Cyclic mechanical pressure-loading alters epithelial homeostasis in a three-dimensional in vitro oral mucosa model: clinical implications for denture-wearers.

Denture-wearing affects the quality and quantity of epithelial cells in the underlying healthy oral mucosa. The physiologic mechanisms, however, are poorly understood. This study aimed to compare histologic changes and cellular responses of an epithelial cell layer to cyclic mechanical pressure-loading mimicking denture-wearing using an organotypic culture system to develop a three-dimensional in vitro oral mucosa model (3DOMM). Primary human oral keratinocytes and fibroblasts were serially grown in a monolayer culture, and cell viability was measured under continuous cyclic mechanical pressure (50 kPa) for 7 days (cycles of 60 min on, 20 s off to degas and inject air). Upon initiation of an air-liquid interface culture for epithelial stratification, the cyclic pressure, set to the mode above mentioned, was applied to the 3DOMMs for 7 days. Paraffin-embedded 3DOMMs were examined histologically and immunohistochemically. In the monolayer culture, the pressure did not affect the viability of oral keratinocytes or fibroblasts. Few histologic changes were observed in the epithelial layer of the control and pressure-loaded 3DOMMs. Immunohistochemical examination, however, revealed a significant decrease in Ki-67 labelling and an increase in filaggrin and involucrin expression in the suprabasal layer of the pressure-loaded 3DOMMs. Pressure-loading attenuated integrin ß1 expression and increased matrix metalloproteinase-9 activity. Incomplete deposition of laminin and type IV collagen beneath the basal cells was observed only in the pressure-loaded 3DOMM. Cyclic pressure-loading appeared to disrupt multiple functions of the basal cells in the 3DOMM, resulting in a predisposition towards terminal differentiation. Thus, denture-wearing could compromise oral epithelial homeostasis.

Serum levels of insulin-like growth factor (IGF); IGF-binding proteins-3, -4, and -5; and their relationships to bone mineral density and the risk of vertebral fractures in postmenopausal women.

We previously found that serum levels of insulin-like growth factor I (IGF-I) and IGF-binding protein (IGFBP)-3, but not IFGBP-2, were associated with bone mineral density (BMD) and the risk of vertebral fractures. The aim of the present study was to investigate the roles of IGFBP-4 and -5 in age-dependent bone loss and vertebral fracture risk in postmenopausal Japanese women and to compare them with those of IGF-I and IGFBP-3. One hundred and ninety-three Japanese women aged 46-88 years (mean 62.5) were enrolled in the cross-sectional study. BMD was measured at the lumbar spine, femoral neck, ultradistal radius (UDR), and total body by dual-energy X-ray absorptiometry. Serum levels of IGFBP-4 and -5 as well as IGF-I and IGFBP-3 were measured by radioimmunoassay. Serum levels of IGF-I, IGFBP-3, and IGFBP-5 declined with age, while serum IGFBP-4 increased with age. Multiple regression analysis was performed between BMD at each skeletal site and serum levels of IGF-I and IGFBPs adjusted for age, body weight, height, and serum creatinine. BMD at the UDR was significantly and positively correlated with all serum levels of IGF-I and IGFBPs measured (P < 0.01), while BMD at the femoral neck was correlated with none of them. Serum IGF-I level was significantly and positively correlated with BMD at all sites except the femoral neck (P < 0.01), while serum IGFBP-3 and -4 levels were significantly and positively correlated with only radial BMD (P < 0.01). Serum IGFBP-5 level was positively correlated with UDR BMD (P < 0.001) and negatively correlated with total BMD (P < 0.05). Serum IGF-I, IGFBP-3, and IFGBP-5 levels were significantly lower in women with vertebral fractures than in those without fractures (mean +/- SD: 97.1 +/- 32.1 vs. 143.9 +/- 40.9 ng/dl, P < 0.0001; 2.18 +/- 1.02 vs. 3.23 +/- 1.07 microg/ml, P < 0.0001; 223.6 +/- 63.3 vs. 246.5 +/- 71.5 ng/ml, P = 0.0330, respectively). When multivariate logistic regression analysis was performed with the presence of vertebral fractures as a dependent variable and serum levels of IGF-I and IGFBPs adjusted for age, body weight, height, serum creatinine, and serum alubumin as independent variables, IGF-I and IGFBP-3 were selected as indices affecting the presence of vertebral fractures [odds ratio (OR) = 0.29, 95% confidential interval (CI) 0.15-0.57 per SD increase, P = 0.0003 and OR = 0.31, 95% CI 0.16-0.61 per SD increase, P = 0.0007, respectively]. To compare the significance values, IGF-I, IGFBP-3, and age were simultaneously added as independent variables in the analysis. IGFBP-3 was more strongly associated with the presence of vertebral fractures than IGF-I and age (P = 0.0006, P = 0.0148, and P = 0.0013, respectively). Thus, after comprehensive measurements of serum levels of IGF-I and IGFBPs, it seems that serum IGF-I level is most efficiently associated with bone mass and that serum IGFBP-3 level is most strongly associated with the presence of vertebral fractures in postmenopausal women among the IGF system components examined.

