Melatonin is a potential drug for the prevention of bone loss during space flight.

Astronauts experience osteoporosis-like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O-methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast-inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

Expression of Non-collagenous Bone Matrix Proteins in Osteoblasts Stimulated by Mechanical Stretching in the Cranial Suture of Neonatal Mice.

We investigated the influence of mechanical stretching on the genetic expression pattern of non-collagenous bone matrix proteins in osteoblasts. The cranial sutures of neonatal mice were subjected to ex vivo mechanical stretching. In the non-stretched control group, as osteoblast differentiation progressed, the successive genetic expression of bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) was detected using in situ hybridization, in that order. In the stretched group, the sutures were widened, and after 24 hr of cultivation, a large number of osteoblasts and abundant new osteoid were observed on the borders of the parietal bones. All new osteoblasts expressed BSP and some of them expressed OPN, but very few of them expressed OCN. After 48 hr, more extensive presence of osteoid was noted on the borders of the parietal bones, and this osteoid was partially mineralized; all osteoblasts on the osteoid surface expressed BSP, and more osteoblasts expressed OPN than those after 24 hr cultivation. Surprisingly, many of the osteoblasts that did not express OPN, expressed OCN. This suggests that when osteoblast differentiation is stimulated by mechanical stress, the genetic expression pattern of non-collagenous proteins in the newly differentiated osteoblasts is affected.

Trabecular bone microstructure and mineral density in human residual ridge at various intervals over a long period after tooth extraction.

BACKGROUND: Long-term changes of trabecular microstructure in human tooth extraction socket have not been investigated. PURPOSE: To examine the trabecular microstructure of human residual ridges at various intervals following tooth extraction, and to determine whether bone remodeling activity can attain points of relative stability and when such points are reached. MATERIALS AND METHODS: Forty-four bone biopsy specimens were obtained from lower molar or premolar regions of residual ridges. Postextraction times ranged from 1.6 to 360 months. Samples were analyzed using micro-computed tomography and three-dimensional bone morphometry with histological analyses. Trabecular bone parameters were plotted against postextraction times, and a stepwise piecewise linear regression analysis was performed to determine at which points of time these parameters either increased or decreased. RESULTS: Using piecewise linear regression, "inflection points" were found in most trabecular bone parameters between 7 and 12 months postextraction. Among the residual ridge samples, woven trabecular structure became mature, consisting of thick lamellar trabeculae with sufficient bone density, under dynamic bone remodeling until the 7th to 12th month post-tooth extraction. After this period, the mature network structure remained stable with low remodeling activity. CONCLUSION: Bone remodeling of trabecular structure in human residual ridge after tooth extraction had a stabilization period.

Histomorphometric Comparison of Transmandibular Implant and Titanium Implant.

This study was a histomorphometric comparison of the transmandibular implant (TMI) system and titanium implant using a confocal laser scanning microscope (CLSM). Two dogs were used. In each dog, 16 implant holes were made in the bilateral mandibular angle. Four cortical screws in the TMI, four titanium plasma spray (TPS) implants, and four hydroxyapatite (HA) coating implants were inserted and four holes were left as a control. Two dogs were sacrificed at 3 and 6 months. The samples were cut longitudinally and examined under CLSM. The contact rate of trabecular bone was smaller than that of cortical bone in all implants. The contact rates in TMI at 3 and 6 months was significantly smaller than those of TPS and HA and there was no difference between TPS and HA. Although the contact rate of TMI was low, only a small contact area may be enough for TMI because of its box form structure.

Differences in vertebral, tibial, and iliac cancellous bone metabolism in ovariectomized rats.

Bone histomorphometry is usually performed on the iliac bone in humans and the tibia or vertebrae in rats. Bone metabolism differences among skeletal sites may be problematic when translating experimental results from rats to humans, but data on such differences in rats are lacking. Therefore, we examined the differences in bone structure and metabolism among skeletal sites using the lumbar vertebra (LV), tibia, and iliac bone obtained from ovariectomized or sham-operated rats preoperatively and at various times from 3 days to 26 weeks postoperatively. The trabeculae were thicker in the LV, where bone metabolism was less active than at other sites, and numerous fine trabeculae were observed in the tibia, where bone metabolism was more active. The iliac bone structure and metabolism were intermediate between those of the tibia and LV. Ovariectomy induced lower bone volume and higher bone metabolism in all skeletal sites, but the changes were greatest and occurred earliest in the tibia, followed by the iliac bone and then LV. Ovariectomy caused changes in bone metabolic markers, which occurred earlier than those in bone tissue. Activation frequency (Ac.f) increased after ovariectomy. At week 26 in ovariectomized rats, Ac.f was highest in the tibia (3.13 N/year) but similar between iliac bone (0.87 N/year) and LV (1.39 N/year). Ac.f is reportedly 0.3-0.4 N/year in the iliac bone of postmenopausal women, suggesting that bone turnover in rats is several times higher than in humans. The reference values reported here are useful for translating experimental results from rats to humans.

