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.

Effects of ovariectomy on trabecular structures of rat alveolar bone.

An association between postmenopausal osteoporosis and tooth loss has been proposed. However, histomorphometrical changes in alveolar bone following estrogen deficiency are rarely reported with data on microtrabecular structural changes. To clarify the relationship between estrogen deficiency and tooth loss, we histomorphometrically analyzed the trabecular structural changes of mandibular alveolar bone in ovariectomized rats. Twenty-four adult female Fischer rats were used. Eight rats were sacrificed on day 0 (baseline). The remaining 16 rats were divided into two groups. One group was ovariectomized bilaterally (OVX) and the other group was subjected to sham surgery (Sham). After administration of tetracycline and calcein, the animals were sacrificed 60 days after surgery. Bone histomorphometry, node-strut analysis and measurement of thickness of alveolar bone proper were performed on the interradicular septum of the first molar on the sagittal surface. The trabecular bone volume and trabecular number of the OVX group were significantly lower than those of the baseline and Sham groups. All of the bone resorptive and formative parameters of the OVX group were significantly higher (about one-and-a-half times) than those of the Sham group. Several osteoclasts were seen lining the irregular, eroded surface facing the bone marrow in the OVX group. Furthermore, the OVX group tended to have low microtrabecular stiffness and showed significantly thinner distal alveolar bone proper than in the baseline and Sham groups. In summary, estrogen deficiency caused osteoporotic changes and thin alveolar bone proper in the interradicular septum of rat first molar. This phenomenon might accelerate destruction of alveolar bone and tooth loss, especially in elderly women affected by periodontal disease.

Immunolocalization of osteoclast differentiation factor in rat periodontium.

The aim here was to observe the immunohistochemical localization of osteoclast differentiation factor (ODF)/receptor activator of NF kappa B ligand (RANKL) in the rat periodontium. Rat mandibles were demineralized and embedded in paraffin, and horizontal and frontal sections were prepared for immunohistochemical analysis. In horizontal sections, immunolocalization of RANKL was marked in the distal area of the periodontium of molars in which osteoclasts appeared, due to physiological tooth drift. In frontal sections, RANKL immunoreactivity was localized on spindle-shaped mesenchymal cells around blood vessels near the bone surface in the periodontium. In addition, immunoreaction for RANKL was detected on structures that appeared to be elongated cell processes near blood vessels in frontal sections. Immunohistochemical examination for the general antigen of nerve-specific protein suggested a similarity between these structures and nerve fibres.

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.

Ultrastructural analysis of bone calcification by using energy-filtering transmission electron microscopy.

In order to elucidate the mechanisms of bone calcification, embryonic rat calvariae treated with chemical or cryo-fixation were observed using transmission electron microscopy by three techniques: fine structures, various cvtochemical localizations including nonspecific proteoglycan, decorin, chondroitin 4-sulfate, hyaluronan, alkaline phosphatase (ALP), and osteonectin, as well as the elemental mapping of calcium and phosphorus by energy-filtering electron microscopy. In the calvariae, the calcification sequence ran as follows crystallization within matrix vesicles, formation of calcified nodules, collagen calcification, and finally the establishment of an expansive calcified matrix. The osteoid contained an abundance of mesh-like fibers of proteoglycans, including decorin, chondroitin 4-sulfate, and hyaluronan, around collagen fibrils approximately 50 nm in diameter. Calcium tended to localize at the proteoglycan sites, while phosphorus was often mapped to the collagen fibril-structures in the osteoid. Calcium/phosphorus co-localization was found in and around the calcified nodules, where ALP and small sized proteoglycans were observed. During this stage, native proteoglycans surrounding the collagen fibrils disappeared, with the collagen fibrils fusing laterally, and attaining a diameter of more than 400nm. The calcified nodules expanded to occupy the entire space made available by the collagen fibril-fusion, following osteonectin accumulation in the calcified nodule/collagen fibril border. In conclusion, crystals present within the matrix vesicles became calcified nodules, in a process induced by the co-localization of calcium and phosphorus. ALP and proteoglycans may participate in the calcium/phosphorus co-localization. Decreases in the native proteoglycans, and the lateral fusion of collagen fibrils are thought to be involved in the expansion of calcified areas, followed by osteonectin-mediated collagen calcification.

Role of stromal cells in osteoclast differentiation in bone marrow.

