Toshihiko Yajima

Osteoporosis and reduction of residual ridge in edentulous patients.

The relationship between the height of the mandibular residual ridge and the severity of osteoporosis in elderly edentulous patients was investigated. The height of the mandibular residual ridge was measured by use of the mental foramen on panoramic radiographs. The severity of osteoporosis was determined by examining frontal and lateral radiographs of the vertebrae. All of the patients received a blood analysis. The correlation coefficient between age and the height of residual ridge was -0.38, which was statistically significant (p < 0.01). The residual ridge in women was lower than that of men, showing a statistical significance (p < 0.01). The correlation coefficient was -0.42 between degree of severity of osteoporosis and the height of the residual ridge, which was significant (p < 0.01). The parathyroid hormone level was high in the patients with a low residual ridge, and the calcitonin (CT) level was low. This study indicates that osteoporosis strongly affects reduction of the residual ridge in edentulous patients.

Effects of epidermal growth factor on osteoblastic cellsin vitro

SummaryThe effect of epidermal growth factor (EGF) on clone MC3T3-El cells that have osteoblastic activity was examined by phase-contrast microscopy and electron microscopy; hydroxyproline content, collagen synthesis, collagen pattern, and alkaline phosphatase (ALP) activity were also determined. We found that EGF (0.4 ng/ml) transformed the cells from their normal polygonal shape to a spindle-like morphology by 8 h. This hormone also caused dose-related suppression of hydroxyproline content and ALP activity which was detectable 2 days and 1 day, respectively, after EGF addition. Indomethacin did not affect hydroxyproline content and ALP activity, suggesting that the effect of EGF on the cells may not be mediated by prostaglandins. Epidermal growth factor at concentrations of 2 to 50 ng/ml significantly decreased collagen synthesis in the cells, whereas protein synthesis was stimulated. Electron microscopy demonstrated that collagen fiber formation was also reduced by EGF; an immature type of fibril was observed compared with the typical cross-striated one in the controls. Moreover, the hormone treatment also resulted in the appearance of type III collagen in addition to the type I already present in the cells. These suppressive effects of EGF on MC3T3-El cellsin vitro suggest that this hormone may be involved in bone remodellingin vivo as well.

Matrix Mineralization as a Trigger for Osteocyte Maturation

The morphology of the osteocyte changes during the cells 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.

Acid phosphatase activity and intracellular collagen degradation by fibroblasts in vitro

SummaryHuman gingival fibroblasts were cultured with collagen fibrils. The precise process of collagen phagocytosis and the relationship between acid phosphatase activity and intracellular degradation of collagen were investigated by cytochemical methods at the ultrastructural level. The collagen fibrils were first engulfed at one end by cellular processes, or the cell membrane wrapped itself around the middle of the fibrils. Collagen phagocytosis induced acid phosphatase activity in the fibroblast Golgi-endoplasmic reticulum-lysosome system. By application of the tracer lanthanum, deposits were observed in the intercellular spaces and along the fibrils being phagocytosed. At this stage, primary lysosomes were seen in close proximity to the collagen being engulfed, but no signs of fusion were observed. When the fibrils had been interiorized in whole or in part, they ultimately became enclosed within phagosomes, and no tracer was observed along the interiorized portion of the fibrils. Primary lysosomes then fused with these collagen-containing phagosomes to form phagolysosomes. Collagen degradation occurred within these bodies even though the end of a fibril might have protruded outside of the cell. These results suggest that selective and controlled phagocytosis of collagen and intracellular digestion of it may play a central role in the physiological remodeling and metabolic breakdown of the collagen of connective tissues.

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.

Immunolocalization of receptor activator of nuclear factor-κB 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‐κB ligand (RANKL) and osteoprotegerin (OPG) in areas of resorption caused by osteoclasts/chondroclasts on embryonic days 14–16 (E14–16) in Meckels cartilage, and compared the results with those in endochondral bones in mice. Intense RANKL and OPG immunoreactivity was detected in the chondrocytes in Meckels cartilage. On E15, when the incisor teeth were closest to the middle portion of Meckels 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 Meckels cartilage. Growth of the incisor teeth may be involved in the time‐ and position‐specific resorption of Meckels 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.

Stretching modulates oxytalan fibers in human periodontal ligament cells

MATERIAL AND METHODS We subjected periodontal ligament fibroblasts to stretching strain to examine the effects on their formation of oxytalan fibers in cell/matrix layers. RESULTS Stretching increased the levels of fibrillin-1 and fibrillin-2 by 25% relative to the control, but did not affect the gene expression level of either type of fibrillin. Immunofluorescence and immunogold electron microscopy analysis revealed that bundles of oxytalan fibers became thicker under stretching conditions. CONCLUSION These results suggest that tension strain functionally regulates microfibril assembly in periodontal ligament fibroblasts and thus may contribute to the homeostasis of oxytalan fibers in periodontal ligaments.

Effects of running exercise on the mandible and tibia of ovariectomized rats

Abstract To examine the effects of running exercise on the mandible and tibia of ovariectomized (OVX) rats, 26-week-old sham-operated (Sham) and OVX rats 1 week post-ovariectomy were subjected to non-exercise (Sham-Cont and OVX-Cont) and exercise (Sham-Exc and OVX-Exc) for 8 weeks. OVX induced a significant decrease in alkaline phosphatase (ALP) and an increase in tartrate-resistant acid phosphatase (TRAP) activity and a reduction of 17β-estradiol in the serum. In OVX-Cont rats, histology and bone mineral density (BMD) showed bone loss in the proximal tibia, and histology, soft X-ray photographs and bone marrow area (BMA) revealed enlargement of the bone marrow cavity in the neck of the condylar process. In OVX-Exc rats, exercise significantly increased ALP activity, decreased TRAP activity and markedly elevated serum progesterone levels. Histology and BMD in the tibia and histology, X-ray photographs and BMA in the mandible were comparable to those in Sham rats. In Sham-Exc rats, unexpected decreases were observed in serum enzymes and hormones, but the histology and BMD in the tibia and histology, X-ray photographs and BMA in the mandible were very similar to those in Sham-Cont rats, suggesting a decrease of bone turnover with no change of bone mass in the tibia and mandible. We conclude that exercise has a beneficial effect not only on bone loss in the tibia, but also on differential changes in the neck of the condylar process, perhaps by increasing serum levels of progesterone in OVX rats.

Gene Expression and Accumulation of Fibrillin-1, Fibrillin-2, and Tropoelastin in Cultured Periodontal Fibroblasts

The elastic system fibers consist of three types—oxytalan, elaunin, and elastic fibers—differing in their relative microfibril and elastin contents. All three types are found in human gingiva, but human periodontal ligaments contain only elastin-free fibers. We examined cultured human gingival fibroblasts (HGF) and cultured human periodontal ligament fibroblasts (HPLF) to determine the gene expression of fibrillin-1 and fibrillin-2 (the major components of microfibrils) and of tropoelastin. In addition, we assessed the degree of accumulation of these proteins in the extracellular matrix. Northern blot analysis revealed that the level of expression of fibrillin-1 and fibrillin-2 was higher in HGF than in HPLF. However, examination of matrix samples from HGF and HPLF cell layers showed that there was no difference in fibrillin-1 accumulation, although fibrillin-2 accumulated to a much greater extent in the HGF-derived matrix. Tropoelastin was expressed only in and around HGF. These results show a correlation between gene expression and the accumulation of tropoelastin and fibrillin-2 in HGF.

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.