Visualization of acidic compartments in cultured osteoclasts by use of an acidotrophic amine as a marker for low pH

Using the acidotrophic amine 3-(2,4-dinitroanillino)-3'-amino-N-methyldipropylamine (DAMP) as a marker for low pH and immunofluorescence cytochemistry, we examined acidic compartments of osteoclasts cultured on cover glasses or bone slices, where they could resorb the bone surface, forming resorptive lacunae. DAMP-positive structures were seen as vesicular and tubular forms in the cytoplasm, indicating lysosomes and endosomes. Not only the osteoclastic cytoplasm but also the extracellular area around the ruffled border and resorptive lacunae were stained with DAMP, suggesting acidic regions. Immunofluorescence was localized predominantly on the substratum side of actively resorbing osteoclasts, whereas an evenly distributed staining pattern was seen in the nonactive cell. The most intensive reaction was seen at the advancing front of resorptive lacunae within the actively resorbing osteoclasts. The distribution pattern of DAMP seemed to be correlated with the osteoclastic activity, since osteoclasts exhibit alternating resorption and migration phases during the bone-remodeling cycle. In this culture system, the resorptive lacunae were left behind after the osteoclasts had completed resorption and migrated along the bone surface. These exposed resorptive lacunae were also stained with DAMP, which were presumably kept at an acidic pH. The effect of treatment with monensin, chloroquine, ammonium chloride, or nigericin was varied in terms of the immunoreactivity for DAMP, but not complete abolition of the staining was obtained. Weak bases such as chloroquine or ammonium chloride inhibited both intra- and extracellular immunoreactivity. Immunoreactivity for the vacuolar type of proton ATPase (V-ATPase) was demonstrable in the cytoplasm of the osteoclasts but was weakened by the addition of bafilomycin. Immunofluorescence of the resorptive lacunae was still retained even after the treatment with bafilomycin and acetazolamide. Besides, both bafilomycin and acetazolamide reversibly inhibited cellular acidity as judged by DAMP immunocytochemistry, which agrees with the fact that ostoeclastic acidification results from the action of vacuolar proton-pump ATPase coupled with carbonic anhydrase.     

In vitro studies of human and rat osteoclast activity on hydroxyapatite,beta-tricalcium phosphate,calcium carbonate

Investigations on the ceramic degradation caused by osteoclasts are designed to assess osteoclast-ceramic interactions and to determine which ceramics are more suitable for use as bone substitute. This study investigated the resorptive activity of osteoclasts on ceramics presenting different solubility rates. Osteoclasts isolated from new-born rat and from human giant cell tumour were cultured on different bioceramics: hydroxyapatite (HA), beta-tricalcium phosphate (TCP) and calcium carbonate (calcite). Cytoskeletal was revealed by actin labelling and ceramic surfaces were observed by scanning electron microscopy (SEM). On all materials, the distribution of actin in typical ring was revealed. SEM examinations showed a clear difference in the shape and the depth of resorption lacunae on different ceramics. On pure HA, a superficial attack, clearly visible but very little extended. Numerous resorption lacunae, deep and well-delimited were observed on pure beta-TCP, but attacks less punctually were detected too. On pure calcite, an attack with form of spikes, very widespread but superficial was revealed. Degradation measurements revealed a significant increase of P release from the phosphocalcic ceramics and of Ca from all ceramics in the presence of osteoclasts. The both cell models found these characteristics, the rat osteoclasts were also an excellent model to study the ceramic resorption.     

Topography of the mineralization front on different surfaces of the body of a vertebra

Peculiarities of the relief of the mineralization front have been investigated on the periostal surface of the human vertebral body in several zones. The material has been obtained from male persons at the age of 20-84 years and studied by means of the light and scanning electron microscopy. The size of lateral surfaces of the vertebral body does not differ essentially from the relief of the periostal surfaces of other bones. In people of middle age certain changes in structure of the mineralized cartilage plate in the area adjoining the nucleus pulposus and the fibrous ring of the intervertebral disc are demonstrated. In persons of elderly and old age a definite decrease in thickness of the cortical layer of the vertebra is noted. At the same time, the plate of the mineralized cartilage adjoining the cortical layer grows thicker and collagene fibers in the spinal column ligaments undergo mineralization. Sometimes, microfractures of the cortical plate of the vertebral body and Schmorl noduli are revealed.     

