Sadakazu Ejiri

Comparison of the effects of intermittent and continuous administration of human parathyroid hormone(1–34) on rat bone

We compared the effects of synthetic human parathyroid hormone (1-34) (hPTH[1-34]) on rat bones when administered by intermittent injection and by continuous infusion for 4 weeks. Continuous infusion of hPTH(1-34) caused a dose-dependent decrease in the dry weight of the femur and a hyperparathyroidism-like condition in the high-dose group (214 ng/kg/h). In this group, although bone morphology was quite abnormal, the metaphyseal trabecular bone volume increased, whereas the epiphyseal trabecular bone volume tended to decrease, enhancing both bone resorption and formation. The diaphyseal cortical bone of the tibia became markedly porous, and this appeared to be the main cause of the decrease in total bone weight. Conversely, the dry weight of the femur increased in a dose-dependent manner in the intermittent-injection groups. Neither porosity of the cortical bone nor abnormal morphology of the trabecular bone was observed. The volume of the metaphyseal trabecular bone increased while retaining its normal morphology. The epiphyseal trabecular bone formation obviously increased without increase in the number of osteoclasts. In other words, it was postulated that the total weight of bone increased because intermittent injection of hPTH(1-34) increased the trabecular bone volume without loss of the cortical bone volume. These results show that either mode of treatment with hPTH(1-34) continuously accelerated bone formation, whereas the continuous infusion was essential for persistently and markedly enhanced acceleration of bone resorption. It appears that the increase in bone volume with intermittent injection of hPTH(1-34) resulted from the prominent manifestation of its bone-formative action rather than from its bone-resorptive action.(ABSTRACT TRUNCATED AT 250 WORDS)

Osteocytic osteolysis observed in rats to which parathyroid hormone was continuously administered.

In order to prove osteocytic osteolysis in vivo, human parathyroid hormone (hPTH (1–34), 749 ng/h), or only solvent of the same volume, was continuously administered to 8-month-old rats by an infusion pump for 4 weeks, and then structural changes in osteocytes in the cortical bones of the tibiae were analyzed morphometrically, histologically, and histochemically. Based on contact microradiography (CMR) observations, the osteocyte lacunae in the PTH group tended to be enlarged, compared with those of the control, while the average lacuna area was 137.0 µm2 in the PTH group versus 93.9 µm2 in the control, suggesting evidence of osteocytic osteolysis. Acid phosphatase enzyme histochemical localization was observed in some osteocytes in the PTH group; therefore, lysosome systems may participate in the osteolytic mechanisms. On histological samples stained with hematoxylin-eosin or toluidine blue, the lacunae of the controls were surrounded by narrow areas of matrices both positive for hematoxylin and metachromatic for toluidine blue, while belt-like areas positive for hematoxylin were observed around the PTH-group lacunae. These findings suggested that, after osteocytic osteolysis, regenerated bone matrices may be added to the walls of osteocytes that possess enlarged lacunae.

Localizational Alterations of Calcium, Phosphorus, and Calcification-Related Organics Such as Proteoglycans and Alkaline Phosphatase During Bone Calcification

To further approach the mechanisms of bone calcification, embryonic rat calvariae were observed at electron microscopic level by the means of fine structures and various cytochemical localizations, including nonspecific proteoglycan (PG) stained by cuprolinic blue (CB), decorin, chondroitin sulfate, hyaluronan, and alkaline phosphatase (ALP), as well as the elemental mapping of calcium (Ca) and phosphorus (P) by energy‐filtering transmission electron microscopy (EFTEM). In the calvariae, calcification advanced as the distance from osteoblasts increased. Closer to the osteoblasts, the osteoid was marked by an abundance of CB‐positive PGs around collagen fibrils. After crystallization within matrix vesicles, calcified nodules formed and expanded, creating a coherent calcified matrix. The sizes of CB‐positive PG‐like structures diminished as calcification proceeded. Although small CB‐positive structures were accumulated in early stage‐calcified nodules, they were localized along the periphery of larger calcified nodules. Cytochemical tests for decorin, chondroitin sulfate, and hyaluronan determined their presence in the areas around collagen fibrils of the osteoid, as well as in and around calcified nodules, whereas ALP was found in the matrix vesicles, as well as in and around the calcified nodules. Ca tended to localize at the PG sites, while P often mapped to the collagen fibril structures, in the uncalcified matrix. In contrast, Ca/P colocalization was visible in and around the calcified nodules, where ALP and smaller CB‐positive structures were observed. The difference in the localization patterns of Ca and P in uncalcified areas may limit the local [Ca2+][PO43−] product, leading to the general inhibition of hydroxyapatite crystallization. The downsizing of CB‐positive structures suggested enzymatic fragmentation of PGs. Such structural alterations would contribute to the preservation and transport of calcium. ALP possesses the ability to boost local phosphate anion concentration. Therefore, structurally altered PGs and ALP may cooperate in Ca/P colocalization, thus promoting bone calcification.

