Minimal model systems for beta sheet secondary structure in proteins

The intracellular mechanisms responsible for inhibition of osteoclast activity are of significant interest in the search for more effective ways of managing bone diseases associated with enhanced bone resorption. Previous studies have suggested that the protein kinase C (PKC) pathway is an important inhibitory second messenger in osteoclasts. We, therefore, investigated the effects of the synthetic peptide fragments, PKC(530-558) and (19-36), which correspond to parts of the catalytic and regulatory domains of PKC, on the activity of isolated osteoclasts. These fragments have been shown to activate and inhibit PKC, respectively, in biochemical studies employing isolated rat brain PKC, but have rarely been employed in studies of cellular activity. PKC(19-36), an enzyme inhibitor (PKC-I), had no effect by itself on osteoclastic bone resorption. However, PKC(530-558), a PKC activator (PKC-A), caused a dose-responsive inhibition of bone resorption, which was accompanied by a rapid and distinctive change in osteoclast morphology. This effect was reversible: (a) upon removal of PKC-A, (b) upon continuous exposure to this fragment for more than 36 h, or (c) in the presence of PKC-I. In conclusion, a short synthetic peptide fragment of PKC (PKC-A) significantly inhibits osteoclastic bone resorption; this, together with the fact that the inhibitory effect is abolished in the presence of PKC-I, provides further evidence for an important physiological role for the PKC pathway in the regulation of osteoclast activity. Selective activation of this pathway may have important therapeutic implications for the management of bone diseases associated with enhanced resorption.     

Immunoglobulin kappa chain receptor editing in systemic lupus erythematosus

Wortmannin (WT) and 17beta-hydroxywortmannin (HWT), which are inhibitors of phosphatidylinositol-3(OH)-kinase (PI3K), have been shown previously to inhibit bone resorption in vitro and in vivo, possibly by interfering with formation of the osteoclast ruffled border. Since migration of osteoclasts also plays an important role in the process of bone resorption, we investigated the effects of these inhibitors on osteoclast morphology and motility. Both HWT and WT caused a sustained decrease in the planar area of osteoclasts in vitro (half maximal effect at 25 and 165 nM, respectively), with the effect of HWT on cell area more readily reversible than WT. These agents also caused accumulation of intracellular vesicles. Time-lapse video microscopy was used to record the migration of osteoclasts in response to macrophage colony-stimulating factor (M-CSF) or vehicle, flowing passively from a micropipette positioned 200-400 microm from the cell. M-CSF caused directed migration of osteoclasts, indicating chemotaxis (over 3 h osteoclasts migrated 96 +/- 14 microm in response to M-CSF vs. 11 +/- 2 microm in control experiments). Both WT (100 or 500 nM) and LY294002 (100 microM), a specific PI3K inhibitor structurally unrelated to WT, significantly inhibited osteoclast chemotaxis in response to M-CSF. Taken together, these effects of WT, HWT, and LY294002 are consistent with an important role for PI3K in regulating cytoskeletal function in osteoclasts. The inhibitory effects of WT and HWT on bone resorption may be due, in part, to impairment of osteoclast motility.     

A combination of osteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro

Both human and murine osteoclasts can be derived in vitro from hematopoietic cells or monocytes that are co-cultured with osteoblasts or marrow-derived stromal cells. The osteoclastogenic stimulus provided by murine osteoblasts and marrow-derived stromal cells is now known to be mediated by osteoclast differentiation factor (ODF), a membrane-bound tumor necrosis factor-related ligand. This study demonstrates that mouse spleen cells and monocytes form osteoclasts when cultured in the presence of macrophage-colony stimulating factor (M-CSF) and a soluble form of murine ODF (sODF). Numerous multinucleated osteoclasts expressing tartrate resistant acid phosphatase (TRAP) and calcitonin receptor (CTR) formed within 7 days of culture and engaged in extensive lacunar bone resorption. Osteoclast number and bone resorption area was dependent on sODF concentration. Long-term cultured human monocytes also formed bone resorbing osteoclasts in response to co-stimulation by sODF and M-CSF, although this required more than 11 days in culture. This human osteoclast differentiation was strongly inhibited by granulocyte-macrophage colony stimulating factor. This study further characterises murine osteoclast differentiation caused by sODF and M-CSF co-stimulation in vitro, and shows that the same co-stimulation causes human osteoclast differentiation to occur. We propose that this methodology can be employed to investigate the direct effects of cytokines and other factors on human osteoclast differentiation.     