Wolff's law and the problem of muscle attachment on resorptive surfaces of bone

During the growth of a bone, outer (periosteal) surfaces in many areas undergo normal remodeling processes involving resorptive removal. Attachments of muscles commonly occur on such outer resorptive surfaces. The cortex in these regions grows in an inward direction by bone deposition on endosteal surfaces. In some areas of a bone, a portion of a muscle can be inserted onto a depository surface, but other parts of the same muscle may be attached onto an adjacent resorptive surface. It has been generally assumed that the pull of a muscle acts to directly stimulate deposition of new bone, and that attachments of muscle are thereby responsible for determining the gross morphology of a whole bone. In view of the foregoing considerations, a re-evaluation and an expansion of this concept is now needed. Muscle pull, in many regions of a bone, can be associated with normal cortical recession (involving surface resorption) as well as with outward bone deposition.     

Morpho-functional correlations of the structure of bone cells and adjoining bone matrix in the developing bone

By means of scanning and transmissive electron microscopy methods structure of the developing bone has been studied. Interconnection of the cell structure and spatial organization of the adjoining matrix has been demonstrated. On the surface of the growing bone not only forming areas have been revealed, where under osteoblasts at various functional states, osteoid layer is determined, but also areas of resorption and completed osteogenesis. This demonstrates an interrupted character of osteogenesis at modelling. At the same time for the remodelling process presence of erosive lacunae is specific; they are filled with a newly deposited collagenous matrix. Therefore, it is possible to suppose that formation of the bone as an organ during the postnatal development includes in itself both mechanisms supporting its form at outgrowth of the osseous matrix volume (modeling) and its continuous rearrangement and adaptation to real conditions of functioning (remodelling).     

Minocycline impairment of both osteoid tissue removal and osteoclastic resorption in a synchronized model of remodeling in the rat

In addition to their antibacterial effects, tetracyclines may inhibit interstitial collagenase activity and bone resorption. These properties were assessed morphometrically using minocycline (25 and 50 mg/kg/day given by the IM route) in a rat model of synchronized remodeling in which osteoclastic resorption peaks 4 days after the activating event (the extractions of the upper molars) along the antagonist mandibular cortex, a zone undergoing physiologically active formation. During the first 2 days of activation, minocycline at the two doses impaired very significantly the disorganization of both the osteoid seam and the layer of osteoblasts, a prerequisite to give osteoclasts access to the mineralized bone surface. The number of readily identifiable osteoblasts decreased slightly during this period, suggesting that minocycline prevented their transformation into lining cells. Their synthetic activity, as estimated by the size of the cells and their nucleus, appeared relatively preserved too, mostly with the higher dose. At the peak of osteoclascia, the bone surfaces undergoing remodeling were significantly decreased in the minocycline-treated groups. The resorption surface was reduced (P < 0.0003) as well as the number of osteoclasts (P < 0.0007), which were also significantly smaller. Their resorbing activity was dramatically affected as well: they excavated lacunae whose area was significantly reduced by over 70%. In addition, formation was still a prominent activity in the treated animals. These data are compatible with the inhibition at the early stages of activation of an osteoblast-secreted collagenase whose action may be the elimination of the osteoid seam. The inhibition of an osteoclast collagenase and/or of a bone matrix bound-collagenase may be responsible for the reduction in lacunar size. A direct effect of minocycline on osteoclast resorptive activity may also participate in the low resorption profile, as tetracyclines are known to interfere with the intracellular [Ca²⁺]. © 1996 Wiley-Liss, Inc.     

The significance of a differential distribution of phosphomonoesterases on bone surfaces after prolonged demineralization

Summary An observed differential distribution of alkaline and acid phosphatase on the surfaces of growing bones may serve to describe transformative processes of bone growth. This conclusion has been reached by comparing the distribution of the two enzymes on the surfaces of fibulae from young rats with the patterns of apposition and resorption on the periosteal surfaces of this bone, revealed by in vivo staining with alizarin red S. Presence of reaction to acid phosphatase is, as shown before, an indication of resorptive surfaces, while the presence of reaction to alkaline phosphatase is an indication of depository surfaces.     

The skeleton in primary hyperparathyroidism: a review focusing on bone remodeling, structure, mass, and fracture.