Statins modulate the levels of osteoprotegerin/receptor activator of NFkappaB ligand mRNA in mouse bone-cell cultures.

Statins stimulate bone formation partly by inducing osteoblast differentiation, although there is controversy about the effects of statins on bone mineral density and fracture risk. Several studies have revealed that statins suppress bone resorption. However, the mechanism by which statins inhibit bone resorption is still unclear. The present study was performed to clarify the effects of statins on osteoclast formation as well as the levels of osteoprotegerin (OPG) and receptor activator of NFkappaB ligand (RANKL) mRNA in mouse bone-cell cultures by semiquantitative RT-PCR. 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] significantly stimulated osteoclast formation and 10(-6) M statins (mevastatin and simvastatin) significantly antagonized osteoclast formation stimulated by 1,25(OH)2D3 in mouse bone-cell cultures, including both osteoblasts and osteoclasts. 10(-6) M mevastatin and simvastatin increased the level of OPG mRNA in mouse bone-cell cultures. On the other hand, 10(-6) M mevastatin and simvastatin inhibited the level of RANKL mRNA in these cultures. In conclusion, the present study demonstrates that statins inhibit osteoclast formation in mouse bone-cell cultures. Moreover, statins also increased and decreased the levels of OPG and RANKL mRNA expression in these cultures, respectively. The modulation of OPG/RANKL may be involved in the inhibition of osteoclast formation by statins.

Testosterone increases osteoprotegerin mRNA expression in mouse osteoblast cells.

The role that androgens play in the regulation of bone metabolism has been substantiated in animals and humans. We previously demonstrated that testosterone inhibits osteoclast differentiation stimulated by parathyroid hormone through the androgen receptor in mouse bone-cell cultures. However, the details of this mechanism are still unknown. The present study was aimed at examining whether testosterone would affect the mRNA levels of osteoprotegerin (OPG) and receptor activator of Nf kappa B ligand (RANKL) in mouse bone-cell cultures as well as mouse osteoblastic cell-line, MC3T3-E1 cells by employing semi-quantitative RT-PCR. Testosterone increased OPG mRNA expression in both mouse bone-cell cultures and MC3T3-E1 cells. 10-8 M PTH-(1-34) as well as 10-8M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibited OPG mRNA expression in mouse bone cells. 10-8 M testosterone antagonized OPG mRNA expression inhibited by 10-8 M PTH-(1-34), but failed to affect OPG mRNA expression inhibited by 10-8 M 1,25(OH)2D3. 10-8 M alpha-dehydrotestosterone, a non-aromatizable androgen, increased OPG mRNA expression. On the other hand, testosterone did not affect RANKL mRNA expression in MC3T3-E1 or mouse bone cells. In conclusion, the present study demonstrated that testosterone increased OPG mRNA expression in mouse bone-cell cultures and the osteoblastic cell line. These effects are likely to take place through the androgen receptor.

Positional dissociation between the genetic mutation responsible for pseudohypoparathyroidism type Ib and the associated methylation defect at exon A/B: evidence for a long-range regulatory element within the imprinted GNAS1 locus.