Cytokine-induced nitric oxide inhibits bone resorption by inducing apoptosis of osteoclast progenitors and suppressing osteoclast activity

Interferon-gamma (IFN-gamma) has been shown to inhibit interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) stimulated bone resorption by strongly stimulating nitric oxide (NO) synthesis. Here we studied the mechanisms underlying this inhibition. Osteoclasts were generated in 10-day cocultures of mouse osteoblasts and bone marrow cells and the effect of cytokine-induced NO on osteoclast formation and activity was determined. Stimulation of the cocultures with IL-1 beta, TNF-alpha and IFN-gamma markedly enhanced NO production by 50- to 70-fold, and this was found to be derived predominantly from the osteoblast cell layer. When high levels of NO were induced by cytokines during early stages of the cocultures, osteoclast formation was virtually abolished and bone resorption markedly inhibited. Cytokine stimulation during the latter stages of coculture also resulted in inhibition of bone resorption, but here the effects were mainly due to an inhibitory effect on osteoclast activity. At all stages, however, the inhibitory effects of cytokines on osteoclast formation and activity were blocked by the NO-synthase inhibitor L-NMMA. Further investigations suggested that the NO-mediated inhibition of osteoclast formation was due in part to apoptosis of osteoclast progenitors. Cytokine stimulation during the early stage of the culture caused a large increase in apoptosis of bone marrow cells, and these effects were blocked by L-NMMA and enhanced by NO donors. We found no evidence of apoptosis in osteoclasts exposed to high levels of cytokine-induced NO at any stage in the culture, however, or of apoptosis affecting mature osteoclasts exposed to high levels of NO, suggesting that immature cells in the bone marrow compartment are most sensitive to NO-induced apoptosis. In summary, these studies identify NO as a potentially important osteoblast-osteoclast coupling factor which has potent inhibitory effects on bone resorption. These actions, in turn, are mediated by inhibition of osteoclast formation probably due to NO-induced apoptosis of osteoclast progenitors and by inhibition of the resorptive activity of mature osteoclasts.     

Histomorphometric evidence for echistatin inhibition of bone resorption in mice with secondary hyperparathyroidism

Echistatin, an RGD-containing peptide, was shown to inhibit the acute calcemic response to exogenous PTH or PTH-related protein (PTH-rP) in thyroparathyroidectomized rats, suggesting that echistatin inhibits bone resorption. In this study: 1) we present histological evidence for echistatin inhibition of bone resorption in mice with secondary hyperparathyroidism, and show that 2) echistatin binds to osteoclasts in vivo, 3) increases osteoclast number, and 4) does not detectably alter osteoclast morphology. Infusion of echistatin (30 microg/kg x min) for 3 days prevented the 2.6-fold increase in tibial cancellous bone turnover and the 36% loss in bone volume, produced by a low calcium diet. At the light microscopy level, echistatin immunolocalized to osteoclasts and megakaryocytes. Echistatin treatment increased osteoclast-covered bone surface by about 50%. At the ultrastructural level, these osteoclasts appeared normal, and the fraction of cells containing ruffled borders and clear zones was similar to controls. Echistatin was found on the basolateral membrane and in intracellular vesicles of actively resorbing osteoclasts. Weak labeling was found in the ruffled border, and no immunoreactivity was detected at the clear zone/bone surface interface. These findings provide histological evidence for echistatin binding to osteoclasts and for inhibition of bone resorption in vivo, through reduced osteoclast efficacy, without apparent changes in osteoclast morphology.     