The mechanisms behind the influence of PHPT on the skeleton are closely connected with bone turnover. Throughout life, the skeleton is continuously renewed by bone remodeling, a process which serves the purpose of repairing damaged bone and adapting the skeleton to changes in physical load. In this process, old bone is removed by osteoclastic resorption and new bone is laid down by osteoblastic formation. Bone mass increases with growth in the first decades of life, and around the age of 30 years the peak bone mass is reached. Thereafter, as a result of mechanisms involving bone remodeling, a net bone loss is seen: 1) A reversible bone loss because of increase in the remodeling space, i.e., the amount of bone resorped but not yet reformed during the remodeling cycle. This mechanism leads to decrease in average trabecular thickness and cortical width, and to increase in cortical porosity. 2) An irreversible bone loss caused by negative bone balance, where the amount of bone formed by the osteoblasts is exceeded by the amount of bone resorbed by the osteoclasts at the same remodeling site. Consequently, progressive thinning of trabecular elements, reduced cortical width and increased cortical porosity is seen. 3) Finally, perforation of trabecular plates by deep resorption lacunae leads to complete irreversible removal of structural bone components. Parathyroid hormone, together with vitamin D, are the principal modulators in calcium homeostasis. The main actions of PTH are executed in bone and kidneys. In the kidneys, PTH increases the tubular re-absorption of calcium, thereby tending to increase serum calcium. PTH also induces increased conversion of 25(OH)-D to 1,25(OH)2-D. This last action, enhances intestinal calcium absorption and increased skeletal calcium mobilization, which further adds to the circulating calcium pool. In bone, the "acute" regulatory actions of PTH on serum calcium are probably accompliced via activation of osteocytes and lining cells. A second mechanism of PTH in bone is the regulation of bone remodeling. The action seems to be an increased recruitment from osteoblastic precursor cells and activation of mature osteoclasts. It is supposed that these responses are predominantly mediated indirectly through actions on osteoblast-like or nonosteoblast-like stromal cells, as osteoclasts themselves to not have PTH receptors. Bone metabolism and bone mass are studied by biochemical bone markers, bone histomorphometry, and densitometry. As bone markers and bone histomorphometry give information on bone metabolism from different points of view, these methods are preferably combined. Histomorphometry gives detailed information about bone turnover on cellular level, the whole remodeling sequence is described, and the bone balance can be calculated. However, they focus on a small volume, and may, therefore, not be representative for the whole skeleton. On the other hand, studies of bone markers supply general information about turnover in the whole skeleton, but they do not give facts on the bone turnover on the cellular or tissue level and bone balance. Bone densitometry is the principal method in studying bone mass, but valuable information concerning bone structure also comes from histomorphometry. Bone remodeling is considerably increased in PHPT. Studies of bone markers show increase in both resorptive and formative markers, and the increases seem to be of equivalent size. This is in agreement with histomorphometric findings and shows that the coupling between resorption and formation is preserved. By histomorphometry on iliac crest biopsies, trabecular bone remodeling is found increased by 50%, judged by the increase in activation frequency; a measure of how often new remodeling is initiated on the trabecular bone surface. In PHPT, such remodeling activity is repeated about once every year. Reconstruction of the whole remodeling sequence does not show major deviations in lengths of the resorptive and formative periods compared to normal. Furthermore, the amount of bone removed by the osteoclasts during the resorptive phase is matched by the amount of new bone formed by the osteoblasts leading to a bone balance very close to zero. Compared with trabecular bone, the turnover rate in cortical bone is considerably lower, around 10%. Remodeling of the cortical bone takes place at the endocortical, the pericortical, and the Haversian surfaces. Endocortical bone remodeling activities are very similar to trabecular remodeling activities with good correlation between individual parameters. Periosteal remodeling activity is negligible in PHPT, as it is in the normal state. Cortical porosity, which reflects the remodeling activity on the Haversian surface, is increased by 30-65% in PHPT. (ABSTRACT TRUNCATED)     

Bone Is Not Essential for Osteoclast Activation

Background

The mechanism whereby bone activates resorptive behavior in osteoclasts, the cells that resorb bone, is unknown. It is known that α_vβ₃ ligands are important, because blockade of α_vβ₃ receptor signaling inhibits bone resorption, but this might be through inhibition of adhesion or migration rather than resorption itself. Nor is it known whether α_vβ₃ ligands are sufficient for resorption the consensus is that bone mineral is essential for the recognition of bone as the substrate appropriate for resorption.

