Effects of ovariectomy on turnover of alveolar bone in the healed extraction socket in rat edentulous mandible.

OBJECTIVE: The number of patients with postmenopausal osteoporosis receiving dental implants because of edentulism is increasing. Since osseointegration around implants requires formation and maintenance of new bone, knowledge of how ovariectomy (OVX) affects turnover of mandibular and maxillary bone is required. In the present study, we investigated the effects of OVX on turnover of alveolar bone in the healed extraction socket of the rat left mandibular incisor. METHODS: The molars and the incisor on left side in 6-month-old Sprague-Dawley female rats (n=38) were extracted and left to heal for 4 months. Animals were then ovariectomized and killed at the time of OVX (baseline) (n=4), 6 weeks (n=10), 6 months (n=12) and 9 months (n=12) post-OVX. Changes in bone mass and bone turnover were assessed using static and dynamic histomorphometric parameters. RESULTS: Bone turnover was increased by ovariectomy (OVX) as reflected by increased static parameters of bone formation and resorption. The changes in dynamic parameters were not statistically significant. Cancellous bone volume/total volume (%) in the post-OVX group decreased more than that in the control group. CONCLUSIONS: Our results suggest that OVX increases the turnover of alveolar bone in the healed extraction socket of rat mandibular incisor, resulting in a decrease of cancellous bone volume with time.

Fibronectin inhibits osteoclastogenesis while enhancing osteoclast activity via nitric oxide and interleukin-1ß-mediated signaling pathways.

Osteoclasts are bone-resorbing cells formed by fusion of mononuclear precursors. The matrix proteins, fibronectin (FN), vitronectin (VN), and osteopontin (OPN) are implicated in joint destruction and interact with osteoclasts mainly through integrins. To assess the effects of these matrix proteins on osteoclast formation and activity, we used RAW 264.7 (RAW) cells and mouse splenocytes differentiated into osteoclasts on tissue culture polystyrene (TCP) or osteologic™ slides pre-coated with 0.01-20 µg/ml FN, VN, and OPN. At 96 h, osteoclast number and multinucleation were decreased on VN and FN compared to OPN and TCP in both RAW and splenocytes cell cultures. When early differentiation was assessed, VN but not FN decreased cytoplasmic tartrate-resistant acid phosphatase activity and pre-osteoclast number at 48 h. OPN had the opposite effect to FN on osteoclast formation. When RAW cells were differentiated on OPN and treated by FN and OPN, osteoclast number only in the FN treated group was 40-60% lower than the control, while the total number of nuclei was unchanged, suggesting that FN delays osteoclast fusion. In contrast to its inhibitory effect on osteoclastogenesis, FN increased resorption by increasing both osteoclast activity and the percentage of resorbing osteoclasts. This was accompanied by an increase in nitric oxide (NO) levels and interleukin-1ß (IL-1ß). IL-1ß production was inhibited using the NO-synthase inhibitor only on FN indicating a FN-specific cross-talk between NO and IL-1ß signaling pathways. We conclude that FN upregulates osteoclast activity despite inhibiting osteoclast formation and that these effects involve NO and IL-1ß signaling.

Bone matrix proteins and extracellular acidification: potential co-regulators of osteoclast morphology.

Osteoclasts are signaled by the bone matrix proteins fibronectin (FN), vitronectin (VN), and osteopontin (OPN) via integrins. To perform their resorptive function, osteoclasts cycle between compact (polarized), spread (non-resorbing) and migratory morphologies. Here we investigate the effects of matrix proteins on osteoclast morphology and how those effects are mediated using RAW 264.7 cells differentiated into osteoclasts on FN, VN, and OPN-coated culture dishes. After 96 h, 80% of osteoclasts on FN were compact while 25% and 16% on VN were in compact and migratory states respectively. In contrast, OPN induced osteoclast spreading. Furthermore, osteoclasts formed on VN and FN were two- to fourfold smaller than those formed on OPN in the 21-30 nuclei/osteoclast group. These effects were not due to defects in cytoskeletal reorganization of osteoclasts on VN and FN, demonstrated by the ability of these cells to spread in response to 35 ng/ml macrophage colony stimulating factor (M-CSF). Conversely, osteoclasts on OPN failed to spread when induced by M-CSF. Moreover, the extracellular pH on FN and VN (7.25 and 7.3, respectively) was significantly lower than that on OPN (∼7.4). We further investigated the role of extracellular pH and found that at pH 7.5 the duration of an osteoclast's compact phase was 25.6 min and that of the spread phase was 62.5 min. Reducing the pH to 7.0 increased the frequency of osteoclast cycling by threefold. These results show that matrix proteins play a role in regulating osteoclast morphology, possibly via altering extracellular and intracellular pH.