Pseudohypoparathyroidism type Ib (PHP-Ib) is a paternally imprinted disorder which maps to a region on chromosome 20q13.3 that comprises GNAS1 at its telomeric boundary. Exon A/B of this gene was recently shown to display a loss of methylation in several PHP-Ib patients. In nine unrelated PHP-Ib kindreds, in whom haplotype analysis and mode of inheritance provided no evidence against linkage to this chromosomal region, we confirmed lack of exon A/B methylation for affected individuals, while unaffected carriers showed no epigenetic abnormality at this locus. However, affected individuals in one kindred (Y2) displayed additional methylation defects involving exons NESP55, AS and XL, and unaffected carriers in this family showed an abnormal methylation at exon NESP55, but not at other exons. Taken together, current evidence thus suggests that distinct mutations within or close to GNAS1 can lead to PHP-Ib and the associated epigenetic changes. To further delineate the telomeric boundary of the PHP-Ib locus, the previously reported kindred F, in which patient F-V/51 is recombinant within GNAS1, was investigated with several new markers and direct nucleotide sequence analysis. These studies revealed that F-V/51 remains recombinant at a single nucleotide polymorphism (SNP) located 1.2 kb upstream of XL. No heterozygous mutation was identified between exon XL and an SNP approximately 8 kb upstream of NESP55, where this affected individual becomes linked, suggesting that the genetic defect responsible for parathyroid hormone resistance in kindred F, and probably other PHP-Ib patients, is located >or=56 kb centromeric of the abnormally methylated exon A/B. A region upstream of the known coding exons of GNAS1 is therefore predicted to exert, presumably through imprinting of exon A/B, long-range effects on G(s)alpha expression.

Skeletal responsiveness to parathyroid hormone in pseudohypoparathyroidism.

BACKGROUND: Although there have been some case reports suggesting that bone in patients with pseudohypoparathyroidism (PHP) might respond to parathyroid hormone (PTH), no information is available as to whether serum PTH concentration is related to bone metabolic markers or to bone mineral density (BMD) in PHP. OBJECTIVE: To address these relationships, by comparing intact serum PTH, bone metabolic markers and BMD in patients with PHP with those in patients with idiopathic hypoparathyroidism (IHP) and postoperative hypoparathyroidism (OHP). METHODS: Intact serum PTH, bone metabolic markers (osteocalcin, tartrate-resistant acid phosphatase, pyridinoline, deoxypyridinoline) and BMD by dual-energy X-ray absorptiometry or single-photon absorptiometry were measured in patients with PHP Ia (n=2) and PHP Ib (n=8). The results were compared with those in patients with IHP (n=5) and OHP (n=14). RESULTS: All bone metabolic markers measured were present in significantly greater amounts in patients with PHP Ib than in those with IHP+OHP. The Z score (standard deviation of average BMD at each age) of the BMD of femoral neck was significantly lower in patients with PHP Ib than in those with IHP+OHP. The Z scores of BMD of lumbar spine and radius were also lower in patients with PHP Ib than in those with IHP+OHP, but the difference was not significant. Moreover, the intact serum PTH concentrations were significantly and positively related to bone metabolic marker levels in all patients, and the intact serum PTH concentrations were significantly and negatively related to BMD of lumbar spine in PHP patients. CONCLUSIONS: These results suggest that PTH stimulates bone turnover in PHP Ib patients, resulting in a relatively lower BMD in PHP Ib patients than in IHP+OHP patients. The present study indicates that bones of most cases of PHP could respond to PTH.

Stimulatory effect of insulin-like growth factor binding protein-5 on mouse osteoclast formation and osteoclastic bone-resorbing activity.