Bone marrow stromal cells have been considered to play an important role in osteoclast differentiation. However, the interaction of these cells in vivo has not been clearly demonstrated. To clarify this, we examined the distribution of alkaline phosphatase (ALPase) and tartrate-resistant acid phosphatase (TRAPase) activities as markers of osteoblastic and osteoclastic cells, respectively. Rat tibiae were fixed and embedded in Technovit 8100 or paraffin. ALPase and TRAPase activities were detected simultaneously on a plastic section by the azo-dye method. ALPase activity was detected on the plasma membranes of osteoblasts and some bone marrow fibroblastic stromal cells. These ALPase-positive cells were connected to each other by cytoplasmic processes, forming a cellular network in bone marrow. The ALPase activity of fibroblastic stromal cells tended to be stronger in those cells close to the bone surface than in the cells in the center of bone marrow. Reticular fibers in bone marrow were found to form a network. The ALPase-positive fibroblastic stromal cells may be reticular cells, because the localization of those cells was in accord with the localization of reticular fibers. The TRAPase-positive mononuclear cells and osteoclasts were mostly observed to be associated with the intensely ALPase-positive fibroblastic stromal cells. Immunoreactivity of osteoclast differentiation factor (ODF) was found in the fibroblastic stromal cells. These findings suggest that the network of ALPase-positive fibroblastic stromal cells in bone marrow serves as a guide for the migration of osteoclast precursor cells toward the bone surface, and may control the differentiation and activity of osteoclasts.

Detection and characterization of glutathione S-transferase activity in rice EF-1betabeta'gamma and EF-1gamma expressed in Escherichia coli.

Plant elongation factor EF-1 consists of four subunits (EF-1alphabetabeta'gamma). EF-1alpha. GTP catalyses the binding of aminoacyl-tRNA to the ribosome. EF-1beta and EF-1beta' catalyze the GDP/GTP exchange on EF-1alpha. GDP. However, the function of EF-1gamma, a subunit detected in eukaryotes, but not in prokaryotes remained unknown. This report demonstrates that rice EF-1betabeta'gamma and recombinant EF-1gamma possess glutathione S-transferase (GST) activity. The EF-1betabeta'gamma- or EF-1gamma-dependent GST activity is about one-fiftieth of the rice GST activity. The Km values of EF-1betabeta'gamma, EF-1gamma, and rice GST for glutathione and 1-chloro-2,4-dinitrobenzene are of about the same order. Although recombinant EF-1gamma is heat labile, active EF-1gamma was obtained by purifying it in the presence of 20% glycerol.

Impact of recombinant human bone morphogenetic protein-2 on residual ridge resorption after tooth extraction: an experimental study in the rat.

Residual ridge resorption begins following tooth extraction and continuously reduces alveolar bone volume, potentially creating a significant problem in dental implant treatment. In this study, the role of recombinant human bone morphogenetic protein-2 (rhBMP-2) in residual ridge resorption after tooth extraction was investigated. A polylactic acid/polyglycolic acid copolymer-coated gelatin sponge carrier was implanted with or without rhBMP-2 (1 microg) in the mesial root sockets after removal of maxillary first molars in male Wistar rats. Fine structural and histomorphologic analyses were conducted 3 to 84 days after implantation. Direct bone formation was first observed after 5 days on the rhBMP-2 side, which was transformed into cortical alveolar ridge with a smooth periosteal layer by 84 days, whereas the control side displayed slower healing. Bone histomorphometry revealed greater total bone area and increased bone height after 14, 28, 56, and 84 days on the rhBMP-2 side compared to the control side, and differences were significant after 14, 28, and 56 days. Larger numbers of proliferating cells and densely populated differentiating mesenchymal cells were observed on the rhBMP-2 side than on the control side in the early stage, and chondrogenesis was not observed. The findings indicate that rhBMP-2 may stimulate proliferation and differentiation of mesenchymal cells in the rat maxillary root socket to preserve cortical bone volume in the socket without any evidence of chondrogenesis.

Cellular distribution of thrombomodulin as an early marker for warm ischemic liver injury in porcine liver transplantation: protective effect of prostaglandin I2 analogue and tauroursodeoxycholic acid.