VEGF-A expression in osteoclasts is regulated by NF-kappaB induction of HIF-1alpha.

Large osteoclasts (10+ nuclei), predominant in rheumatoid arthritis and periodontal disease, have higher expression of proteases and activating receptors and also have increased resorptive activity when compared to small (2-5 nuclei) osteoclasts. We hypothesized that large and small osteoclasts activate different signaling pathways. A Signal Transduction Pathway Finder Array was used to compare gene expression of large and small osteoclasts in RAW 264.7-derived osteoclasts. Expression of vascular endothelial growth factor A (Vegfa) was higher in large osteoclasts and this result was confirmed by RT-PCR. RT-PCR further showed that RANKL treatment of RAW cells induced Vegfa expression in a time-dependent manner. Moreover, VEGF-A secretion in conditioned media was also increased in cultures with a higher proportion of large osteoclasts. To investigate the mechanism of Vegfa induction, specific inhibitors for the transcription factors NF-kappaB, AP-1, NFATc1, and HIF-1 were used. Dimethyl bisphenol A, the HIF-1alpha inhibitor, decreased Vegfa mRNA expression, whereas blocking NF-kappaB, AP-1, and NFATc1 had no effect. Furthermore, the NF-kappaB inhibitor gliotoxin inhibited Hif1alpha mRNA expression. In conclusion, VEGF-A gene and protein expression are elevated in large osteoclasts compared to small osteoclasts and this increase is regulated by HIF-1. In turn, Hif1alpha mRNA levels are induced by RANKL-mediated activation of NF-kappaB. These findings reveal further differences in signaling between large and small osteoclasts and thereby identify novel therapeutic targets for highly resorptive osteoclasts in inflammatory bone loss.

IL-1alpha and IL-1beta have different effects on formation and activity of large osteoclasts.

Interleukin 1 (IL-1) is a proinflammatory cytokine upregulated in conditions such as rheumatoid arthritis and periodontal disease. Both isoforms, IL-1alpha and IL-1beta, have been shown to activate osteoclasts (OCs), the cells responsible for resorbing bone. Inflammatory conditions are also characterized by increased bone loss and by the presence of large OCs (10+ nuclei). We and others have previously shown that large OCs are more likely to be resorbing compared to small OCs (2-5 nuclei). Moreover, large OCs express higher levels of the IL-1 activating receptor IL-1RI, integrins alphav and beta3, RANK, and TNFR1, while small OCs have higher levels of the decoy receptor IL-1RII. We hypothesized that IL-1 would have different effects on large and small OCs due to these distinct receptor expression patterns. To test this hypothesis, RAW 264.7 cells were differentiated into populations of small and large OCs and treated with IL-1alpha or IL-1beta (1 and 10 ng/ml). In the presence of sRANKL, both IL-1alpha and IL-1beta increased total OC number and resorptive activity of large OCs. IL-1alpha stimulated formation of large OCs and increased the number of resorption pits, while IL-1beta changed the morphology of large OCs and integrin-beta3 phosphorylation. No effects were seen in small OCs in response to either IL-1 isoform. These results demonstrate that IL-1 predominantly affects large OCs. The dissimilarity of responses to IL-1alpha and IL-1beta suggests that these isoforms activate different signaling pathways within the two OC populations.

Comparison of the effect of ovariectomy on bone mass in dentate and edentulous mandibles of adult rats.