Recording of mitochondrial transmembrane potential and volume in cultured rat osteoclasts by confocal laser scanning microscopy

Osteoclasts are multinuclear bone-resorbing cells which contain abundant mitochondria. Morphological studies have suggested that a correlation may exist between mitochondrial concentration and bone resorption by osteoclasts. However, investigation of mitochondrial transmembrane potential (delta psi) and volume has been hampered by the difficulty in obtaining a sufficient number of osteoclasts for assessing these characteristics by flow cytometric analysis. In this study, we have used confocal laser scanning microscopy after loading the cells with Rhodamine 123 and 10-nonyl Acridine Orange to record mitochondrial delta psi and volume, respectively, in isolated rat osteoclasts cultured on bovine bone slices. Optimal staining conditions were found to be 10 micrograms ml-1 for 40 min for Rhodamine, and 1 microM for 10 min for the 10-nonyl Acridine Orange derivative. Two osteoclast populations, whose shape seemed to reflect bone resorption and migratory functions, were identified depending on their shape and on the distribution of the two dye probes. 'Round-shaped' osteoclasts had significantly higher mitochondrial delta psi and volume in the apical regions than in the basolateral portions (p < 0.00001). In contrast, mitochondrial delta psi and volume in 'irregular-shaped' osteoclasts were rather evenly distributed in both these regions (p > 0.05). Our results indicate that there is an apical polarization of mitochondria in osteoclasts corresponding to the energy demands associated with bone resorption.     

5-(5,6-Dichloro-2-indolyl)-2-methoxy-2,4-pentadienamides: novel and selective inhibitors of the vacuolar H+-ATPase of osteoclasts with bone antiresorptive activity

The vacuolar H+-ATPase (V-ATPase), located on the ruffled border of the osteoclast, is a proton pump which is responsible for secreting the massive amounts of protons that are required for the bone resorption process. With the aim to identify new agents which are able to prevent the excessive bone resorption associated with osteoporosis, we have designed a novel class of potent and selective inhibitors of the osteoclast proton pump, starting from the structure of the specific V-ATPase inhibitor bafilomycin A1. Compounds 3a-d potently inhibited the V-ATPase in chicken osteoclast membranes (IC50 = 60-180 nM) and were able to prevent bone resorption by human osteoclasts in vitro at low-nanomolar concentrations. Notably, the EC50 of compound 3c in this assay was 45-fold lower than the concentration required for half-maximal inhibition of the V-ATPase from human kidney cortex. These results support the validity of the osteoclast proton pump as a useful molecular target to produce novel inhibitors of bone resorption, potentially useful as antiosteporotic agents.     

Expansions of T-cell subsets expressing particular T-cell receptor variable regions in chronic beryllium disease

Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of metabolic bone diseases. Although the molecular targets of bisphosphonates have not been identified, these compounds inhibit bone resorption by mechanisms that can lead to osteoclast apoptosis. Bisphosphonates also induce apoptosis in mouse J774 macrophages in vitro, probably by the same mechanisms that lead to osteoclast apoptosis. We have found that, in J774 macrophages, nitrogen-containing bisphosphonates (such as alendronate, ibandronate, and risedronate) inhibit post-translational modification (prenylation) of proteins, including the GTP-binding protein Ras, with farnesyl or geranylgeranyl isoprenoid groups. Clodronate did not inhibit protein prenylation. Mevastatin, an inhibitor of 3-hydroxy-3-methylglutatyl (HMG)-CoA reductase and hence the biosynthetic pathway required for the production of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, also caused apoptosis in J774 macrophages and murine osteoclasts in vitro. Furthermore, alendronate-induced apoptosis, like mevastatin-induced apoptosis, could be suppressed in J774 cells by the addition of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, while the effect of alendronate on osteoclast number and bone resorption in murine calvariae in vitro could be overcome by the addition of mevalonic acid. These observations suggest that nitrogen-containing bisphosphonate drugs cause apoptosis following inhibition of post-translational prenylation of proteins such as Ras. It is likely that these potent antiresorptive bisphosphonates also inhibit bone resorption by preventing protein prenylation in osteoclasts and that enzymes of the mevalonate pathway or prenyl protein transferases are the molecular targets of the nitrogen-containing bisphosphonates. Furthermore, the data support the view that clodronate acts by a different mechanism.     