Insulin-like growth factor binding protein-5 (IGFBP-5) stimulates osteoblast proliferation directly or indirectly through IGF-I action, but its effects on osteoclast formation and osteoclastic activity are unknown. We tested the effects of IGFBP-5 on osteoclastic activity and osteoclast formation. IGFBP-5 significantly stimulated pit formation by pre-existent osteoclasts in mouse bone cell cultures and its stimulatory effect was completely blocked by IGF-I antibody (Ab). However, IGFBP-5 did not affect the bone-resorbing activity of isolated rabbit osteoclasts. When IGFBP-5 was added to unfractionated bone cells after degeneration of pre-existent osteoclasts, IGFBP-5 (77 pM-7.7 nM) dose-dependently stimulated osteoclast-like cell formation, irrespective of the presence of IGF-I Ab. Moreover, osteoclast-like cells newly formed by IGFBP-5 from unfractionated bone cells possessed the ability to form pits on dentine slices. We next examined the direct effect of IGFBP-5 on osteoclast precursors in the absence of stromal cells, using hemopoietic blast cells derived from spleen cells. IGFBP-5 dose-dependently stimulated osteoclast-like cell formation from osteoclast precursors, irrespective of the presence of IGF-I Ab. Growth hormone (GH) as well as IGF-I significantly stimulated bone resorption by pre-existent osteoclasts in mouse bone cell cultures and these stimulatory effects were completely blocked by IGF-I Ab. GH as well as IGF-I stimulated osteoclast-like cell formation from unfractionated bone cells and this stimulatory effect of GH was significantly but partially blocked by IGF-I Ab. The direct stimulatory effect of GH on osteoclast-like cell formation from hemopoietic blast cells was not affected by IGF-I Ab. The present data indicate that IGFBP-5 stimulates bone resorption both by stimulation of osteoclast formation in an IGF-I-independent fashion and by IGF-I-dependent activation of mature osteoclasts, possibly via osteoblasts, in vitro.

Involvement of osteoprotegerin/osteoclastogenesis inhibitory factor in the stimulation of osteoclast formation by parathyroid hormone in mouse bone cells.

OBJECTIVE: Recently, osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) has been shown to inhibit osteoclast differentiation. On the other hand, we have reported that parathyroid hormone (PTH) stimulated osteoclast formation, presumably through a PTH-responsive cAMP-dependent protein kinase (PKA) pathway, in mouse bone cells. DESIGN AND METHODS: The present study was performed to examine how OPG/OCIF expression is regulated by PTH and to further investigate the possible involvement of OPG/OCIF in the stimulation of osteoclast formation by PTH in mouse bone cells. OPG/OCIF mRNA expression was analyzed by Northern hybridization after 24h treatments of mouse whole bone cells and mouse stromal cell line, ST2 cells with PTH or various second messenger analogs. RESULTS: Human (h) PTH(1-34) (10(-10) and 10(-8)mol/l) but not 10(-8)mol/l hPTH(3-34) down-regulated OPG/OCIF mRNA expression in mouse bone cells. Dibutyryl cAMP, but not phorbol ester, an activator of protein kinase C, or A23187, a calcium ionophore, down-regulated it. The same was also observed in ST2 cells, suggesting that stromal cells are responsible for the inhibitory effect of PTH and cAMP analogs on OPG/OCIF mRNA expression in mouse bone cells. CONCLUSIONS: The present study indicates that PTH down-regulates OPG/OCIF mRNA expression through the PKA pathway in stromal cells, which would result in the stimulation of osteoclast formation.

New partition technique for two-chamber pressure casting unit for titanium.

Although titanium has been widely used in prosthetic appliances, such as cast removable partial denture frameworks, high quality castings are still difficult to obtain. This study proposes a new partition technique using a metal foil for improving castability of a two-chamber pressure casting unit. The metal foil was formed beforehand to bring it in contact with the mold crucible wall so that no clearance was left behind it. Using this formed foil, the mold cavity can be completely isolated from the upper chamber and can be maintained in a vacuum even after the rupture of the foil. Accordingly, a large casting force is generated very quickly, and as a result, castability is expected to be significantly improved. This expectation was confirmed by the experiments using wax patterns including thin plate and mesh plate.

High extracellular calcium inhibits osteoclast-like cell formation by directly acting on the calcium-sensing receptor existing in osteoclast precursor cells.