Methodology/Principal Findings

Vitronectin- but not fibronectin-coated coverslips induced murine osteoclasts to secrete tartrate-resistant acid phosphatase, as they do on bone. Osteoclasts incubated on vitronectin, unlike fibronectin, formed podosome belts on glass coverslips, and these were modulated by resorption-regulating cytokines. Podosome belts formed on vitronectin-coated surfaces whether the substrates were rough or smooth, rigid or flexible. We developed a novel approach whereby the substrate-apposed surface of cells can be visualized in the scanning electron microscope. With this approach, supported by transmission electron microscopy, we found that osteoclasts on vitronectin-coated surfaces show ruffled borders and clear zones characteristic of resorbing osteoclasts. Ruffles were obscured by a film if cells were incubated in the cathepsin inhibitor E64, suggesting that removal of the film represents substrate-degrading behavior. Analogously, osteoclasts formed resorption-like trails on vitronectin-coated substrates. Like bone resorption, these trails were dependent upon resorbogenic cytokines and were inhibited by E64. Bone mineral induced actin rings and surface excavation only if first coated with vitronectin. Fibronectin could not substitute in any of these activities, despite enabling adhesion and cell spreading.

Conclusions/Significance

Our results show that ligands α_vβ₃ are not only necessary but sufficient for the induction of resorptive behavior in osteoclasts; and suggest that bone is recognized through its affinity for these ligands, rather than by its mechanical or topographical attributes, or through a putative ‘mineral receptor’.     

Bone Canopies in Pediatric Renal Osteodystrophy

Pediatric renal osteodystrophy (ROD) is characterized by changes in bone turnover, mineralization, and volume that are brought about by alterations in bone resorption and formation. The resorptive and formative surfaces on the cancellous bone are separated from the marrow cavity by canopies consisting of a layer of flat osteoblastic cells. These canopies have been suggested to play a key role in the recruitment of osteoprogenitors during the process of bone remodeling. This study was performed to address the characteristics of the canopies above bone formation and resorption sites and their association with biochemical and bone histomorphometric parameters in 106 pediatric chronic kidney disease (CKD) patients (stage 2–5) across the spectrum of ROD. Canopies in CKD patients often appeared as thickened multilayered canopies, similar to previous reports in patients with primary hyperparathyroidism. This finding contrasts with the thin appearance reported in healthy individuals with normal kidney function. Furthermore, canopies in pediatric CKD patients showed immunoreactivity to the PTH receptor (PTHR1) as well as to the receptor activator of nuclear factor kappa-B ligand (RANKL). The number of surfaces with visible canopy coverage was associated with plasma parathyroid hormone (PTH) levels, bone formation rate, and the extent of remodeling surfaces. Collectively, these data support the conclusion that canopies respond to the elevated PTH levels in CKD and that they possess the molecular machinery necessary to respond to PTH signaling.     

Calcium sensing and cell signaling processes in the local regulation of osteoclastic bone resorption

The skeletal matrix in terrestrial vertebrates undergoes continual cycles of removal and replacement in the processes of bone growth, repair and remodeling. The osteoclast is uniquely important in bone resorption and thus is implicated in the pathogenesis of clinically important bone and joint diseases. Activated osteoclasts form a resorptive hemivacuole with the bone surface into which they release both acid and osteoclastic lysosomal hydrolases. This article reviews cell physiological studies of the local mechanisms that regulate the resorptive process. These used in vitro methods for the isolation, culture and direct study of the properties of neonatal rat osteoclasts. They demonstrated that both local microvascular agents and products of the bone resorptive process such as ambient Ca2+ could complement longer-range systemic regulatory mechanisms such as those that might be exerted through calcitonin (CT). Thus elevated extracellular [Ca2+], or applications of surrogate divalent cation agonists for Ca2+, inhibited bone resorptive activity and produced parallel increases in cytosolic [Ca2+], cell retraction and longer-term inhibition of enzyme release in isolated rat osteoclasts. These changes showed specificity, inactivation, and voltage-dependent properties that implicated a cell surface Ca2+ receptor (CaR) sensitive to millimolar extracellular [Ca2+]. Pharmacological, biophysical and immunochemical evidence implicated a ryanodine-receptor (RyR) type II isoform in this process and localized it to a unique, surface membrane site, with an outward-facing channel-forming domain. Such a surface RyR might function either directly or indirectly in the process of extracellular [Ca2+] sensing and in turn be modulated by cyclic adenosine diphosphate ribose (cADPr) produced by the ADP-ribosyl cyclase, CD38. The review finishes by speculating about possible detailed models for these transduction events and their possible interactions with other systemic mechanisms involved in Ca2+ homeostasis as well as the possible role of the RyR-based signaling mechanisms in longer-term cell regulatory processes.     