Microstructural Observation with MicroCT and Histological Analysis of Human Alveolar Bone Biopsy from a Planned Implant Site: A Case Report.

The subject was a 53-year-old male. An alveolar bone sample was obtained from the site of the lower left first molar, before dental implant placement. Although the details of the trabecular structure were not visible with conventional computed tomography, micro-computed tomography (microCT) three-dimensional images of the alveolar bone biopsy sample showed several plate-like trabeculae extending from the lingual cortical bone. Histological observations of the bone sample revealed trabeculae, cuboidal osteoblasts, osteoclasts and hematopoietic cells existing in the bone tissue at the implantation site. Bone metabolic markers and calcaneal bone density were all within normal ranges, indicating no acceleration of the patient's bone metabolism. Using microCT, and histological and histomorphometrical techniques, a great deal of valuable information about the bone tissue was obtained from a biopsy sample extracted from the patient's planned implant site.

Static and dynamic hypergravity responses of osteoblasts and osteoclasts in medaka scales.

Fish scales are a form of calcified tissue similar to that found in human bone. In medaka scales, we detected both osteoblasts and osteoclasts and subsequently developed a new scale assay system. Using this system, we analyzed the osteoblastic and osteoclastic responses under 2-, 3-, and 4-gravity (G) loading by both centrifugation and vibration. After loading for 10 min, the scales from centrifugal and vibration loading were incubated for 6 and 24 hrs, respectively, after which the osteoblastic and osteoclastic activities were measured. Osteoblastic activity significantly increased under 2- to 4-G loading by both centrifugation and vibration. In contrast, we found that osteoclastic activity significantly decreased under 2- and 3-G loading in response to both centrifugation and vibration. Under 4-G loading, osteoclastic activity also decreased on centrifugation, but significantly increased under 4-G loading by vibration, concomitant with markedly increased osteoblastic activity. Expression of the receptor activator of the NF-κB ligand (RANKL), an activation factor of osteoclasts expressed in osteoblasts, increased significantly under 4-G loading by vibration but was unchanged by centrifugal loading. A protein sequence similar to osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, was found in medaka using our sequence analysis. The ratio of RANKL/OPG-like mRNAs in the vibration-loaded scales was significantly higher than that in the control scales, although there was no difference between centrifugal loaded scales and the control scales. Accordingly, medaka scales provide a useful model by which to analyze bone metabolism in response to physical strain.

Correlations between alveolar bone microstructure and bone turnover markers in pre- and post-menopausal women.

OBJECTIVE: Alveolar cancellous bone biopsy samples were extracted during dental implant preparation for investigating microstructural changes due to menopause and relationships between these changes and bone turnover markers. STUDY DESIGN: Subjects were 18 women receiving mandibular implants: premenopausal (n = 5), early postmenopausal (≤5 years; n = 3), and late postmenopausal (>5 years; n = 10). Bone turnover markers were measured and the samples analyzed using microscopic computerized tomography and 3-dimensional bone morphometry. RESULTS: The alveolar bone volume was significantly less in late postmenopausal women than in premenopausal ones. The trabeculae in early postmenopausal women were more separated and rod-like than in premenopausal ones (P < .05). Each alveolar bone parameter was significantly (P < .05) related to at least 1 bone turnover marker. CONCLUSIONS: Alveolar cancellous bone structure begins changing even in early postmenopausal women, and this structure varies in close relationship to bone turnover markers.

Prostaglandin E2 increases both osteoblastic and osteoclastic activity in the scales and participates in calcium metabolism in goldfish.

Using our original in vitro assay system with goldfish scales, we examined the direct effect of prostaglandin E2 (PGE2) on osteoclasts and osteoblasts in teleosts. In this assay system, we measured the activity of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) as respective indicators of each activity in osteoblasts and osteoclasts. ALP activity in scales significantly increased following treatment at high concentration of PGE2(10⁻7 and 10⁻6 M) over 6 hrs of incubation. At 18 hrs of incubation, ALP activity also significantly increased in the PGE2 (10⁻9 to 10⁻6 M)-treated scale. In the case of osteoclasts, TRAP activity tended to increase at 6 hrs of incubation, and then significantly increased at 18 hrs of incubation by PGE2 (10(-7) to 10⁻6 M) treatment. At 18 hrs of incubation, the mRNA expression of osteoclastic markers (TRAP and cathepsin K) and receptor activator of the NF-κB ligand (RANKL), an activating factor of osteoclasts expressed in osteoblasts, increased in PGE2 treated-scales. Thus, PGE2 acts on osteoblasts, and then increases the osteoclastic activity in the scales of goldfish as it does in the bone of mammals. In an in vivo experiment, plasma calcium levels and scale TRAP and ALP activities in the PGE2-injencted goldfish increased significantly. We conclude that, in teleosts, PGE2 activates both osteoblasts and osteoclasts and participates in calcium metabolism.