Although it has recently been suggested that high extracellular calcium ([Ca(2+)](e)) inhibits osteoclast function via a calcium-sensing receptor (CaSR) in mature osteoclasts, the role of CaSR in the regulation of osteoclast formation remains unknown. The present study was performed to investigate whether osteoclast precursor cells possess CaSR and to clarify the possible role of CaSR in the regulation of osteoclast formation. Immunocytochemistry detected CaSR in osteoclast precursor cells derived from spleen cells as well as in osteoblastic MC3T3-E1 cells. The use of reverse-transcription polymerase chain reaction (RT-PCR) with CaSR-specific primers, followed by nucleotide sequencing of the amplified products, also identified CaSR transcripts in osteoclast precursor cells derived from spleen cells as well as in MC3T3-E1 cells. High [Ca(2+)](e) (3 to 5 mM) concentration dependently inhibited 1,25(OH)2D3- or human parathyroid hormone (hPTH) (1-34)-induced osteoclast-like cell (Ocl) formation from osteoclast precursor cells derived from spleen cells. Further, the CaSR agonist neomycin also concentration dependently inhibited 1,25(OH)2D3- or hPTH(1-34)-induced Ocl formation. Moreover, a calcimimetic which mimics or potentiates the effects of [Ca(2+)](e) at the CaSR NPS R-467 (1-100 microM) concentration dependently inhibited Ocl formation stimulated by 1,25(OH)2D3 or hPTH(1-34). These findings first demonstrated that osteoclast precursor cells possess CaSR very similar, if not identical, to those in the parathyroid and kidney. Furthermore, the CaSR in osteoclast precursor cells could play a key role in regulating Ocl formation by sensing local changes in [Ca(2+)](e) at the resorptive sites.

Estrogen via the estrogen receptor blocks cAMP-mediated parathyroid hormone (PTH)-stimulated osteoclast formation.

Several lines of evidence indicate that estrogen inhibits parathyroid hormone (PTH)-induced bone resorption in vivo and in vitro. However, its precise mechanism remains unknown. The present study was performed to investigate whether osteoclast precursor cells possess the receptors for PTH/PTH-related protein (PTHrP) and/or estrogen and to clarify the mechanism by which estrogen affects PTH-induced osteoclast-like cell (Ocl) formation. The polymerase chain reaction (PCR) product corresponding in size to the mouse PTH/PTHrP receptor cDNA was detected in mouse hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as in osteoblastic MC3T3-E1 cells. The nucleotide sequence of the PTH/PTHrP receptor PCR product of hemopoietic blast cells was found to be 95.4% identical to that of PTH/PTHrP receptor cDNA of rat osteoblastic ROS cells. The PCR product corresponding in size to the mouse estrogen receptor cDNA was detected in mouse hemopoietic blast cells supported by GM-CSF as well as in MC3T3-E1 cells. The nucleotide sequence of the estrogen receptor PCR product of hemopoietic blast cells was completely identical to that of mouse estrogen receptor cDNA. 17Beta-estradiol (17beta-E2) but not 17alpha-E2 dose dependently antagonized Ocl formation stimulated by human (h) PTH(1-34) at a minimal effective concentration of 10(-10) M in the hemopoietic blast cell culture. 17Beta-E2 also significantly inhibited Ocl formation stimulated by 10(-8) M hPTHrP(1-34), while it did not affect 1,25-dihydroxyvitamin D3-induced Ocl formation. However, 10(-8) M 17beta-E2 significantly inhibited Ocl formation stimulated by dibutyryladenosine cAMP (10(-4) M) and Sp-cAMPS (10(-4) M), an activator of cAMP-dependent protein kinase (PKA) as well as forskolin (10(-5) M). In contrast, 17beta-E2 did not affect Ocl formation by either phorbol myristate acetate (10(-7) M), an activator of protein kinase C (PKC), or A23187 (10(-7) M), a calcium ionophore. The pretreatment with 17beta-E2 significantly inhibited Ocl formation induced by the combined treatment with PTH and PKC inhibitors (H7 or staurosporine), while it did not affect Ocl formation stimulated by the combined treatment with PTH and Rp-cAMPS, a PKA inhibitor. The present data indicate that estrogen inhibits PTH-stimulated Ocl formation by directly acting on hemopoietic blast cells, possibly through blocking a PKA pathway but not a calcium/PKC pathway.

Insulin-like growth factor-I mediates osteoclast-like cell formation stimulated by parathyroid hormone.