We investigated the effect of ovariectomy (OVX) on bone changes in the edentulous and dentate mandibles and compared these to changes in tibiae and femorae using dual energy x-ray absorptiometry (DEXA) and histomorphometric measurements. One hundred and fifteen female rats had their molars and the incisor on one side of the mandible extracted at six months of age and allowed to heal for 4 months. At 10 months of age, animals were divided into an experimental group, which underwent bilateral ovariectomy, and a control group of intact animals. Sixteen OVX and sixteen control animals were sacrificed at 6-weeks, and at 6- and 9-months post-OVX. In addition 12 animals were sacrificed at the time of OVX to act as an internal control group. Bone mineral density measurements (BMD) using DEXA and bone volume measurements on undecalcified sections evaluated under backscattered electron microscopy were investigated in the edentulous and dentate mandibles as well as in tibiae and femorae. At 6-weeks post-OVX, there were no significant differences between the edentulous mandibles, dentate mandibles, tibiae and femorae of OVX and control groups. At 6 months post-OVX, BMD and cancellous bone volume were significantly decreased in the edentulous mandibles (-15.9%; -21.8%), proximal tibiae (-15.6%; -38.5%) and proximalfemorae (-9.7%; -39.6%) of OVX group as compared to the control group. At 9 months post-OVX, BMD and cancellous bone volume were further decreased in the edentulous mandibles (-17.2%; -24%), proximal tibiae (-16.1%; -58.9%) and proximal femorae (-10.3%; -49.9%) of the OVX group as compared to the control group. On the other hand, OVX had no effect on the BMD and cancellous bone volume of the dentate mandibles at 6-weeks and at 6- and 9-months post-OVX. These results show that loss of bone mass in the edentulous mandible of OVX animals is similar to that occurring in the tibiae and femorae, while lack of a significant effect of OVX on bone mass in the dentate mandible suggests that functional loading related to biting force prevented bone loss in the dentate mandible.

Effects of Vitaxin, a novel therapeutic in trial for metastatic bone tumors, on osteoclast functions in vitro.

The integrin alphavbeta3 mediates cell-matrix interactions. Vitaxin(R), a humanized monoclonal antibody that blocks human and rabbit alphavbeta3 integrins, is in clinical trials for metastatic melanoma and prostate cancer. alphavbeta3 is the predominant integrin on osteoclasts, the cells responsible for bone resorption in health and disease. Here, we report the first investigation of Vitaxin's effects on osteoclast activity. Vitaxin (100-300 ng/ml) decreased total resorption by 50%, but did not alter resorptive activity per osteoclast. Vitaxin (300 ng/ml) decreased osteoclast numbers on plastic by 35% after 48 h. Similarly, attachment after 2 h was reduced by 30% when osteoclasts were incubated with Vitaxin (300 ng/ml) for 25 min prior to plating; however, the rate of fusion of osteoclast precursors in Vitaxin-treated and control groups was equal. Using time-lapse microscopy, we evaluated the effect of Vitaxin on osteoclast morphology and found a significant reduction in osteoclast planar area only when cells were pretreated with macrophage colony stimulating factor (M-CSF). Extracellular Ca(2+) and M-CSF have opposite effects on alphavbeta3 conformation. Elevation of extracellular Ca(2+) eliminated the inhibitory effect of Vitaxin on osteoclast attachment. In contrast, the effect of Vitaxin was enhanced in cells pretreated with M-CSF. This action of M-CSF was suppressed by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin, suggesting that M-CSF increases Vitaxin's inhibitory effect by inside-out activation of alphavbeta3. In conclusion, Vitaxin decreases resorption by impairing osteoclast attachment, without affecting osteoclast formation and multinucleation. Our data also show that Vitaxin's inhibitory effects on osteoclasts can be modulated by factors known to alter the conformation of alphavbeta3.

Increased expression of activating factors in large osteoclasts could explain their excessive activity in osteolytic diseases.

Large osteoclasts (>or=10 nuclei) predominate at sites of pathological bone resorption. We hypothesized this was related to increased resorptive activity of large osteoclasts and have demonstrated previously that larger osteoclasts are 8-fold more likely to be resorbing than small osteoclasts (2-5 nuclei). Here we ask whether these differences in resorptive activity can be explained by differences in expression of factors involved in osteoclast signaling, fusion, attachment, and matrix degradation. Authentic rabbit osteoclasts and osteoclasts derived from RAW264.7 cells showed similar increases in c-fms expression (1.7- to 1.8-fold) in large osteoclasts suggesting that RAW cells are a viable system for further analysis. We found 2- to 4.5-fold increases in the expression of the integrins alpha(v) and beta(3), the proteases proMMP9, matMMP9 and pro-cathepsinK, and in activating receptors RANK, IL-1R1, and TNFR1 in large osteoclasts. In contrast, small osteoclasts had higher expression of the fusion protein SIRPalpha1 and the decoy receptor IL-1R2. The higher expression of activation receptors and lower expression of IL-1R2 in large osteoclasts suggest they are hyperresponsive to extracellular factors. This is supported by the observation that the resorptive activity in large osteoclasts was more responsive to IL-1beta, and that this increased activity was inhibited by the IL-1 receptor antagonist, IL-1ra. This increased responsiveness of large osteoclasts to IL-1 may, in part, explain the pathological bone loss noted in inflammatory diseases. The heterogeneity in receptor expression and the differential response to cytokines and their antagonists could prove useful for selective inhibition of large osteoclasts actively engaged in pathological bone loss.