Effects of intermittent parathyroid hormone treatment on new bone formation during distraction osteogenesis in the rat mandible.

OBJECTIVE: The effect of intermittent parathyroid hormone (PTH[1-34]) treatment on bone regeneration in a rat model of mandibular distraction was evaluated using microcomputed tomography. STUDY DESIGN: After a 5-day latency period, mandibles of 18 rats were distracted at 0.2 mm/12 hours for 10 days, and rats in the PTH and control groups received subcutaneous injections of PTH(1-34) at a dosage of 60 µg/kg body weight or a vehicle only, respectively, 3 times a week. The animals were humanely killed after 10 days of distraction and after 1 week and 3 weeks of consolidation. RESULTS: In reconstructed 3-dimensional images of the distracted mandible, mean bone volumes of the desired region of interest in the PTH group were significantly larger than those in the control group at all time points. CONCLUSIONS: Intermittent PTH(1-34) treatment enhances new bone formation during mandibular distraction in a rat model, and it may be effective for shortening the consolidation period.

Matrix remodeling and cytological changes during spontaneous cartilage repair.

During the repair of articular cartilage, type I collagen (COL1)-based fibrous tissues change into a mixture of COL1 and type II collagen (COL2) and finally form hyaline cartilaginous tissues consisting of COL2. In order to elucidate the changes that occur in the matrix during cartilage repair and the roles of fibroblasts and chondrocytes in this process, we generated a minimal cartilage defect model that could be spontaneously repaired. Defects of 0.3 mm were created on the patellofemoral articular cartilage of rats using an Er:YAG laser and were observed histologically, ultrastructurally and histochemically. At week 2 after this operation, fibroblastic cells were found to be surrounded by COL1 throughout the area of the defect. These cells became acid phosphatase positive by week 4, both taking in and degrading collagen fibrils. Thereafter, the cells became rounded, with both COL1 and 2 evident in the matrix, and showed immunolocalized matrix metalloproteinase-1 or -9. In the region of the bone marrow, the cells became hypertrophic and were surrounded mainly by COL2 and proteoglycans. By the eighth week, the cartilaginous matrix was found to contain abundant COL2, in which collagen fibrils of various diameters were arranged irregularly. These morphological changes suggested that the fibroblastic cells both produce and resolve the matrix and undertake remodeling to become chondrocytes by converting from a COL1- into a COL2-dominant matrix. This process eventually forms new articular cartilage, but this is not completely identical to normal articular cartilage at the ultrastructural level.

Radiological and histologic studies of the mandibular cortex of ovariectomized monkeys.

OBJECTIVE: The objective was to study the radiological and histologic changes in the mandibular cortices of ovariectomized monkeys. STUDY DESIGN: Twelve female, adult, Cynomolgus monkeys (Macaca fascicularis) were used. Under anesthesia, 1 group was bilaterally ovariectomized (OVX), and the other (control group) underwent sham surgery. Seventy-six weeks after surgery, the monkeys were humanely killed, their mandibles were excised, and their mandibular inferior cortices (MIC) and adjacent cortices were examined histologically and with panoramic radiographs and micro computed tomography. RESULTS: Striped shadows were seen on the endosteal side of the OVX cortices on panoramic radiographs. Histologic observation revealed many enlarged pores with eroded surfaces and calcein labeling (indicating osteon remodeling) in the OVX cortices. CONCLUSIONS: In the MIC and adjacent cortices of OVX monkeys, enlarged Haversian canals were seen and there were indications of a high rate of bone turnover. The enlarged Haversian canals resulted in striped shadows and unclear endosteal margins on radiographic images.

Histologic study of the cellular events during rat mandibular distraction osteogenesis.

OBJECTIVE: The cellular events, underlying bone regeneration through rat mandibular distraction osteogenesis (DO) was examined using micro computerized tomography (microCT), histology, and histochemistry. STUDY DESIGN: After 5-day latency, mandibles were distracted at 0.2 mm/12 h for 10 days, and fixed at latency 5 days (L5D), distraction 3, 6, 10 days (D3D, D6D, D10D), and consolidation 1, 3, 6, 10 weeks (C1W, C3W, C6W, C10W). RESULTS: The microCT demonstrated radiopacity at the distraction gap (DG) during C1W, which was filled with new bone at C6W and C10W. At D3D, collagen fibers were aligned along the axis of the distraction vector. At D6D, alkaline phosphatase-positive osteoblasts and intramembranous ossification was observed. Collagen bundles became thicker with new bony trabeculae at D10D. Type II collagen-immunopositive areas first appeared at C1W. At C3W, cartilage tissue and endochondral ossification were found. By C6W, the entire DG had been bridged by new bone. The C10W specimens showed mature lamellar bone. CONCLUSION: Mandibular DO produces bone through both intramembranous and endochondral ossification.