There have been several lines of evidence that parathyroid hormone (PTH) stimulates production of insulinlike growth factor I (IGF-I) in bone and that IGF-I stimulates osteoclast formation. Thus, the present study was performed to clarify the possible role of IGF-I in PTH-stimulated osteoclastlike cell formation and the role of PTH-responsive dual signal transduction systems (cyclic [c] AMP-dependent protein kinase [PKA] and calcium/protein kinase C [PKC]) in its mechanism. Treatment with anti-IGF-I antibody (1-10 micrograms/ml) partially but significantly blocked hPTH-(1-34)-stimulated osteoclastlike cell formation in unfractionated mouse bone cell cultures, although it did not affect osteoclastlike cell formation stimulated by 1,25-dihydroxyvitamin D3. Rp-cAMP5 (10(-4) M), a direct PKA inhibitor, as well as two types of PKC inhibitors, H-7 (10 microM) and staurosporine (3 nM), and dantrolene (10(-5) M), an inhibitor of calcium mobilization from intracellular calcium stores, all significantly blocked PTH-stimulated osteoclastlike cell formation. Anti-IGF-I antibody (3 micrograms/ml) significantly blocked osteoclastlike cell formation stimulated by 10(-4) M dbcAMP, 10(-4) M Sp-cAMPS, a direct PKA activator, and 10(-5) M forskolin in mouse bone cell cultures. Dibutyryl cAMP, forskolin, and hPTH-(1-34) significantly stimulated mRNA expression of both IGF-I and IGF-binding protein 5 (IGFBP-5) in these cultures, but neither 10(-7) M PMA, a PKC activator, nor 10(-7) M A23187 did. Moreover, anti-IGF-I antibody significantly blocked osteoclastlike cell formation stimulated by the conditioned medium from MC3T3-E1 cells pretreated with 10(-8) PTH-(1-34), which induced IGF-I and IGFBP-5 mRNA expression in these cells. In conclusion, the present study indicates that IGF-I mediates osteoclastlike cell formation stimulated by PTH and that the PKA pathway is involved in its mechanism. However, IGF-I does not seem to be the sole effector molecule to be active in this system.

Dexamethasone stimulates osteoclast-like cell formation by directly acting on hemopoietic blast cells and enhances osteoclast-like cell formation stimulated by parathyroid hormone and prostaglandin E2.

Although an excess of glucocorticoid induces secondary osteoporosis, the mechanism still remains unclear, particularly in regard to glucocorticoid-stimulated bone resorption. We examined the effects of dexamethasone (Dex) on osteoclast-like cell formation and bone-resorbing activity by employing mouse bone and spleen cell cultures and further investigated whether Dex would modulate osteoclast-like cell formation stimulated by several bone-resorbing factors. Dex stimulated osteoclast-like cell formation in stromal cell-containing mouse bone cell cultures in a concentration-dependent manner. Also, Dex significantly stimulated osteoclast-like cell formation from hemopoietic blast cells in spleen cell cultures derived from 5-fluorouracil-pretreated mice. In contrast, Dex (10(-8) M) did not affect the bone-resorbing activity of mature osteoclasts. Pretreatment with 10(-8) M Dex significantly enhanced osteoclast-like cell formation in unfractionated mouse bone cell cultures stimulated by 10(-8) M human (h) parathyroid hormone (PTH) (1-34), 10(-8) M hPTH-related protein (1-34) and 10(-6) M prostaglandin E2, but not by 10(-8) M 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Moreover, pretreatment with 10(-8) M Dex significantly enhanced osteoclast-like cell formation stimulated by both forskolin and dbcAMP. In contrast, pretreatment with 10(-8) M Dex significantly inhibited osteoclast-like cell formation in mouse spleen cell cultures stimulated by both 10(-8) M hPTH(1-34) and 10(-8) M 1,25(OH)2D3. These findings suggest that Dex stimulates osteoclast-like cell formation, at least in part by directly acting on hemopoietic blast cells. They further suggest that Dex enhances osteoclast-like cell formation stimulated by PTH and prostaglandin E2 through an indirect pathway via cells other than hemopoietic blast cells.

Possible segregation caused by centrifugal titanium casting.