Effect of age on progesterone receptor expression, and osteoprogenitor proliferation and differentiation in female rat vertebral cell populations.

In the present investigation, we evaluated whether the capacity for proliferation and differentiation of progesterone (Prog)-dependent osteoprogenitors in the female rat skeleton is related to the level of Prog receptors (PRs) and/or the level of circulating estrogen. We confirmed that in rats, estrogen levels at 18 months of age are higher than those at 3 months, and higher again in rats of 22.5, 25.5, and 26 months of age. Prog levels in rats of ages between 18 and 25.5 months were lower than those at 26 and 3 months of age. PR-A levels were tenfold higher than those of PR-B in cell populations where PR-B was detectable; PR-B receptors were not detectable in all cell populations. In populations derived from 22.5 to 26 months old rats, the basal levels of PR-A were higher than those derived from 3 and 18 months old rats by five- and twofold respectively. Prog treatment enhanced PR-A expression in animals of all ages. Estrogen enhanced the effect of Prog on PR-A expression in cell populations from the 3 and 18 months old rats, but had no effect on PR-A expression in cell populations from 22.5, 25.5, and 26 months old rats. This might be related to the high basal expression of PR in 22.5-26 months old rats (the 'persistent estrous' phase). Our results also confirm our previous observation that in rats, the number of Prog- and dexamethasone (Dex)- dependent osteoprogenitors, and the effect of estrogen on the response to Prog do not decrease with age. In conclusion, we have shown that the basal level of PR-A was increased in old rats, and that this correlated with increased serum estrogen levels, but not with the number of detectable Prog-dependent osteoprogenitors. We also found that Prog upregulates the expression of its own receptors and that estrogen enhances this in young rats but not in rats over 22.5 months of age, in which estrogen levels are elevated.

Effects of human a3 and a4 mutations that result in osteopetrosis and distal renal tubular acidosis on yeast V-ATPase expression and activity.

V-ATPases are multimeric proton pumps. The 100-kDa "a" subunit is encoded by four isoforms (a1-a4) in mammals and two (Vph1p and Stv1p) in yeast. a3 is enriched in osteoclasts and is essential for bone resorption, whereas a4 is expressed in the distal nephron and acidifies urine. Mutations in human a3 and a4 result in osteopetrosis and distal renal tubular acidosis, respectively. Human a3 (G405R and R444L) and a4 (P524L and G820R) mutations were recreated in the yeast ortholog Vph1p, a3 (G424R and R462L), and a4 (W520L and G812R). Mutations in a3 resulted in wild type vacuolar acidification and growth on media containing 4 mM ZnCl2, 200 mM CaCl2, or buffered to pH 7.5 with V-ATPase hydrolytic and pumping activity decreased by 30-35%. Immunoblots confirmed wild type levels for V-ATPase a, A, and B subunits on vacuolar membranes. a4 G812R resulted in defective growth on selective media with V-ATPase hydrolytic and pumping activity decreased by 83-85% yet with wild type levels of a, A, and B subunits on vacuolar membranes. The a4 W520L mutation had defective growth on selective media with no detectable V-ATPase activity and reduced expression of a, A, and B subunits. The a4 W520L mutation phenotypes were dominant negative, as overexpression of wild type yeast a isoforms, Vph1p, or Stv1p, did not restore growth. However, deletion of endoplasmic reticulum assembly factors (Vma12p, Vma21p, and Vma22p) partially restored a and B expression. That a4 W520L affects both Vo and V1 subunits is a unique phenotype for any V-ATPase subunit mutation and supports the concerted pathway for V-ATPase assembly in vivo.

