Microfibril-associated glycoprotein-1 and fibrillin-2 are associated with tropoelastin deposition in vitro.

Elastic system fibers consist of microfibrils and tropoelastin. During development, microfibrils act as a template on which tropoelastin is deposited. Microfibril-associated glycoprotein-1 (MAGP-1) and fibrillin-2, the major components of microfibrils, provide the likely template for tropoelastin deposition. In this study, we used the RNA interference (RNAi) technique to establish MAGP-1 and fibrillin-2 gene-specific knock-downs individually in elastin-producing cells (human gingival fibroblasts). We then examined the extracellular deposition of tropoelastin by western blotting. These two genes were specifically suppressed to < 30% of the control level, and this was responsible for the diminution of tropoelastin deposition. An immunofluorescence study also confirmed that RNAi-mediated down-regulation of MAGP-1 or fibrillin-2 led to the loss of tropoelastin immunoreactivity. These results suggest that MAGP-1 and fibrillin-2 are, directly or indirectly, associated with the extracellular deposition of tropoelastin during elastic fiber formation in human gingival fibroblasts in vitro.

Induction of fibulin-5 gene is regulated by tropoelastin gene, and correlated with tropoelastin accumulation in vitro.

Fibulin-5 (also known as DANCE) is an elastin-binding protein that is thought to play a role in elastogenesis. We examined the relationship between the gene expression of fibulin-5 and the gene expression and accumulation of tropoelastin by comparing elastin-producing cells (human gingival fibroblasts) with non-elastin-producing cells (human periodontal ligament fibroblasts) by Northern blot analysis. Fibulin-5 gene induction was found only in elastin-producing cells. Induction of the fibulin-5 gene in elastin-producing cells occurred after induction of the tropoelastin gene, and the fibulin-5 level was reduced upon RNA interference-mediated down-regulation of tropoelastin. Fibulin-5 gene induction was also correlated with a rapid increase of tropoelastin accumulation within the cell layer. These results may suggest that the fibulin-5 gene induction is directly or indirectly regulated by tropoelastin gene expression and plays a role in the accumulation of elastic fibers within matrices.

Expression of bHLH transcription factors NSCL1 and NSCL2 in the mouse olfactory system.

We examined the expression of basic helix-loop-helix transcription factors NSCL1 and NSCL2 in the olfactory epithelium (OE) and the vomeronasal organ (VNO) during development. As detected by in situ hybridization, at embryonic day (E) 10 NSCL1 was weakly expressed in the entire olfactory placodes. From E12 to postnatal day (P) 3, NSCL1 was expressed in olfactory receptor neurons (ORNs) and receptor neurons of the VNO. The expression pattern of NSCL2 was similar to that of NSCL1. By Northern blot analysis, strong expression of NSCL1 was detected in the OE from E12 to P7, but the expression there was low in the adult (P35). NSCL2 mRNA was detected in the E12 and P1 OE, but its level was very low in the P7 and adult OE. The spatial pattern of expression suggests that NSCL1 and NSCL2 contribute to the maturation of ORNs (VNO receptor neurons) or maintenance of their differentiated state. Moreover, the temporal pattern of expression suggests that NSCL1 and NSCL2 may function during development rather than in the adult stage.

Osteoclast differentiation in ectopic bone formation induced by recombinant human bone morphogenetic protein 2 (rhBMP-2).

Osteoclast differentiation in the process of ectopic bone formation induced by recombinant human bone morphogenetic protein 2 (rhBMP-2) was examined to clarify the relationship between osteoclast development and rhBMP-2-induced bone formation. A combination of rhBMP-2 with a porous microsphere (PMS) and blood clot was implanted subcutaneously on the bilateral chest muscles of rats. Tartrate-resistant acid phosphatase (TRAPase) activity, cathepsin K (cath K), and calcitonin receptor (CTR), as markers of osteoclasts and their precursors, were examined using enzyme and immunohistochemical analysis up to 7 days after implantation. Mononuclear cells positive for TRAPase, cath K, and CTR first appeared on day 3 in connective tissue surrounding the PMS after implantation of rhBMP-2. Simultaneously, alkaline phosphatase activity became detectable in mesenchymal cells in the connective tissue. Electron microscopy demonstrated some mononuclear cells with abundant mitochondria and poorly developed rough endoplasmic reticulum in the proximity of mesenchymal cells. However, there was no evidence of cartilage or bone matrix formation on day 3. Osteoclasts in various stages of development, classified by the pattern of immunoreactivity for cath K, were observed by day 7. The polarized intracellular distribution of cath K was found only in osteoclasts attached to bone matrix. In conclusion, we have demonstrated for the first time the appearance of osteoclast precursors before bone matrix formation induced by rhBMP-2, suggesting that bone matrix is not a prerequisite for osteoclast precursor recruitment. Furthermore, we suggest that differentiation into polarized functional osteoclasts is accomplished when the osteoclasts attach to the bone matrix.