BACKGROUND: Warm ischemia of the graft from non-heart-beating donors is considered a risk factor for posttransplant graft dysfunction. The early administration of cytoprotective agents may help improve graft dysfunction. METHODS: Four groups of 10 pigs each underwent orthotopic liver transplantation. Prostaglandin I2 analogue, OP-41483, was administered intraportally 30 min before warm ischemic insult in donors and after reperfusion in recipients in one group. In the other study group, additional intravenous tauroursodeoxycholic acid (TUDC) was given before the warm ischemic insult in donors and after reperfusion, then maintained continuously until postoperative day (POD) 7. RESULTS: Exposure of liver grafts to warm ischemia resulted in severe congestion with the disappearance of thrombomodulin (Tm) from the sinusoidal endothelial cells (SECs) and smooth muscle cells (SMCs) around biliary epithelial cells (BEpCs) 2 hr after reperfusion, followed by positive immunoreactivity of Tm in BEpCs with hyperbilirubinemia, which was related to high mortality. Combined administration of OP-41483 and TUDC had a protective effect, demonstrated by sustained immunoreactivity of Tm from SECs and SMCs until POD 7, without that reactivity in BEpCs. This was associated with reduced congestion and hyperbilirubinemia, similar to the control group not subjected to warm ischemia. CONCLUSIONS: These findings suggest that negative immunoreactivity of Tm in SECs and SMCs surrounding BEpCs and positive in BEpCs may be an early marker for ischemic liver injury, and that OP-41483 and TUDC may protect against the microcirculatory and biliary derangement.

Organic components of crystal sheaths in bones.

Crystals in bones are enveloped within organic crystal sheaths of 5-10 nm widths. In order to analyse their components, we investigated the immunolocalizations of chondroitin 4- and 6-sulphate, keratan sulphate, bone sialoprotein and osteopontin. All of these, except chondroitin 6-sulphate, were found in bone matrix. Although the localizations of chondroitin 4-sulphate and keratan sulphate tended to focus within calcified nodules, bone sialoprotein and osteopontin were widely distributed in the area, being linearly arranged along electron-dense structures corresponding to crystal sheaths. These two proteins possess the ability to affect nucleation or elongation of hydroxyapatite, positively or negatively, in vitro. Our results suggested that bone sialoprotein and osteopontin may combine to form the crystal sheaths which are thought to control crystal formation and growth, using the seemingly opposite functions of bone sialoprotein and osteopontin.

Replantation of very thumb distal amputations with pre-osteosynthesis interpositional vein graft.

Replantation of distal thumb amputations is difficult because of the anatomic orientation of the thumb and the location and size of vessels. A simplified technique is presented, in which interposition vein grafts are anastomosed to a terminal branch of the digital artery and a subcutaneous vein in the amputated thumb tip. This technique is convenient, relatively easy and reliable.

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+][PO4(3-)] 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.

Observation of human dentine by focused ion beam and energy-filtering transmission electron microscopy.

Molar dentine was sliced into 100 nm ultrathin sections, by means of a focused ion beam, for observation by energy-filtering transmission electron microscopy (EFTEM). Within the matrix, crystals approximately 10 nm wide and 50-100 nm long were clearly observed. When carbon and calcium were mapped in electron spectroscopic images by EFTEM, carbon failed to localize in crystals. However, it was found in other regions, especially those adjacent to crystals. Because carbon localizations were thought to reflect the presence of organic components, carbon concentration in regions near crystals suggested the interaction of crystals and organics, leading to organic control of apatite formation and growth. Ca was present in almost all regions. The majority of Ca localizing in regions other than crystals may be bound to organic substances present in dentine matrix. These substances are thought to both accumulate Ca and act as reservoirs for crystallization of apatite in dentine.

Molded bone augmentation by a combination of barrier membrane and recombinant human bone morphogenetic protein-2.

OBJECTIVES: To provide the histological background to a new method of local bone augmentation, we examined the events occurring beneath a barrier membrane applied with recombinant human bone morphogenetic protein-2 (rhBMP-2). MATERIALS AND METHODS: The effects on bone augmentation of rhBMP-2, applied with a membrane mold (BMP-Memb), over surgically-induced bone defects in rat calvaria were examined histologically, and the results compared with those from application of rhBMP-2 (BMP) alone, or of a molded membrane (Memb) alone. RESULTS: At postoperative week 2, the BMP group showed the most marked bone formation. However, the bone diminished in size by week 8. The Memb group showed slow but continuous bone formation by week 8. In the BMP-Memb group, bone filled the space in the mold at week 2, and this was maintained until week 8. Moreover, the soft tissue that had intervened between newly formed bone and the membrane in the Memb group was not evident in the BMP-Memb group, in which bone had formed directly on the membrane. CONCLUSIONS: The results suggest that the combination of rhBMP-2 and barrier membrane has advantages in producing and maintaining bone in the intended shape by inducing osteoblasts directly on the inner surface of the membrane.