Distinctive Subdomains in the Resorbing Surface of Osteoclasts

We employed a novel technique to inspect the substrate-apposed surface of activated osteoclasts, the cells that resorb bone, in the scanning electron microscope. The surface revealed unexpected complexity. At the periphery of the cells were circles and crescents of individual or confluent nodules. These corresponded to the podosomes and actin rings that form a ‘sealing zone’, encircling the resorptive hemivacuole into which protons and enzymes are secreted. Inside these rings and crescents the osteoclast surface was covered with strips and patches of membrane folds, which were flattened against the substrate surface and surrounded by fold-free membrane in which many orifices could be seen. Corresponding regions of folded and fold-free membrane were found by transmission electron microscopy in osteoclasts incubated on bone. We correlated these patterns with the distribution of several proteins crucial to resorption. The strips and patches of membrane folds corresponded in distribution to vacuolar H+-ATPase, and frequently co-localized with F-actin. Cathepsin K localized to F-actin-free foci towards the center of cells with circular actin rings, and at the retreating pole of cells with actin crescents. The chloride/proton antiporter ClC-7 formed a sharply-defined band immediately inside the actin ring, peripheral to vacuolar H+-ATPase. The sealing zone of osteoclasts is permeable to molecules with molecular mass up to 10,000. Therefore, ClC-7 might be distributed at the periphery of the resorptive hemivacuole in order to prevent protons from escaping laterally from the hemivacuole into the sealing zone, where they would dissolve the bone mineral. Since the activation of resorption is attributable to recognition of the αVβ3 ligands bound to bone mineral, such leakage would, by dissolving bone mineral, release the ligands and so terminate resorption. Therefore, ClC-7 might serve not only to provide the counter-ions that enable proton pumping, but also to facilitate resorption by acting as a ‘functional sealing zone’.     

Osteoclast activation: Potent inhibition by the bisphosphonate alendronate through a nonresorptive mechanism

Alendronate, an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its mechanism of action is unknown. Because it localizes to bone surfaces, we compared the sensitivity of components of the resorptive process to incubation on alendronate-coated bone surfaces. We found that bone resorption by osteoclasts isolated from neonatal rat bone was unaffected by alendronate (10^-4 M). Osteoclast production in bone marrow cultures, as assessed by the production of calcitonin-receptor positive cells, was observed even at 10^-4 M, but bone resorption in these cultures was almost completely abolished by 10^-6 M alendronate. The greater sensitivity of osteoclast activation to inhibition by alendronate that these results suggest was supported by similar inhibition of osteoblast-mediated activation of osteoclasts from neonatal rat bone. Thus, activation of osteoclasts by osteoblastic/stromal cells is apparently the most sensitive component of the pathway whereby bone resorption is affected. Moreover, the ability of alendronate to suppress osteoclastic activation does not depend on resorption-mediated release of alendronate from bone surfaces. This ability extends the range of cell types and processes that might be affected by alendronate, beyond those in the immediate vicinity of resorbing cells, to include any cell that comes into contact with alendronate-coated bone surfaces. J. Cell. Physiol. 172:79–86, 1997. © 1997 Wiley-Liss, Inc.     