Effects of hind limb unloading and reloading on nitric oxide synthase expression and apoptosis of osteocytes and chondrocytes.

In rat bone, the absence of mechanical load results in a reduction in bone formation, inhibition of longitudinal growth, and a decrease in the number of osteoblasts and osteoprogenitors in cancellous bone. Unloading has also been linked to an increase in apoptosis of osteocytes and chondrocytes through production of nitric oxide (NO) and increased expression of NO synthases (NOS). Reloading results in recovery of bone volume within 14 days, although osteoblast and osteoclast numbers remain below control values, suggesting decreased bone turnover. This study was designed to evaluate the effects of hind limb unloading and subsequent reloading on apoptosis, NOS expression, and histomorphometric parameters in trabecular and cortical bone, articular cartilage, and growth plate cartilage of the proximal tibia of the hind limbs. Compared to ambulatory controls, 2 weeks of unloading resulted in a 66% increase in the percentage of apoptotic osteocytes in the trabecular metaphysis, a 14% increase in osteoclast number and a 48% decrease in bone volume. The percentage of eNOS- or iNOS-positive osteocytes was unchanged. Upon reloading, the percentage of apoptotic osteocytes and bone volume returned to baseline whereas the percentage of iNOS-positive osteocytes increased by 50% and osteoclast number decreased by 30% compared to ambulatory controls. More striking changes were observed in articular and growth plate cartilage. Unloading resulted in a 230% increase in apoptotic chondrocytes, a 400% increase in iNOS-positive chondrocytes, and a 17% reduction in width in articular cartilage. Reloading for 2 weeks resulted in partial recovery. Chondrocytes in the proliferative and hypertrophic zones of the growth plate responded similarly to those in the articular cartilage. In summary, we observed that 14 days of unloading increased apoptosis of osteocytes and chondrocytes. This was associated with an increase in the proportion of iNOS-positive chondrocytes whereas the proportion of iNOS-positive osteocytes remained unchanged. Reloading for 14 days restored osteocyte apoptosis to control levels but the percentage of iNOS- and eNOS-positive osteocytes increased in reloaded bone compared to controls. This was associated with a decrease in osteoclast number. In cartilage, reloading for 2 weeks did not result in a return to baseline in any of the parameters measured, suggesting that the effects of unloading on articular cartilage and the growth plate last longer than those in bone and may have prolonged effects on joint biomechanics and longitudinal bone growth.

The effect of reloading on bone volume, osteoblast number, and osteoprogenitor characteristics: studies in hind limb unloaded rats.

Skeletal unloading during space flight results in bone loss. In astronauts the extent to which bone is lost varies greatly between different bones of the skeleton as well as between different individuals. Following return to earth, recovery of bone mass during reloading also varies between different bones and different individuals. Due to this variability and the limited number of subjects it is difficult to study the effects of unloading/reloading on bone in humans. A viable alternative is to use the rat model of hind limb unloading developed at NASA. We have previously demonstrated that, in 6-week-old male rats, 14 days of unloading result in a decrease in osteoprogenitor number in cell populations isolated from the proximal femur. The goal of the present study was to determine the number of osteoprogenitor cells present in cell populations derived from the proximal femur of young rats after 14 days of unloading followed by 14 days of reloading and to characterize their proliferative capacity. To do this, we determined the number of alkaline phosphatase-positive colony forming units (CFU-AP) and osteoblast CFU (CFU-O). To establish whether the effects of unloading and reloading were specific for cells of the osteoblast lineage, we also determined the number of fibroblastic CFU (CFU-F). Effects on proliferation were evaluated by measuring the size of CFU-O. Unloading resulted in a 66% reduction in CFU-AP. CFU-O numbers were decreased by 76% and mean colony size was 33% less than controls. The decrease in osteogenic and osteoprogenitor cells in vitro paralleled the decrease in bone volume (- 50%), osteoblast number (- 35%), and bone formation rate (- 46%) observed in the proximal tibial metaphysis of unloaded rats. Unloading had no effect on osteoclast number or surface. Subsequent reloading for 14 days restored CFU-AP. CFU-O numbers were only partially restored at 14 days (83% of controls) but nodule size was 1.2-fold greater than controls. Neither unloading nor reloading had an effect on the total number of progenitors (CFU-F). Reloading restored bone volume back to control values, but osteoblast number and bone formation rate were still lower than those in corresponding controls. Both osteoclast number and surface were lower in reloaded animals than in age-matched controls. Our results indicate that 14 days of unloading result in a decrease in osteoprogenitor number and that reloading for 14 subsequent days completely restores CFU-AP and bone volume to control levels, while the number of CFU-O in vitro and osteoblasts in vivo were partially recovered but still lower than corresponding controls. Strikingly, osteoclastic bone resorption after 14 days of reloading was greatly reduced compared to controls, suggesting a significant contribution of this to the recovery process.