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.

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.

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.

Histological and histomorphometrical changes in rat alveolar bone following antagonistic tooth extraction and/or ovariectomy.

OBJECTIVE: To examine changes appearing in the alveolar bone following the removal of the mechanical stress of occlusal loading, as well as the added influence of estrogen deficiency on such changes. DESIGN: The right mandibular molars of female rats were extracted. After 8 weeks, 12 animals were ovariectomized (OVX), and the other 12 were subjected to sham surgery (sham). Four weeks after surgery, all rats were sacrificed. The left-half and right-half maxillas of the sham group (the sham-occluded side and the sham-extruded side, respectively) and right-half maxilla of OVX group (the OVX-extruded side) were examined by histological observation and bone histomorphometry. RESULTS: The vertical height of alveolar bone in the sham-extruded and the OVX-extruded sides increased as compared with that of the sham-occluded side. In both extruded sides, active bone formation occurred on the surface of the alveolar bone facing the periodontal ligament, but the bone marrow was expanded and the bone volume had decreased in the internal area of the alveolar bone. In the OVX-extruded side, the bone marrow expanded more remarkably than that of the sham-extruded side, and the highest percentage of osteoclast surface was detected. CONCLUSIONS: Around the extruded teeth, there were regional differences in bone dynamics between the internal area of the alveolar bone and the bone surface facing the periodontal ligament, and estrogen deficiency seems to have caused further loss of bone volume in the interior of the alveolar bone supporting the extruded tooth.

Peri-implant stress analysis in simulation models with or without trabecular bone structure.

Most 3-dimensional (3D) finite element analyses (FEAs) simplify the cancellous bone to a block and completely ignore its trabecular structure. Thus, a 3D FEA was performed to compare the peri-implant stress distribution of a model in which the trabecular structure was accurately simulated (precise model) with that of a model with a homogenous cancellous bone component (simplified model). In contrast to the simplified model, the distribution patterns and higher stresses in the precise model may explain the overall bone resorption at the implant-bone interface in load-related implant failures. Further studies using data from the jawbone and a more detailed implant simulation are planned.

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 microm2 in the PTH group versus 93.9 microm2 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.

Calcitonin-induced change in serum calcium levels and its relationship to osteoclast morphology and number of calcitonin receptors.

It has been shown that, in live subjects, the ability of calcitonin (CT) to decrease serum calcium (Ca) levels can be lost in response to its continued or repeated administration. The present study investigated the relationship between such changes of in vivo serum Ca levels and the response of osteoclasts to CT administration, including the downregulation of their CT receptors (CTRs). Rats were either given a single injection of CT or repeated injections at either 6- or 24-h intervals, after which their serum Ca levels were evaluated. Their parietal bones were dissected, and the amount of 125I-labeled elcatonin (125I-eCT) binding to their osteoclasts measured using autoradiography. Ultrastructural changes in the osteoclasts were also examined. Twenty-four hours after a single CT administration, serum Ca levels had dropped, and there was an absence of ruffled borders on the osteoclasts. Less 125I-eCT binding to the osteoclast was found than in the control group. Forty-eight and 72 h after CT administration, serum Ca levels had almost returned to control levels, and the osteoclasts showed ruffled borders once again. The amount of 125I-eCT binding to the osteoclast also recovered to control levels. When these osteoclasts were then incubated in CT, their ruffled borders once again disappeared. In the 6-h interval multiple CT administration schedule subjects, upon inspection 72 h after their first administration (6 h following the final one), serum Ca levels were found to have almost returned to control levels with the presence of osteoclast ruffled borders. The amount of 125I-eCT binding to these osteoclasts was remarkably limited, and no disappearance of the ruffled borders occurred in response to additional CT incubation. In the 24-h interval multiple administration schedule subjects, upon inspection 72 h after their first CT administration (24 h following the final one), there was less 125I-eCT binding than in the single-dose subjects tested 24 h after their injection, and the ability of CT to lower their serum Ca levels was reduced. The ability of CT to lower serum Ca levels was therefore related to the response of osteoclasts to the CT (the disappearance of the ruffled borders), and this response was related to the amount of CTRs available for binding with CT on the osteoclast surface. Furthermore, the reduced effectiveness of CT in response to repeated CT administration was found to be related to the downregulation of the CTRs on the osteoclast surface.