Study of immunoelectron microscopic localization of cathepsin K in osteoclasts and other bone cells in the mouse femur

The localization of cathepsin K protein in mouse osteoclasts was examined by immunolight and immunoelectron microscopy using the avidin-biotin-peroxidase complex method with anti-cathepsin K (mouse) antibody. With light microscopy, a strong immunoreaction for cathepsin K was found extracellularly along the bone and cartilage resorption lacunae and detected intracellularly in vesicles, granules, and vacuoles throughout the cytoplasm of multinuclear osteoclasts and chondroclasts attached to the surface of the bone or cartilage. Mononuclear cells, probably preosteoclasts, some distance from the bone also contained a few cathepsin K-positive vesicles and granules. Cathepsin K was sometimes found in the cisternal spaces of the rough endoplasmic reticulum and vesicles of the Golgi apparatus with electron microscopy of the basolateral region of the osteoclasts. Cathepsin K-positive vesicles and granules as lysosomal compartments were present in various stages of fusion with vacuoles as endosomal compartments that contained fragmented cathepsin K-negative fibril-like structures. Some of the vacuoles (endolysosomes), which seemed to be formed by this process of fusion, contained cathepsin K-positive vesicles and fibril-like structures that did not show the regular cross striation of type I collagen fibrils. In the apical region of the osteoclasts, cathepsin K-positive vesicles and pits had already fused with or were in the process of fusing with the ampullar extracellular spaces. There were large deposits of cathepsin K on fragmented fibril-like structures without regular cross striation in the extracellular spaces, as well as on and between the cytoplasmic processes of the ruffled border. There were also extensive deposits of cathepsin K on the type I collagen fibrils with cross striation in the bone resorption lacunae. Osteoblasts and osteocytes were negative for cathepsin K. In the immunocytochemical controls, no immunoreaction was found in the osteoclasts or preosteoclasts, or on the collagen fibrils in the resorption lacunae. The results indicate that cathepsin K is produced in mature osteoclasts attached to the bone and secreted into the bone resorption lacunae. The findings suggest that cathepsin K participates in the extracellular degradation of collagen fibrils in the resorption lacunae and in the subsequent degradation of the fragmented fibrils in the endolysosomes. It is also suggested that cathepsin K degrades the organic cartilage matrix.     

Mitochondrial granules in chondrocytes

Rat and mice epiphyseal growth plates were studied with the electron microscope. A gradient of mitochondrial electron-dense granules was found. Chondrocytes in the proliferative zone had few granules, while those of the succeeding zones showed a gradual increase in number and density until the zone of provisional calcification was reached. This zone showed a peripheral distribution of mitochondria and a decrease in the number and density of mitochondrial granules. Isotopic⁴⁷calcium was used autoradiographically to determine the location of calcium in these cells. Grains were found over the endoplasmic reticulum membranes and over most mitochondria. The demonstration of a gradient of these granules and their spatial relation to the mineralization front suggests a possible involvement of mitochondria in the onset of matrix calcification.This work was supported in part by a grant No. D.E. 224-01 from the National Institutes of Health, United States Public Health Service.     

Calcium flux and endogenous calcium content in isolated mammalian growth-plate chondrocytes, hyaline-cartilage chondrocytes, and hepatocytes

The role of chondrocyte mitochondria in endochondral ossification has been the subject of intensive investigation and controversy. The purpose of this study was to quantitate the endogenous calcium content and the maximum capacity for calcium accumulation and release in isolated mammalian growth-plate chondrocytes and hyaline-cartilage chondrocytes. The results indicated that the mitochondria of the isolated growth-plate and hyaline-cartilage chondrocytes possess a greater endogenous calcium content, a greater capacity for calcium accumulation, and a larger labile Ca+2 pool than do the mitochondria of hepatocytes. Growth-plate and hyaline-cartilage mitochondria had an endogenous calcium content of 908 and 142 nanomoles of Ca+2 per milligram of mitochondrial protein. The growth-plate mitochondria had a maximum calcium capacity of 5249 nanomoles of Ca+2 per milligram of mitochondrial protein. In comparison, the mitochondria of hepatocytes had a much smaller endogenous-calcium content and a smaller maximum Ca+2 capacity: twenty-one and 3262 nanomoles of Ca+2 per milligram of mitochondrial protein, respectively. The mitochondrial labile-calcium pool in both growth-plate and hyaline-cartilage chondrocytes was twofold greater than that in the mitochondria of hepatocytes. Chondrocyte mitochondria released approximately 2400 nanomoles of Ca+2 per milligram of mitochondrial protein, whereas hepatocyte mitochondria released 1200 nanomoles of Ca+2 per milligram. These results suggest that the chondrocyte mitochondria are specialized for calcium transport and are important in the calcification of the extracellular matrix of the growth plate.     

Effects of cyclosporine on osteoclast activity: inhibition of calcineurin activity with minimal effects on bone resorption and acid transport activity.