Influence of metal ion solutions on rabbit osteoclast activities in vitro

The purpose of the present study was to compare the effects of various metal ions (aluminium, chromium, cobalt, gold, iron, strontium, titanium and vanadium) on rabbit osteoclast activities, with respect to their number, size, resorptive capacity and their capacity to release proteinases. Marked heterogeneous osteoclastic behaviour was observed early in culture with metal ions (24 h) in term of resorption parameters. In contrast, protease activities (cysteine-proteinase and metalloproteinase activities) were not modulated in our culture conditions. Aluminium, iron, gold and titanium reduced the number of osteoclasts significantly. Aluminium and gold had no effect on osteoclast-mediated resorption on dentin-slices, although aluminium induced a greater number of very small lacunae. Titanium reduced only the mean surface area per lacunae, cobalt reduced the mean surface area of lacunae and increased their number, and iron reduced both parameters. Strontium had no effect on osteoclast formation and on total dentin slice surface resorbed. However, strontium increased the number of small lacunae formed on dentin-slices by osteoclasts. Chromium had no effect on osteoclast activities. These findings indicate that metal ions induce very early effects on osteoclasts, which can contribute to periprosthetic pathologies via different cellular mechanisms.     

Immunohistochemical localization of cathepsins B,D and L in the rat osteoclast

Immunohistochemical localization of cathepsins B, D and L in the osteoclasts of rat alveolar and femoral bones was investigated by using the avidin-biotin-peroxidase complex method for semithin, 1-m-thick cryosections. Extracellular immunoreactivity for cathepsins B and L was clearly demonstrated along the bone resorption lacunae; the intensity of the extracellular immunoreactivity of cathepsin L was stronger than that of cathepsin B. However, the intracellular immunoreactivity of both cathepsins was weak compared with that of cathepsin D. The intracellular immunoreactivity of cathespin D in the osteoclasts was clearly observed in the granules and/or vacuoles, but extracellular cathepsin D immunoreactivity was either negligible or not detected along the resorption lacunae. In the adjacent sections stained with anti-cathepsin L or D, extensive extracellular deposition of cathepsin L was found along the bone resorption lacunae, with or without osteoclasts, although the intracellular reactivity of cathepsin L was weak. This is the first morphological study in which cathepsins B and L have been demonstrated to be produced in the osteoclasts and extensively secreted into resorption lacunae, and in which cathepsin D was found to be present in the cells but scantily secreted into the lacunae. These findings suggest that cathepsins B and L directly and effectively participate in the degradation of the bone matrix.     

Osteoclast biology in the osteopetrotic (op) rat

Osteopetrosis is a metabolic bone disease characterized by reduced bone resorption. From experimental studies of various osteopetrotic mutations has emerged the hypothesis that each is unique with respect to mechanisms whereby osteoclast development and/or function are reduced. The osteopetrotic (op) mutation in the rat was discovered in Fatty/ORL stock over a decade ago. The paucity of data about osteoclast biology in this mutation prompted this study of cytological, cytochemical, and ultrastructural features of osteoclasts. In op rats, osteoclasts are significantly reduced in number, but are larger and more vacuolated than in normal littermates. Mutant osteoclasts can form ruffled borders and clear zones, but their ability to fragment and excavate bone surfaces is greatly impaired. Cytoplasmic vacuoles in op osteoclasts are randomly distributed and greatly enlarged, and they stain weakly for two cytochemical characteristics of osteoclasts, tartrate-resistant acid phosphatase and acid ATPase. These findings suggest that an abnormality in the lysosomal/vacuolar system, an important component of the resorptive mechanism, may be involved in the interception of osteoclast function in this mutation.     

The in vitro effect of pH on osteoclasts and bone resorption in the cat: implications for the pathogenesis of FORL

Dental disease due to osteoclast over-activity reaches epidemic proportions in older domestic cats and has also been reported in wild cats. Feline osteoclastic resorptive lesions (FORL) involve extensive resorption of the tooth leaving it liable to root fracture and subsequent tooth loss. The aetio-pathogenesis of FORL is not known. Recent work has shown that systemic acidosis causes increased osteoclast activation and that loci of infection or inflammation in cat mouth are likely to be acidotic. To investigate this, we generated osteoclasts from cat blood and found that they formed in large numbers (approximately 400) in cultures on bovine cortical bone slices. Acidosis caused an increase in the size of cells-in cultures maintained up to 14 days at basal pH 7.25, mean osteoclast area was 0.01 +/- 0.003 mm(2), whereas an 8.6-fold increase was observed in cells cultured between 11 and 14 days at pH 7.15 (0.086 +/- 0.004 mm(2)). Acidosis caused a modest increase in the number of osteoclasts. Exposure to pH 6.92 exhibited a 5-fold increase in the area of bone slices covered by resorption lacunae ( approximately 70% bone slice resorbed). In line with this finding, significant increases were observed in the expression of cathepsin K and proton pump enzymes (both approximately 3-fold) that are key enzymes reflective of resorptive activity in osteoclasts. These results demonstrate that acidosis is a major regulator of osteoclast formation and functional activation in the cat, and suggest that local pH changes may play a significant role in the pathogenesis of FORL.     