Dynamical quark effects on light quark masses

Light quark masses are calculated in lattice QCD with two degenerate flavors of dynamical quarks. The calculations are made with improved actions with lattice spacing a = 0.22-0.11 fm. In the continuum limit we find m(M&Smacr;)(ud)(2 GeV) = 3.44(+0.14)(-0.22) MeV using the pi and rho meson masses as physical input, and m(M&Smacr;)(s)(2 GeV) = 88(+4)(-6) MeV or 90(+5)(-11) MeV with the K or straight phi meson mass as additional input. The quoted errors represent statistical and systematic combined, the latter including those from continuum and chiral extrapolations, and from renormalization factors. Compared to quenched results, two flavors of dynamical quarks reduce quark masses by about 25%.

Quenched light hadron spectrum

We present results of a large-scale simulation for the flavor nonsinglet light hadron spectrum in quenched lattice QCD with the Wilson quark action. Hadron masses are calculated at four values of lattice spacing in the range a approximately 0.1-0.05 fm on lattices with a physical extent of 3 fm at five quark masses corresponding to m(pi)/m(rho) approximately 0.75-0.4. The calculated spectrum in the continuum limit shows a systematic deviation from experiment, though the magnitude of deviation is contained within 11%. Results for decay constants and light quark masses are also reported.

Localization of alkaline phosphatase and osteopontin during matrix mineralization in the developing cartilage of coccygeal vertebrae.

We observed the manner in which alkaline phosphatase (ALPase) and osteopontin were localized in the cartilage and intramembranous bone of coccygeal vertebrae during matrix mineralization, shedding considerable light on the manner in which they develop. In the cartilage matrix of coccygeal vertebrae, we observed the localization of ALPase activity in the boundary of the proliferative and the hypertrophic zones. Granular nodules of mineralization were consistently found in the boundary of both zones, and increased in size when close to the hypertrophic zone. While osteopontin was rarely present in the early stages of mineralization, its localization along the margins of mineralized matrices in the hypertrophic zone was prominent. In contrast to cartilage, mineralized nodules in the intramembranous bone in the mid-portion of the vertebra displayed osteopontin-immunoreactivity, indicating its early synthesis and subsequent accumulation to early-stage mineralized nodules. When blood vessels, accompanied by osteoblastic and osteoclastic cell populations, invaded the cartilage, osteopontin was localized in the lower region of the hypertrophic zone, despite its maintaining the localization of ALPase and early-stage mineralization. Thus, our investigation demonstrated ALPase activity consistent with early-stage mineralization in the cartilage matrix. However, the fact that osteopontin-localization could not be pinpointed might account for its multifunctionality as concerns both the regulation of mineralization and the attachment of migrating osteogenic and osteoclastic cells to the mineralized matrix.

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.

Effect of intermittent administration of human parathyroid hormone (1-34) on the mandibular condyle of ovariectomized rats.

Intermittent administration of human parathyroid hormone (1-34) (PTH) increases bone mass in lumbar vertebrae and long bones of osteoporotic experimental animals. However, whether PTH has the same effect on jaw bones remains unclear. This study determined the effect of intermittent administration of PTH on rat mandibular condyle affected by estrogen deficiency. Fifty 6-month-old rats were either sham operated or ovariectomized, then divided into five groups depending on surgical procedure and hormone administration: sham + vehicle (SV), OVX + vehicle (OV), OVX + PTH 6 micrograms/kg once per week (OP6-1), OVX + PTH 60 micrograms/kg once per week (OP60-1), and OVX + PTH 20 micrograms/kg three times per week (OP20-3). PTH or vehicle was injected intermittently for 6 months in 5 rats of each group either immediately after surgery in a preventive administration experiment, or injected starting 6 months after surgery in a therapeutic administration experiment. The mandibles were excised, and bone morphometry was performed using confocal laser scanning microscopy and soft X-ray images. In both experiments, the bone volume of the OV groups was significantly lower than that of the SV group (P < 0.01); also, depending on dose and frequency, the bone volume of the OP group was higher than that of the OV group, particularly in the OP20-3 group. The value of mineralized surface of the OP groups was significantly higher than that of the OV group (P < 0.01), whereas the value of eroded surface of the OP groups was not significantly higher than that of the OV group. This study indicates that preventive and therapeutic intermittent administration of PTH in ovariectomized rats increase the bone formation in rat mandibular condyle without accelerating bone resorptive activity. This anabolic effect was best induced by the injection mode of 20 micrograms/kg three times per week.