Pregnancy-associated plasma protein-A proteolytic activity in rat vertebral cell cultures: stimulation by dexamethasone--a potential mechanism for glucocorticoid regulation of osteoprogenitor proliferation and differentiation.

Glucocorticoids (GCs) at physiological concentrations stimulate osteoprogenitor proliferation and differentiation in rat bone cell populations, and this is mediated in part by an increased response to insulin-like growth factors (IGFs). Since IGF binding proteins (IGFBPs) modulate IGF actions, we evaluated whether the increased IGF responsiveness might be associated with decreased inhibitory IGFBP-4 peptide levels. Rat vertebral cells were cultured for up to 20 days with or without dexamethasone (Dex). Cell layer proteins were extracted at day 6, 8, 14, and 20, conditioned media (CM) collected at day 8, 14, and 20, and total RNA isolated at day 14 and 20 of culture. Western blotting showed that cell layer IGFBP-4 levels were lower, while IGFBP-4 protease activity in CM was higher, in Dex-treated cultures. Addition of pregnancy-associated plasma protein-A (PAPP-A) antibody to CM abrogated IGFBP-4 proteolysis. PAPP-A mRNA levels were the same in control and Dex-treated cultures as evaluated by RT-PCR. Our data demonstrate that activity of the IGFBP-4 protease, PAPP-A, in rat bone cell cultures is increased by Dex via post-transcriptional mechanisms. Since IGFBP-4 mRNA levels in Dex-treated cultures were the same as in controls at day 8, slightly lower than in controls at day 14, and higher than in controls at day 20 as shown previously, the decreased IGFBP-4 peptide levels in Dex-treated cultures likely result from increased IGFBP-4 proteolysis by the elevated PAPP-A enzymatic activity. Our findings underscore a novel mechanism whereby GCs increase IGF responses in rat bone cells via PAPP-A-induced IGFBP-4 proteolysis.

Number, frequency, self-renewal, and expansion of osteoprogenitor cells (CFU-O) in subcultured female rat vertebral cell populations.

The present study was undertaken to determine whether the frequency and/or number of dexamethasone- and progesterone-responsive osteoprogenitors in cell populations derived from vertebrae of 6-week-old female rats could be increased relative to that of other progenitors. Frequencies and numbers of both progenitor types were determined for up to six subcultures using continuous subculturing, limiting dilution analysis, and colony assays. In dexamethasone-containing medium, subculturing resulted in an eightfold increase in the total number of dexamethasone-responsive osteoprogenitors and a 14-fold increase in progesterone-responsive osteoprogenitors in second subculture cells over first subculture cells without a significant increase in the frequency of these progenitors. From the third subculture onward, the frequency of both classes of osteoprogenitors decreased in a linear manner and none were observed after six subcultures. Similar results were obtained in progesterone-containing medium. Limiting dilution analysis in the presence of dexamethasone indicated that 2.61 % of cells represented a colony forming unit-fibroblast and 0.28 % represented an osteoprogenitor in first subculture cells, while in second subculture cells, these frequencies increased to 5.56 % and 0.40 %, respectively. Results show that while the frequency of colony forming unit-osteoprogenitor is not increased in the second subculture over the first, the total number of osteoprogenitors is greatly increased because of expansion of the total progenitor cell pool.

Quantitative assessment of post-extraction healing and alveolar ridge remodelling of the mandible in female rats.