Localization of osteopontin in resorption lacunae formed by osteoclast-like cells: a study by a novel monoclonal antibody which recognizes rat osteopontin

The characteristics of a monoclonal antibody produced against osteoclast-like multinucleated cells (MNCs) formed in rat bone marrow cultures were examined immunohistochemically and biochemically. The in vitro immunization was performed using as immunogen the MNCs from rat bone marrow cell culture, which revealed many characteristics of osteoclasts. After screening and cloning of hybridomas, the monoclonal antibody HOK 1 was obtained. This antibody reacted weakly with stromal cells and intensely with both MNCs and their putative migratory traces on culture dishes. Immunofluorescent examination of paraffin sections revealed intense reactivity on the epithelium of the choroid plexus, the ileum and the proximal-convoluted tubules of the kidney, and also on bone cells such as osteocytes, osteoblasts, and osteoclasts. Western blotting using purified rat osteopontin verified that the antigen recognized by HOK 1 was osteopontin. Positive HOK 1 immunoreactivity was further observed in the resorption lacunae formed by a culture of MNCs on human tooth slices and on the surface of osteoclasts. The present data suggested that osteopontin is preferentially present on the resorption lacunae in resorbing calcified matrices and that osteoclasts under a specific state might trap this protein on their cell surface.     

Resorption of vital or devitalized bone by isolated osteoclasts in vitro

Summary The maintainance of resorptive capability towards vital or devitalized bone in osteoclasts isolated from the medullary bone of laying hens and cultured for five days in vitro has been investigated morphologically with the aid of light and transmission electron microscopy. Devitalized bone particles ranging in size from 50 to 100 m, added to cultures of osteoclasts, were rapidly surrounded by the osteoclasts which, in transmission electron microscopy, showed ruffled borders and clear zones at the surfaces of contact with bone — features typical of resorptive activity. Alternatively osteoclasts were added onto the endosteal surfaces of vital or devitalized diaphyses of quail femurs after removal of the endosteal and periosteal cell layers. The results indicated that, when the vital or devitalized bone surfaces were devoid of cells, the osteoclasts adhered and resorbed bone (as confirmed by transmission electron microscopy). When vital bone of quail was cultured for 24 h before the addition of osteoclasts a new cell layer was formed; it enveloped all bone surfaces and precluded the access of osteoclasts to bone. The role of these lining cells, ultrastructurally indistinguishable from resting osteoblasts, is discussed.     

Vitronectin receptor has a role in bone resorption but does not mediate tight sealing zone attachment of osteoclasts to the bone surface

During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption lacunae. Vitronectin receptor has previously been shown to be expressed in osteoclasts and it has been suggested that it mediates the tight attachment at the sealing zone. In this study we have shown that glycine-arginine-glycine-aspartic acid- serine pentapeptide inhibits bone resorption by isolated osteoclasts and drastically changes the morphology of the osteoclasts. When the vitronectin receptor was localized by immunofluorescence in rat and chicken osteoclasts cultured on bone slices, it was found to be distributed throughout the osteoclast cell membrane except in the sealing zone areas. Immunoperoxidase staining of rat bone sections at the light microscopical level also revealed intense staining of the cell membrane with occasional small unstained areas, probably corresponding to the sealing zones. Immunoelectron microscopy confirmed the results obtained by light microscopy showing specific labeling only at the ruffled borders and basolateral membranes (0.82 and 2.43 gold particles/microns of membrane, respectively), but not at the sealing zone areas (0.06 gold particles/microns of membrane). Both alpha v and beta 3 subunits of the vitronectin receptor were similarly localized. These results strongly suggest that, although the vitronectin receptor is important in the function of osteoclasts, it is not mediating the final sealing zone attachment of the osteoclasts to the mineralized bone surface.