Ultrastructural study of synovitis induced by trauma to the rat temporomandibular joint (TMJ).

BACKGROUND: Electron microscopy was used to examine the histologic effect of trauma on the rat temporomandibular joint synovial membrane. METHODS: Trauma to the TMJ in male Wister rats (100-200 g) was introduced through repeated forced condylar hypermobility. Ultrastructural observations were made 5 days and 6 weeks after the trauma. RESULTS: The early response of the synovial membrane was synovial hyperplasia, type A synovial cell loss, dilation of the r-ER in the type B synovial cells and fibrin deposition on the synovial surfaces. The late response included degeneration of synovial cells with swollen mitochondria and cell projections, and cell fragmentation. Large amount of fibrin deposition on opposing surface layers was also noticed. CONCLUSION: The type A cell loss and fibrin deposition followed by the occurrence of fibrinous materials at opposing surface layers of the synovial membrane suggest that traumatic synovitis causes synovial adhesions.

Afferent and efferent lymph-collecting vessels of the submandibular nodes with special reference to the lymphatic route passing through the mylohyoid muscle.

BACKGROUND: Although metastasis of cancer in the oral region to the submandibular node is well described, there has been no anatomic representation of lymph vessels penetrating the oral floor and draining into the node. MATERIALS AND METHODS: Ninety specimens were obtained from formalin-fixed, donated cadavers. Histologic observations using serial sections followed the macroscopic observations. RESULTS: In 19 of 90 specimens, we found afferent collecting lymph vessels exiting from the mylohyoid surface and draining into the preglandular submandibular node. In 3 of the 19 specimens, collecting vessels passing through the narrow muscle gap with or without arteries, veins, and nerves were identified histologically. The postglandular submandibular node was not evident in the drainage route. CONCLUSIONS: Although it carries a low incidence, because of the direct lymphatic route or pathway between the oral region and preglandular submandibular node, the pathologically positive supraomohyoid node sometimes seems to be found even in elective neck dissection. However, we speculate that sentinel node investigation would reveal the much more critical role of the jugulodigastric node not only as the actual sentinel node but also as the common terminal node along the various drainage routes from the oral region.

Apoptosis induced by generated OH radicals inside cells after irradiation.

OH radicals play a major role in radiation-induced DNA and cell membrane damage. These types of damage can also induce death by apoptosis through activation of a pro-apoptosis pathway. We attempted to detect OH radicals inside human promyelocytic leukemia (HL60) cells and estimate the relationship between radiation-induced apoptosis and OH radicals generated inside the cells. Electron spin resonance spectroscopy showed that OH radicals were generated by X-rays within irradiated cell pellets and the relative signal intensities of OH radicals increased with the radiation dose. Agarose gel electrophoresis revealed that the death of HL60 cells by apoptosis was accompanied by internucleosomal DNA fragmentation at 2 h after irradiation with 10-30 Gy. On ultrastructure evaluation by transmission electron microscopy, certain irradiated HL60 cells demonstrated condensed chromatin forms at the nuclear membrane and nuclear fragmentation. The frequency of apoptotic cells with condensation and fragmentation of nuclear chromatin increased with radiation dose in semithin sections. The increase of quantitative DNA fragmentation and percentage of non-living cells also correlated with radiation dose. These results suggest that OH radicals are generated inside cells before apoptosis occurs. The amount of OH radicals generated correlates with apoptotic cell death.

Calcitonin gene-related peptide (CGRP)-containing nerve fibers in bone tissue and their involvement in bone remodeling.

Bone remodeling is a process of bone renewal accomplished by osteoclastic bone resorption and osteoblastic bone formation. These two activities are regulated by systemic hormones and by local cytokines and growth factors. Moreover, the nervous system and certain neuropeptides seem to be involved in regulation of bone remodeling. In this paper, we focus on the distribution of CGRP-containing nerve fibers and their dynamics, and discuss the role of these fibers as a possible mechanism for nervous system involvement in regulation of bone remodeling. CGRP-immunoreactive nerve fibers are widely distributed in bone tissue, such as periosteum and bone marrow, and show apparent regional distribution with different densities. They are often associated with blood vessels and show a beaded appearance. The wide distribution of CGRP-immunoreactive nerve fibers in bone tissue and the changes in distribution during bone development and regeneration suggest the involvement of these fibers in bone remodeling. The effect of CGRP on bone remodeling could partly be through its action on blood vessels, thereby regulating local blood flow. Moreover, in vitro biochemical data and the localization of CGRP-immunoreactive nerve fibers in the vicinity of bone cells suggest that they are directly involved in local regulation of bone remodeling by elevating the concentration of CGRP in the microenvironment around bone cells, especially during bone growth or repair.