The cellular actions of interleukin-11 on bone resorption in vitro

The pleiotropic cytokine interleukin-11 (IL-11) stimulates osteoclast formation in vitro, but it is not known whether it influences other steps in the bone-resorptive cascade. Using a variety of in vitro model systems for studying bone resorption we have investigated the effects of IL-11 on 1) osteoclast formation, fusion, migration, and activity; and 2) osteoblast-mediated osteoid degradation. The involvement of matrix metalloproteinases (MMPs) and products of arachidonic acid metabolism in IL-11-mediated resorption were also assessed. We first examined the bone-resorptive effects of IL-11 by assessing 45Ca release from neonatal mouse calvarial bones. IL-11 dose-dependently stimulated bone resorption with an EC50 of 10(-10) M. The kinetics of IL-11-mediated 45Ca release demonstrated that it was without effect for the first 48 h of culture, but by 96 h, it stimulated 45Ca release to the same level as that produced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (a hormone that stimulates osteoclast formation and activity). IL-11 also produced a dose-dependent increase in osteoblast-mediated type I collagen degradation with a maximum of 58.0 +/- 6.2% at 5 x 10(-9) M; this effect of IL-11 was less than that produced by 1,25-(OH)2D3 (76.5 +/- 7.1%) and was prevented by an inhibitor of MMPs, but not those blocking arachidonic acid metabolism. We then tested the effects of IL-11 on isolated mouse osteoclasts cultured on ivory slices in the presence and absence of primary mouse osteoblasts. IL-11 had no effect on isolated osteoclast activity even in coculture with primary osteoblasts. We then examined the effects of IL-11 on the formation of osteoclast-like multinucleate cells in mouse bone marrow cultures and the resorptive activity of such cultures using ivory as a substrate. IL-11 dose-dependently increased 1) the number of tartrate-resistant acid phosphatase-positive osteoclast-like multinucleate cells and 2) the surface area of lacunar resorption, although the effects were less than that of 1,25-(OH)2D3. The effect of IL-11 on bone marrow lacunar resorption was prevented by a combination of inhibitors of 5-lipoxygenase and cyclooxygenase. In 17-day-old metatarsal bones, IL-11 prevented the migration of (pre)osteoclasts to future resorption sites, whereas their fusion was unaffected. These results provide strong evidence that IL-11 stimulates bone resorption by enhancing osteoclast formation and osteoblast-mediated osteoid degradation rather than stimulating osteoclast migration and activity. Our data also suggest that the stimulatory effects of IL-11 involve both MMPs and products of arachidonic acid metabolism.     

Cloning and identification of human Sca as a novel inhibitor of osteoclast formation and bone resorption

Increased osteoclast activity is responsible for the enhanced bone destruction in postmenopausal osteoporosis, Paget's disease, bone metastasis, and hypercalcemia of malignancy. However, the number of known inhibitory factors that block osteoclast formation and bone resorption are limited. Therefore, we used an expression-cloning approach to identify novel factors produced by osteoclasts that inhibit osteoclast activity. A candidate clone was identified and isolated from a human osteoclast-like multinucleated cell (MNC) cDNA library, named osteoclast inhibitory peptide-1 (OIP-1), and the cDNA sequence was determined. This sequence matched that of the recently identified human stem cell antigen, was structurally similar to the mouse Ly-6 gene family, and the sequence predicted it was a glycosyl phosphatidyl inositol (GPI)-anchored protein that had a cleavable COOH-terminal peptide. Western blot analysis of conditioned media from 293 cells transfected with the OIP-1 cDNA clone confirmed that OIP-1 was released into the media as a membrane-bound GPI-linked protein. Interestingly, both recombinant OIP-1 expressed in Escherichia coli (which does not have GPI linker) and OIP-1 expressed by mammalian cells significantly reduced osteoclast-like MNC formation induced by 1,25-dihydroxyvitamin D3 or PTH-related protein in mouse and human bone marrow cultures, and inhibited 45Ca release from prelabeled bone in fetal rat organ cultures. In contrast, recombinant OIP-1 did not inhibit the growth of a variety of other cell types. These data indicate that OIP-1 is a novel, specific inhibitor of osteoclast formation and bone resorption.     