Cyclosporine results in rapid and profound bone loss in transplant patients, an effect ascribed to osteoclasts. Cyclosporine, complexed with the appropriate immunophilin, inhibits calcineurin (the calcium/calmodulin dependent serine/threonine phosphatase) activity. We tested the hypothesis that cyclosporine inhibits calcineurin activity in osteoclasts, resulting in stimulation of osteoclast activity. We compared the effects of cyclosporine A and the calmodulin antagonist, tamoxifen, on bone resorption by avian osteoclasts. Tamoxifen inhibits bone resorption approximately 60%, whereas cyclosporine A only inhibited bone resorption 12%. One-hour treatment with 100 nM cyclosporine inhibited osteoclast calcineurin activity 70% in whole cell lysates, whereas 10 microM tamoxifen only inhibited calcineurin activity 25%. We compared the effects of cyclosporine A and tamoxifen on acid transport activity in isolated membrane vesicles and in isolated membrane vesicles obtained from osteoclasts treated with cyclosporine A or tamoxifen under conditions that inhibit calcineurin activity. Direct addition of cyclosporine A in the acid transport assay, or pretreatment of cells with cyclosporine A followed by membrane isolation, had no effect on acid transport activity in membrane vesicles. In contrast, direct addition of tamoxifen to membranes inhibits acid transport activity, an effect that can be prevented by addition of exogenous calmodulin. Furthermore, acid transport activity was also inhibited in membrane vesicles isolated from cells treated with tamoxifen. In conclusion, cyclosporine A inhibits osteoclast calcineurin activity; however, calcineurin inhibition does not correspond to a significant effect on acid transport activity in isolated membrane vesicles or bone resorption by osteoclasts.     

Diphyllin, a novel and naturally potent V-ATPase inhibitor, abrogates acidification of the osteoclastic resorption lacunae and bone resorption.

Dissolution of the inorganic phase of bone by the osteoclasts mediated by V-ATPase and ClC-7 is a prerequisite for bone resorption. Inhibitors of osteoclastic V-ATPase or ClC-7 are novel approaches for inhibition of osteoclastic bone resorption. By testing natural compounds in acidification assays, diphyllin was identified. We characterized diphyllin with respect to the pharmacological effects on osteoclasts. INTRODUCTION: Osteoclastic acidification of the resorption lacuna and bone resorption requires activity of both V-ATPase and the chloride channel ClC-7. Inhibition of these processes represents a novel approach for treatment of bone metabolic disorders. We identified diphyllin, a novel inhibitor of V-ATPase, and characterized this natural compound with respect to activity in human osteoclasts. MATERIALS AND METHODS: Diphyllin was tested in the acid influx assay and V-ATPase assay using bovine chromaffin granules. Human osteoclasts were generated from CD14+ monocytes cultured with macrophage-colony stimulating factor (M-CSF) and RANKL. The effect of diphyllin on lysosomal acidification in human osteoclasts was studied using acridine orange. The effect of diphyllin on bone resorption by osteoclasts was measured as release of C-terminal cross-linked telopeptide of type I collagen (CTX-I) and calcium into the supernatants and by scoring pit area. Osteoclast number, TRACP activity, and cell viability were measured. Furthermore, the effect of diphyllin on bone nodule formation was tested using the mouse osteoblast cell line MC3T3-E1. RESULTS: In the acid influx assay, diphyllin potently inhibited the acid influx (IC50 = 0.6 nM). We found that diphyllin inhibited V-ATPase with an IC50 value of 17 nM, compared with 4 nM for bafilomycin A1. Moreover, diphyllin dose-dependently inhibited lysosomal acidification in human osteoclasts. Furthermore, we found that diphyllin inhibited human osteoclastic bone resorption measured by CTX-I (IC50 = 14 nM), calcium release, and pit area, despite increasing TRACP activity, numbers of osteoclasts, and cell viability. Finally, diphyllin showed no effect on bone formation in vitro, whereas bafilomycin A1 was toxic. CONCLUSIONS: We identified a natural compound that potently inhibits V-ATPase and thereby lysosomal acidification in osteoclasts, which leads to abrogation of bone resorption. Because recent studies indicate that inhibition of the osteoclastic acidification leads to inhibition of resorption without inhibiting formation, we speculate that diphyllin is a potential novel treatment for bone disorders involving excessive resorption.     

Effect of ethane-1-hydroxy-1,1-diphosphonate (EHDP) administration on duodenal calcium transport

Ethane-1-hydroxy-1,1-diphosphonate (EHDP) given subcutaneously to rats in high doses (10 mg P/Kg body weight daily×10) inhibited the transport of calcium by rat duodenal gut sacs, when⁴⁵Ca serosal/mucosal concentration ratios were used to measure active transport. Electron microscopic studies revealed the accumulation of electron-dense granules in the microvilli, representing a bound form of calcium and a paucity of such granules in mitochondria, changes identical to those seen in rachitic rats. These results suggest that the defect is mediated in part by interference in transport of calcium from microvilli to other intracellular sites.Baylor College of Dentistry     

Ultracytochemical investigation of calcium-activated adenosine triphosphatase (Ca++-ATPase) in chick tibia