The possibility of the segregation under solidification process using a centrifugal casting machine was investigated using an electron probe microanalyzer with elemental distribution map, line analysis and quantitative analysis. When a very small quantity of platinum was added to local molten titanium during the casting process, macroscopic segregation was observed under conditions of density difference of 0.1 g/cm3 at the most, confirming that the centrifugal force of the casting machine is extremely strong. When a Ti-6Al-4V alloy was cast, however, no macroscopic segregation was observed. The centrifugal force of the casting machine examined in the present study hardly results in the body-force segregation in this titanium alloy.

High extracellular calcium stimulates osteoclast-like cell formation and bone-resorbing activity in the presence of osteoblastic cells.

The present study was performed to examine the effect of the high concentration of extracellular calcium ([Ca2+]e) on osteoclast-like cell formation and bone-resorbing activity in the presence or absence of osteoblasts. High [Ca2+]e (3 and 5 mM) significantly stimulated osteoclast-like cell formation in osteoblast-containing mouse bone cell cultures, although high [Ca2+]e did not affect the formation of osteoclast-like cells from hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor in mouse spleen cell cultures. The osteoclast-like cells, newly formed by high [Ca2+]e in the presence of osteoblasts, possessed the ability to form pits on the dentine slices. The conditioned medium from osteoblastic MC3T3-E1 cells treated with high [Ca2+]e (5 mM) significantly increased the formation of osteoclast-like cells from hemopoietic blast cells, compared with the control medium. Dantrolene, an inhibitor of calcium mobilization from the intracellular calcium pool, and indomethacin significantly blocked high [Ca2+]e-stimulated osteoclast-like cell formation in the presence of osteoblasts, although voltage-dependent calcium channel blockers and anti-insulin-like growth factor I antibody did not affect it. High [Ca2+]e, however, significantly stimulated the bone-resorbing activity of mature osteoclasts in osteoblast-containing mouse bone cell cultures, although high [Ca2+]e inhibited bone-resorbing activity in isolated rabbit osteoclasts. An increase in the extracellular magnesium concentration (5 mM) affected neither osteoclast-like cell formation nor bone-resorbing activity. In conclusion, high [Ca2+]e stimulated osteoclast-like cell formation and bone-resorbing activity of mature osteoclasts, presumably via osteoblasts.

Surface contamination of titanium by abrading treatment.

This study investigated the contamination of abraded Ti surfaces. Using a polishing machine, specimens were abraded with waterproof SiC grit papers under water cooling. The abraded surfaces were examined using element analysis, X-ray diffraction, and hardness tests. Contaminant deposits with dimensions reaching about 30 microns were observed throughout the surface. In these deposits, Ti was apparently reduced by about 10% and replaced by Si and O. The chemical bond state of the Si was similar to that of SiC or a titanium silicide. The O was solute in Ti, which increased the surface hardness. The contaminant deposits were amorphous or very thin. The contamination of Ti, the extent of which was related to hardness, resulted from a reaction with abrasives.

Estrogen blocks parathyroid hormone (PTH)-stimulated osteoclast-like cell formation by selectively affecting PTH-responsive cyclic adenosine monophosphate pathway.

Several lines of evidence have previously indicated that estrogen inhibits PTH-induced bone resorption in vivo and in vitro. However, its mechanism remains unknown. Therefore, the present study was performed to investigate the effect of estrogen on PTH-stimulated osteoclast-like cell formation and clarify its mechanism. 17 beta-estradiol (17 beta-E2) significantly antagonized osteoclast-like cell formation stimulated by 10(-8) M human (h) PTH-(1-34) as well as 10(-8) M hPTH-related peptide (PTHrP)-(1-34)in osteoblast-containing mouse bone cell cultures. The conditioned medium derived from osteoblastic SaOS-2 cells or MC3T3-E1 cells pretreated with both PTH-(1-34) (10(-8)M) and 17 beta-E2(10(-8)M) stimulated osteoclast-like cell formation from hemopoietic blast cells more weakly than conditioned medium from cells pretreated with PTH-(1-34) alone. Moreover, 10(-8) M 17 beta-E2 significantly blocked the formation of osteoclast-like cells stimulated by 10(-8) M hPTH-(1-34) in spleen cell cultures derived from 5-fluorouracil-pretreated mice. On the other hand, 10(-8) M 17 beta-E2 significantly inhibited osteoclast-like cell formation stimulated by dbcAMP (10(-4)M) and Sp-cAMPS (10(-4)M), as well as forskolin (10(-5)M) in mouse bone cell cultures. In contrast, 10(-8)M 17 beta-E2 did not affect PMA (10(-7)M)-, A23187 (10(-7)M)-, or BAYK-8644 (5 x 10(-6) M)-stimulated osteoclast-like cell formation. In conclusion, the present study demonstrated that estrogen inhibits PTH-stimulated osteoclast-like cell formation by directly acting on hemopoietic blast cells as well as by indirectly acting on them via osteoblasts. The inhibitory effects of estrogen on PTH-stimulated osteoclast-like cell formation seemed to be mediated through blocking the cAMP-dependent protein kinase pathway but not by blocking calcium/protein kinase C.