Molecular and functional evidence for calcineurin-A alpha and beta isoforms in the osteoclast: novel insights into cyclosporin A action on bone resorption

We provide the first molecular evidence for the presence of a functional serine/threonine phosphatase, calcineurin-A (CN-A), in the osteoclast. Polymerase chain reaction (PCR) of an osteoclast cDNA library, together with restriction mapping, revealed two isoform sequences, alpha and beta. We then examined the functionality of the detected CN-A by assessing the effect of a classical antagonist, cyclosporin A (CsA), in the osteoclast resorption (pit) assay. CsA (0.1 and 1 microg ml-1) potently inhibited bone resorption. The presence of lymphocytes, with or without prior exposure to CsA in vivo, failed to reverse the CsA-induced resorption-inhibition. Expectedly, CsA had no direct effect on cytosolic Ca2+ levels in fura-2-loaded osteoclasts. These studies are a prelude to further investigations into the possible role of CN-A in osteoclast regulation. Finally, mechanistic studies on the bone effects of CsA, a widely used immunosupressant, should proceed from these observations.     

Stimulation of osteoclast formation by 1,25-dihydroxyvitamin D requires its binding to vitamin D receptor (VDR) in osteoblastic cells: studies using VDR knockout mice

Previous studies have shown that 1,25-dihydroxyvitamin D [1,25(OH)2D] plays important roles in the formation of osteoclasts through its actions on osteoblastic cells. We have generated mice lacking vitamin D receptor (VDR) by gene targeting (VDR-/-). These mice had tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and exhibited similar levels of parameters for bone resorption to those in wild type mice. The present studies were undertaken to clarify whether effects of 1,25(OH)2D on osteoclast formation require VDR in osteoblasts, and to examine mechanisms of the formation of osteoclasts without VDR-mediated actions using VDR-/- mice. When wild-type calvarial osteoblasts and spleen cells were co-cultured with 1,25(OH)2D, TRAP-positive osteoclasts were formed regardless of the genotypes of spleen cells. In contrast, when osteoblasts from VDR-/- mice were co-cultured, no osteoclasts could be formed even with wild-type spleen cells. Parathyroid hormone and interleukin-1alpha stimulated osteoclast formation by co-cultures from VDR-/- mice, and the generated osteoclasts showed resorbing activity. These results demonstrate that VDR-mediated actions of 1,25(OH)2D in osteoblasts are essential for osteoclast formation by 1,25(OH)2D, and that functionally intact osteoclasts can be formed without 1,25(OH)2D actions under stimulations by other agents. It is suggested that osteoclastic bone resorption can be maintained without 1,25(OH)2D actions by other stimulatory agents.     

Development of osteoclasts from embryonic stem cells through a pathway that is c-fms but not c-kit dependent