OBJECTIVE: To quantitatively evaluate the healing and bone changes in the mandible of adult female rats following unilateral extraction of the mandibular molars and the incisor. METHODS: Six-month old female rats had their mandibular molars and the incisor on one side of the mandible extracted. Nine rats were sacrificed at 0, 14, 28, 56 and 112 days post-extraction. Bone mineral density (BMD) as observed by dual energy X-ray absorptiometry (DEXA) and histomorphometric measurements of total bone volume (TBV/TV%) as well as changes in size; height and width on backscattered electron microscopy images of cross-sections of the mandible were evaluated. RESULTS: There was a total increase of 28% in BMD of the body of the mandible and 35.1% increase in TBV/TV% at 112 days post-extraction. A maximal increase of 25% in BMD was observed at 14 days post-extraction. TBV/TV% increased by 9.5% at 14 days post-extraction and further increased by 15.9% (P < 0.001) from 14 to 28 days and by 9.2% (P < 0.001) from 28 to 56 days. A further slight but non-significant increase of 6% (P = 0.108) occurred from 56 to 112 days post-extraction. Regression equations demonstrated that the maximal increase in TBV/TV% and BMD occurred between 0 and 28 days, which subsequently slowed down between 28 and 56 days and further declined between 56 and 112 days post-extraction. Healing was associated with a reduction in cross-sectional area (32.89%), height (21%) and width of the mandible (12.84%). CONCLUSIONS: BMD of the edentulous mandible following extraction of mandibular molars and the incisor on one side of the mandible increases up to 56 days, but that total bone volume increases up to 112 days post-extraction. This indicates that bone volume measurement is more sensitive than BMD measurements in detecting small increase in bone formation at later stages of healing, possibly because of changes in geometry of the edentulous mandible following teeth extraction. The edentulous mandible undergoes a significant reduction in size as a result of reduction in both height and width up to 112 days post-extraction.

Effect of ovariectomy on dexamethasone- and progesterone-dependent osteoprogenitors in vertebral and femoral rat bone cell populations.

We have found previously that the skeleton of adult female rats contains dexamethasone (Dex)- and progesterone (Prog)-dependent osteoprogenitors, and that estrogen treatment in vitro upregulates proliferation and differentiation of the Prog-dependent but not of the Dex-dependent osteoprogenitors (Bone 1997;20:17-25). The purpose of the present study was to determine whether ovariectomy (OVX) would have different effects on these two classes of osteoprogenitors. Six-month-old Sprague-Dawley rats underwent OVX and the lumbar vertebrae and proximal femurs were collected 1.5, 3, and 6 months after OVX. Cells were obtained from outgrowths of explant cultures and grown in alpha-MEM with 10% FBS, 50 microg/ml ascorbic acid, and 5 mM beta-glycerophosphate. Osteoprogenitors were identified by their ability to generate a colony of osteoblastic cells forming bone (bone nodule). We also evaluated the number of colony-forming units-fibroblast (CFU-F) and of alkaline phosphatase (AP)-positive CFU-F. In cell populations obtained from vertebrae of rats ovariectomized for 1.5, 3, and 6 months and their corresponding control rats, both Dex (1-100 nM) and Prog (1-10 microM) dose-dependently stimulated nodule formation. Both Dex- and Prog-induced nodule formation were higher in cell populations from control rats than in those from ovariectomized rats (P < 0.001). Numbers of CFU-F and AP-positive CFU-F were also higher in cell populations from control rats compared with those from ovariectomized rats. Estrogen (10 nM) enhanced Prog-dependent bone nodule formation but decreased Dex-dependent bone nodule formation in populations from both control and ovariectomized rats. In femoral populations, the responses to Dex (10 nM), Prog (3 microM), and estrogen (10 nM) were similar to those of the vertebral populations in both control and ovariectomized rats. Our results demonstrate that ovariectomy in rats results in a dramatic decrease in the number of both Dex- and Prog-dependent osteoprogenitors in cell populations from vertebrae and proximal femurs. In addition, we confirmed our previous observation that estrogen upregulated proliferation and differentiation of Prog-dependent progenitors, but found here that estrogen clearly downregulated proliferation and differentiation of the Dex-dependent progenitors.