Bone structure and mineralization demonstrated using synchrotron radiation computed tomography (SR-CT) in animal models: preliminary findings.

We investigated the ability of synchrotron radiation computed tomography (SR-CT) to demonstrate trabecular microstructure, detail of trabecular surfaces, and mineralization of bones. Eight rat vertebrae, six rat tibiae, and eight minipig vertebrae were scanned using SR-CT at the synchrotron radiation facility Super Photon ring-8GeV (SPring-8). Images obtained using conventional micro-CT, scanning electron microscopy (SEM), and contact microradiography (CMR) were compared with the SR-CT images. SR-CT showed high image quality without visible partial volume effect. Three-dimensional SR-CT revealed shallow concavities in the bone surface, which were considered to correspond to osteoclastic resorption areas, as well as the connectivity, anisotropy, and shape (rod- or platelike) of trabeculae. Two-dimensional SR-CT showed different density along the surface of the trabecular bone, indicating the degree of bone mineralization. In conclusion, SR-CT seems to be a useful tool for delineating trabecular surfaces, evaluating bone mineralization, and revealing precise trabecular structure.

Reduction in bone formation and elevated bone resorption in ovariectomized rats with special reference to acute inflammation

Changes in bone modeling and remodeling in the tibia of growing rats within 30 days of ovariectomy (ovx) were evaluated by histomorphometric, mechanical; and biochemical means. Three days after ovx, suppressed bone formation was seen. This was shown by reduced osteoid volume, osteoblast surface, and bone formation rate in the secondary spongiosa, and a reduced longitudinal growth rate in the growth plate. In addition, the alkaline phosphatase and tartrate-resistant acid phosphatase activity in bone marrow supernatants was suppressed in conjunction with elevated serum sialic acid levels, indicating inflammation. Although estrogen deprivation itself may provoke the inflammatory process, the serum sialic acid level in the ovx group returned to the baseline level within 5 days after surgery, while that of estradiol in the ovx group remained consistently lower. This suggests that surgical stress, not estrogen deprivation, is the primary cause of the inflammatory response shortly after ovx. A significant difference (p < 0.01) between the ovx and sham rats was seen in the osteoclast surface, which peaked on day 7 in the ovx rats. On day 14 postovariectomy, the bone formation rate peaked and remained constant until day 30. In the ovx rats, there was a sustained reduction in the serum albumin level until day 30. Estrogen deprivation may be the primary cause of these changes, because both surgical ovx and medical oophorectomy with gonadotropin-releasing hormone agonist (G(nRHa) reduce the serum albumin level. In numerous studies dealing with changes after ovx in rats, we have observed: 1) a transient reduction in bone formation in relation to inflammatory changes evoked by ovx surgery, and 2) a sustained reduction in the serum albumin level for at least 30 days after ovx that is possibly due to estrogen deprivation.

Regulation of osteoclast polarization

Osteoclast function consists of several processes: recognition of mineralized tissues, development of ruffled borders and sealing zones, secretion of acids and proteolytic enzymes into the space beneath the ruffled border, and incorporation and secretion of bone degradation products using the transcytosis system. One of the most important questions concerning osteoclast function is how osteoclasts recognize bone and polarize. During the past decade, new approaches have been taken to investigate the regulation of osteoclast polarization. Attachment of osteoclasts to some proteins containing the Arg-Gly-Asp sequence motif through vitronectin receptors is the first step in inducing the polarization of osteoclasts. Physical properties of bone such as hardness or roughness are also required to induce osteoclast polarity. Osteoclasts cultured even on plastic dishes secrete protons toward the dish surface, suggesting that osteoclasts recognize plastic as a mineralized matrix and secrete protons. This notion was supported by the recent findings that bisphosphonates and reveromycin A were specifically incorporated into polarized osteoclasts cultured even on plastic dishes. On the other hand, a sealing zone, defined as a thick band of actin, is induced in osteoclasts adherent only on an apatite-containing mineralized matrix. These results suggest that osteoclasts recognize physical properties of the mineralized tissue to secrete protons, and also sense apatite itself or components of apatite to form the sealing zone. Here, we review recent findings on the regulation of osteoclast polarization. We also discuss how osteoclasts recognize mineralized tissues to form the sealing zone.