Osteoclasts are hematopoietic cells essential for bone resorption. To study the derivation of these interesting cells, we developed a stepwise culture system where stromal cells promote embryonic stem (ES) cells to differentiate into mature osteoclasts. Three phases to this differentiation process include (1) induction of hematopoiesis, along with the generation of osteoclast precursors, (2) expansion of these precursors, and (3) terminal differentiation into mature osteoclasts in the presence of 1alpha,25-dihydroxyvitamine D3 . Although the transition of ES cells to the hematopoietic lineage was not blocked by an antibody to c-fms, later phases were dependent on a signaling through this transmembrane receptor as indicated by the finding that anti-c-fms treatment of cells in the second and third phases reduced the number of osteoclasts produced by 75% and more than 99%, respectively. Blockade of signaling through another tyrosine kinase-type receptor, c-kit, did not affect any stages of osteoclastogenesis, although generation of other hemopoietic lineages was reduced to less than 10% of untreated. When small numbers of ES cells were directly cultured under conditions that promote osteoclast differentiation, tartrate-resistant acid phosphatase-positive multinucleated cells were observed at the edge but not inside of colonies. This suggests that some types of cell-cell interactions may inhibit development of mature osteoclasts. The culture system developed here provides an important tool for osteoclast biology.     

Ibuprofen controlled-release formulation. A clinical trial in dental impaction pain

The effects of 17 beta-estradiol (E2) and treadmill exercise on tibial bone mass, tibial uptake of 45Ca, and proximal tibia osteoblast and osteoclast cell number were determined in adult ovariectomized rats. Female rats aged 10 months were ovariectomized and divided into five groups: (1) sham-operated; (2) ovariectomized; (3) ovariectomized, given 10 micrograms E2 biweekly; (4) ovariectomized, trained to exercise on a treadmill daily; and (5) ovariectomized, given E2 and exercised. E2 and/or exercise interventions were started 2 months following surgery and continued for 4 months. The calcium content of the tibial metaphysis and diaphysis and the proximal cancellous bone (BV/TV) were lower in the ovariectomized than in the sham-operated controls 6 months after ovariectomy. This lower bone content was associated with a greater bone uptake of 45Ca and a greater number of osteoblasts and osteoclasts in the proximal tibia compared to the control rats. The metaphyseal calcium content was higher and the 45Ca uptake and osteoblast and osteoclast number were lower in the E2-treated rats than in the nontreated rats. In the exercised group, higher diaphyseal calcium content and proximal cancellous bone were associated with lower bone resorption parameters without a significant effect on bone formation. This study demonstrates that E2 primarily influences tibial cancellous bone of the ovariectomized rat and a positive adaptation to exercise occurs in both cancellous and cortical bone. Under estrogen deficiency, E2 replacement suppresses increased bone formation and resorption; exercise suppresses mainly bone resorption. The effects of E2 replacement and exercise training are independent and additive.     

The effect of intravenous and oral fluconazole on the pharmacokinetics and pharmacodynamics of intravenous alfentanil

The growth-promoting activity of PDGF-BB was studied on the adult osteoclasts in the present study. The PDGF receptor beta was detected on the osteoclast membrane through immunohistochemistry (LSAB method) and immunomicroscopy. The PDGF-BB was exerted on the osteoclasts that adhered to the bone slice at concentrations of 0, 10, 20, 30, and 40 ng/ml. The volume of Howship's lacuna augmented significantly and the number of resorption pits also increased with its dose (p < 0.01). The activity of both total acid phosphatase (ACP) and tartrate-resistant acid phosphatase (TRAP) increased significantly. These results suggest that PDGF-BB promotes adult osteoclastic bone resorption directly through PDGF receptor beta and is believed to play important roles in the bone healing process and reconstruction.     

Macrophage activation results in bone resorption

Monocytes or macrophages from important accessory cells in the regulation of bone metabolism and destruction. Cells of the mononuclear phagocyte lineage form the precursor cells of the osteoclasts. Soluble products produced by activated macrophages regulate progenitor cell proliferation, recruitment, differentiation, and activity of osteoblasts and osteoclasts. After osteoclasts are removed from the resorption site, macrophages process bone surfaces and create a cement line before osteoblasts enter to form new bone. Although osteolysis associated with normal bone remodeling is seen as an osteoclast driven process, it may be that in chronic inflammation macrophage activation and vascular derangements lead to low pH, local bone demineralization (acid attack), and H+ mediated stimulation of the primary afferent nociceptive nerve fibers (bone pain). Osteoclasts are not able to attach to demineralized bone or to osteoid surfaces. However, if macrophages degrade the demineralized organic bone matrix, chemotactic factors and attachment sites for osteoclasts are produced. In such a scenario, the osteoclast-osteoblast mediated activation, resorption, and formation cycle would be secondarily activated. Such events may play a role in the most common orthopaedic problem related to macrophage activation, aseptic loosening of orthopaedic joint implants, which is secondary to a chronic foreign body reaction and to micromovement.     