The a3 isoform of the 100-kDa V-ATPase subunit is highly but differentially expressed in large (>or=10 nuclei) and small (

Osteoclasts dissolve bone through acidification of an extracellular compartment by means of a multimeric vacuolar type H+-ATPase (V-ATPase). In mammals, there are four isoforms of the 100-kDa V-ATPase "a" subunit. Mutations in the a3 isoform result in deficient bone resorption and osteopetrosis, suggesting that a3 has a unique function in osteoclasts. It is thus surprising that several studies show a basal level of a3 expression in most tissues. To address this issue, we have compared a3 expression in bone with expression in other tissues. RNA blots revealed that the a3 isoform was expressed highest in bone and confirmed its expression (in decreasing order) in liver, kidney, brain, lung, spleen, and muscle. In situ hybridization on bone tissue sections revealed that the a3 isoform was highly expressed in multinucleated osteoclasts but not in mononuclear stromal cells, whereas the a1 isoform was expressed in both cell types at about the same level. We also found that a3 expression was greater in osteoclasts with 10 or more nuclei as compared with osteoclasts with five or fewer nuclei. We hypothesize that these differences in a3 expression may be associated with previously demonstrated differences between large and small osteoclasts with reference to their resorptive activity.

Expression of insulin-like growth factor system constituents in differentiating rat osteoblastic cell populations.

The synthetic glucocorticoid dexamethasone (Dex) and insulin-like growth factor-I and -II (IGF-I and -II) stimulate osteoprogenitor proliferation and differentiation in bone cell populations isolated from adult rat vertebrae. Since glucocorticoids have been shown to regulate gene expression of IGFs and IGF binding proteins (IGFBPs) in several experimental models, we investigated whether Dex-stimulated osteoprogenitor proliferation and differentiation was associated with changes in mRNA levels of the IGF system components (i.e., IGF-I and -II, the type 1 and 2 IGF receptor, the insulin receptor and six IGFBPs). Osteoprogenitor-containing bone cell populations were isolated from the outgrowth of vertebral explant cultures of 3-month-old female rats and cultured for 20 days. Total RNA was extracted at day 8, 14, and 20, and mRNA levels of the IGF system constituents were compared between differentiating (Dex-treated) and non-differentiating (control) cultures. Northern hybridization data from 8- and 20-day cultures showed that mRNA levels of IGF-I were markedly lower in Dex-treated cultures than in control cultures at day 8 and 20. At day 20, mRNA levels of IGFBP-3 were also lower in Dex-treated cultures. Signals of IGFBP-5 mRNA were undetectable. To increase the sensitivity of our detection methods and therefore evaluate mRNA levels of all the components of the IGF system, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNA extracted at day 8, 14, and 20 of culture. In agreement with the Northern data, IGF-I mRNA levels in Dex-treated cultures were lower than in control cultures at all three time points, and IGFBP-3 levels were lower in Dex-treated cultures at day 20 of culture. However, at day 8 and 14, IGFBP-3 mRNA levels were higher in Dex-treated cultures than in controls. Levels of the 2 IGF receptor mRNA and the insulin receptor mRNA were lower in Dex-treated cultures. Dex-treated cultures also had decreased levels of IGFBP-1 mRNA but increased levels of IGFBP-2 mRNA at all three time points. IGFBP-4 levels were lower at day 14 in Dex-treated cultures than in controls but higher at day 20. IGF-II and IGFBP-5 mRNA levels in control and Dex-treated cultures were similar. Signals for IGFBP-6 were undetectable. Our findings show that glucocorticoid-induced osteoprogenitor proliferation and differentiation in adult rat bone cell populations are associated with significant changes in the mRNA levels of virtually all components of the IGF system. Some of these changes are dependent on the stages of development (e.g., regulation of IGFBP-3 and -4) and some remain similar trends at all stages (e.g., regulation of IGF-I and the three receptors).

ATPase pumps in osteoclasts and osteoblasts.

Osteoblasts, osteocytes and osteoclasts are specialised cells of bone that play crucial roles in the formation, maintenance and resorption of bone matrix. Bone formation and resorption critically depend on optimal intracellular calcium and phosphate homeostasis and on the expression and activity of plasma membrane transport systems in all three cell types. Osteotropic agents, mechanical stimulation and intracellular pH are important parameters that determine the fate of bone matrix and influence the activity, expression, regulation and cell surface abundance of plasma membrane transport systems. In this paper the role of ATPase pumps is reviewed in the context of their expression in bone cells, their contribution to ion homeostasis and their relation to other transport systems regulating bone turnover.