Estrogen deficiency and its effect on the jaw bones

Estrogen deficiency-induced postmenopausal osteoporosis has become a worldwide problem, inducing low bone mass and microarchitectural deterioration of the bone scaffolding in the vertebrae and long bones. With the prevalence of such osteoporosis on the increase, the influence of this estrogen deficiency on the jaw bones has drawn the attention of researchers and clinicians in the field of dentistry. The aim of this article is therefore to review the microstructural changes occurring after ovariectomy in the jaw bones of animal subjects. Induced estrogen deficiency clearly led to structural changes in the jaw bones and alveolar bone of animal subjects (rats and monkeys). Severe bone loss in the rat alveolar bone was principally caused by high bone resorptive activity. This activity accelerated greatly immediately after ovariectomy, and was then followed by more moderate resorptive activity, which continued over an extended period. Additionally, occlusal hypofunction further greatly accelerated the fragility of the alveolar bone structure in ovariectomized rats. Microstructural damage also seen in the alveolar bone of ovariectomized monkeys was found to be directly connected to their systemic osteoporosis. Recent investigations of the relationship in humans between systemic osteoporosis and jaw bone loss have also suggested that a connection may exist between these two. However, more research is required to confirm this connection in humans as well.

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.

Effects of oestrogen deficiency on osteoclastogenesis in the rat periodontium.

This study was designed to investigate quantitative changes in osteoclast generation in rat periodontium following ovariectomy. Wistar rats, aged 9 weeks, were subjected to either ovariectomy (OVX) or sham surgery. Osmotic pumps were implanted in 24 rats and either 17beta-oestradiol or vehicle solution were infused continuously. The rats were assigned to one of the following groups: (1) OVX+vehicle; (2) sham+vehicle; or (3) OVX+ 17beta-oestradiol. On the days 7 and 14 after surgery, four rats in each group were killed. Mandibles were demineralized and embedded in paraffin. Frontal sections of alveolar bone in the region of the first molar were cut for enzyme histochemistry and immunohistochemistry. On day 7, there was no significant difference in the number of tartrate-resistant acid phosphatase (TRAP)-positive cells located on bone surfaces in either group. However, the number of TRAP-positive mononuclear cells that were separated from the bone surface was significantly higher in group 1 than in groups 2 and 3. On the day 14, the number of TRAP-positive cells in group 1, which were attached to the bone surface, was significantly higher than had been apparent on day 7. There were also significant increases in the number of nuclei of TRAP-positive cells attached to the bone in group 1 compared with groups 2 and 3 on day 14. These findings demonstrate that oestrogen deficiency induces of osteoclastogenesis in the rat periodontium and that quantitative changes in osteoclastogenesis could be prevented by E2 infusion.

Relationship between Porotic Changes in Alveolar Bone and Spinal Osteoporosis

Epidemiological studies have shown that post-menopausal women who do not use an estrogen supplement have fewer teeth than those who do. We hypothesized that changes in the dentition of post-menopausal women might be due to alveolar bone alterations by estrogen deficiency. To clarify this, we analyzed the microstructural alveolar bone changes in ovariectomized monkeys and compared these with their lumbar bone mineral density. The % of baseline bone mineral density showed a significant decrease in the ovariectomized group as compared with the controls. The second-molar interradicular septa in ovariectomized monkeys showed a significantly decreased nodes number, cortices number, and an increased structural model index value. More pores were seen in the ovariectomized group at the top of the septa. This study demonstrated that, in such monkeys, estrogen deficiency led to fragility of the trabecular structure of the molar alveolar bone, and such fragility was inversely correlated with lumbar bone mineral density.