Effects of parathyroid hormone (PTH)-related protein and PTH on osteoclasts and osteoclast precursors in vivo

Increased production of PTH-related protein (PTHrP) and PTH is frequently responsible for hypercalcemia and its associated morbidity. However, it is unclear whether these peptides produce identical effects on cells in the osteoclast lineage in vivo. To examine the effects of continuous in vivo exposure to these factors on both the osteoclast precursors and mature osteoclasts, we inoculated Chinese hamster ovarian cells expressing PTH-(1-84), PTHrP-(1-141), or nontransfected Chinese hamster ovarian cells into nude mice. The effects of these tumors on blood ionized calcium, plasma PTH and PTHrP concentrations, and osteoclast formation were then determined. PTH and PTHrP tumor-bearing mice became hypercalcemic (1.90 +/- 0.04 and 1.97 +/- 0.16 mmol/liter, respectively) compared with control mice (1.29 +/- 0.015 mmol/liter). After 4 days of hypercalcemia, mice were killed, and bone marrow cells were harvested to examine cells at three discrete stages of osteoclast development: multipotent osteoclast precursors, the granulocyte/macrophage colony-forming unit; more committed marrow mononuclear osteoclast precursors; and mature osteoclasts. Neither PTH nor PTHrP had an effect on granulocyte/macrophage colony-forming unit, but similarly increased the number of more committed mononuclear osteoclast progenitors as well as mature osteoclasts in the calvaria. No differences were detected between the effects of PTH and PTHrP on cells in the osteoclast lineage in vivo. Thus, PTH and PTHrP appear to affect only more differentiated cells in the osteoclast lineage, and the differences in osteoclastic bone resorption between primary hyperparathyroidism and humoral hypercalcemia of malignancy are probably due to mechanisms other than effects on osteoclast precursor cells in vivo.     

Later orthodontic appliance reactivation stimulates immediate appearance of osteoclasts and linear tooth movement

BACKGROUND: Delays in the appearance of osteoclasts at compression sites occur after orthodontic appliance reactivation, when this is done during both the period of osteoclast recruitment and the peak expansion in the osteoclast population. This experiment examines osteoclasts and tooth movement in alveolar bone after appliance reactivation coinciding with alveolar bone formation and the time when reactivation osteoclasts first appear (ie, 10 days after initial appliance activation). METHODS: Bilateral orthodontic appliances were activated to mesially tip maxillary molars with 40 cN in 144 rats. After 10 days, all rats were randomized into two groups of 72. Group I had appliances reactivated in precisely the same manner as the first activation. Group II had appliances sham-reactivated. Nine to 12 rats were then sacrificed at 1, 3, 5, 7, 10, and 14 days in both groups (eg, day 1 represents an interval of 11 days after the first appliance activation and 1 day after either sham or real reactivation). Orthodontic movement was measured cephalometrically; changes in osteoclasts and root resorption were assessed at both compression and tension sites histomorphometrically. RESULTS: Teeth in the reactivated group (Group I) displayed linear tooth movement (62.6 micrometers/day), and 0.9 mm tooth movement by day 10. Significant increases in osteoclast numbers, osteoclast surface percentage, and surface per individual osteoclast were evident in these animals by 1 day postreactivation (P <.01). Significant treatment-related increases in root resorption were not evident at compression sites at any time. CONCLUSIONS: These findings indicate that, after appliance reactivation during the time when reactivation osteoclasts appear, a second cohort of osteoclasts can be recruited immediately, along with immediate and substantial tooth movement and no greater risk of root resorption.