Stimulatory effect of growth hormone on bone resorption and osteoclast differentiation.

Although the actions of GH on osteoblasts have been extensively investigated, its effects on osteoclasts remain unknown. In the present study, the effects of GH on bone resorption and osteoclast differentiation were examined in vitro. Bovine GH (bGH; 1-100 ng/ml) significantly stimulated bone resorption by preexistent osteoclasts in stromal cell-containing mouse bone cell cultures, whereas it did not affect the bone-resorbing activity of isolated rabbit osteoclasts. When bGH was added to unfractionated bone cells after degeneration of preexistent osteoclasts, it concentration dependently stimulated osteoclast-like cell formation. GH also enhanced 1,25-dihydroxyvitamin D3-induced osteoclast-like cell formation. Moreover, osteoclast-like cells newly formed from unfractionated bone cells in the presence of bGH possessed the ability to form pits on dentine slices. The conditioned medium from osteoblastic MC3T3-E1 cells or MC3T3-G2/PA-6 stromal cells pretreated with bGH stimulated osteoclast-like cell formation from mouse hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor. On the other hand, the PCR products corresponding in size to the mouse GH receptor were detected in mouse hemopoietic blast cells as well as liver. GH concentration dependently stimulated osteoclast-like cell formation from these hemopoietic blast cells in the absence of stromal cells, and these osteoclast-like cells formed pits on dentine slices in the presence of MC3T3-G2/PA-6 stromal cells. The present study indicated for the first time that GH stimulates osteoclastic bone resorption through both its direct and indirect actions on osteoclast differentiation and through its indirect activation of mature osteoclasts, possibly via stromal cells, including osteoblasts.

Prostaglandin E2 stimulates osteoclast-like cell formation and bone-resorbing activity via osteoblasts: role of cAMP-dependent protein kinase.

Prostaglandin E2 (PGE2) is an important local regulator in bone. The present study was performed to investigate the effect of PGE2 on osteoclast-like cell formation and bone-resorbing activity of mature osteoclasts in the presence or absence of osteoblasts, PGE2 (10(-8) to 10(-6) M) significantly stimulated osteoclast-like cell formation in osteoblast-containing mouse bone cell cultures, although it did not affect osteoclast-like cell formation from hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor in osteoblast-free mouse spleen cell cultures. The conditioned medium from osteoblastic UMR-106 cells pretreated with PGE2 (10(-8) and 10(-6) M) significantly stimulated osteoclast-like cell formation from hemopoietic blast cells. PGE2 also significantly stimulated the bone-resorbing activity of mature osteoclasts in osteoblast-containing mouse bone cell cultures. In contrast, PGE2 significantly inhibited the bone-resorbing activity and osteopontin mRNA expression in isolated rabbit osteoclasts. Rp-cAMPS, a direct protein kinase (PKA) antagonist, significantly inhibited PGE2-stimulated osteoclast-like cell formation and the bone-resorbing activity of mature osteoclasts, although protein kinase C inhibitors, dantrolene (an inhibitor of calcium release from the intracellular calcium pool) and voltage-dependent calcium channel blockers did not affect PGE2-stimulated osteoclast-like cell formation. In conclusion, PGE2 stimulated osteoclast-like cell formation and bone-resorbing activity in mouse bone cell cultures presumably through osteoblasts. The activation of PKA is linked to PGE2-stimulated osteoclast-like cell formation and bone-resorbing activity.