The ultrastructural distribution of Ca++-ATPase in bone cells of growing chick tibia was investigated by a cytochemical method in order to gain insight into possible sites of calcium ion translocation. Both osteoclasts and osteoblasts showed a polar distribution of reaction product along the plasma membrane. In osteoclasts, enzymatic activity occurred along the portion of the plasma membrane facing the marrow but not along the ruffled border or clear zone. The reaction product in these cells was due solely to Ca++-ATPase action. In osteoblasts, the plasma membrane facing away from bone (apical and lateral membrane) was very intensely stained, whereas the basal membrane was unstained. The reaction product in these cells appeared to be the result of both Ca++-ATPase and Ca++,Mg++-ATPase. In osteocytes, no plasma membrane staining was detectable. Mitochondrial staining in all three types of cells was more sensitive to fixation than was the plasma membrane enzyme, suggesting that mitochondrial and plasma membrane Ca++-ATPases are chemically distinct, as biochemical studies have shown. In general, mitochondria in osteoclasts stained more intensely than those in osteoblasts or osteocytes. Mitochondrial and vesicular sites of activity may be related to intracellular calcium storage, whereas calcium ATPases of the plasma membrane are presumed to be involved in calcium efflux from the cells. Calcitonin treatment did not alter the enzymatic distribution or intensity in osteoclasts. The striking polar distribution of both osteoclast and osteoblast plasma-membrane activity suggests that directional calcium pumping by these cells may be of importance in bone-forming and bone-resorbing mechanisms.     

In vivo biological performance of composites combining micro-macroporous biphasic calcium phosphate granules and fibrin sealant

Introduction Fibrin glues are currently used by surgeons and can facilitate the handling of biomaterials. Combining fibrin glue with calcium phosphate bioceramics gives a mouldable composite that cements the granules into the implantation site. In addition to the mechanical aspect of the composite, it has been suggested that the mixture also promotes wound healing. These human blood derivatives contain natural (aprotinin) or synthetic (tranexamic acid) antifibrinolytic substances. We compared the bioactivity of two composites combining calcium phosphate granules with two different types of fibrin glue, one with aprotinin and the other with tranexamic acid.Materials and methods The composite was composed of fibrin glue (Tissucol) and 1 to 2 mm granules of biphasic calcium phosphate granules (MBCP) with a volume ratio of 1 for 2. Bone cavities were drilled in 12 New Zealand rabbits and filled with a composite with aprotinin-fibrin glue on the right condyle and one with tranexamic acid-fibrin glue on the left condyle. The rabbits were randomized into two groups: 3 and 6 weeks of delay. Light microscopy, scanning electron microscopy and image analysis were performed.Results No adverse reactions were observed in either sample. Bony ingrowth associated with bioceramic resorption by osteoclastic TRAP-positive cells was noted. No significant difference was observed between the two composites. The bony ingrowth and ceramic resorption were qualitatively and quantitatively similar with both composites.Conclusion This study demonstrated that the choice of a natural (aprotinin) or synthetic (tranexamic acid) antifibrinolytic agent in the fibrin sealant associated with calcium phosphate granules and used as a bone substitute had no effect on the bioactivity of the composite. It remained efficient in bone reconstruction, no adverse effects were observed, and the bony ingrowth was qualitatively and quantitatively equivalent with the two types of fibrin sealant.     

A sensitiviein vitro method for studying the induction and inhibition of bone resorption

A sensitive method has been developed for studying the early effects of hormones and other agents on the movement of calcium into and out of bone. Half-calvariae from 6-day-old mice that have been pulsed four days previously with⁴⁵Ca, are explanted into separate dishes of liquid medium and maintainedin vitro; one half serves as control and the other for experimentation. The time course of action of any agent is followed by removing small samples aseptically from the media and analysing for isotope. Both parathyroid hormone and vitamin A cause extensive bone resorption, and as compared with controls an increased release of⁴⁵Ca from the treated bones into the medium can be detected within 2 hours. Calcitonin rapidly inhibits the release of⁴⁵Ca from resorbing bones; the time course is similar to that for its actionin vivo in lowering serum calcium. Our results indicate that agents that induce bone resorption increase both the number of multinucleate osteoclasts and the effectiveness of the existing cells in mobilising calcium. Calcitonin prevents the formation of new multinucleate osteoclasts, and also prevents existing osteoclasts from mobilising bone mineral. Calcitonin inhibits the release of⁴⁵Ca from living explants maintained in control medium. This reduction is attributed to the suppression of the endogenous resorption that is in progress when the bones are explanted; exchange of isotope still occurs, as in a dead explant.     

Elevated concentrations of synthetic fluorinated glucocorticoid analogs transiently increase the intracellular exchangeable calcium in cultured bone cells

Summary This study investigated the influence of various glucocorticoids on the transport and accumulation of Ca²⁺ in cultured bone cells. For measuring changes in the amount of intracellular exchangeable Ca²⁺, cultures were initially preincubated with⁴⁵Ca for 48 h thereby achieving a steady state. Triamcinolone acetonide induced a transient increase in the cells' content of exchangeable Ca²⁺—an effect that lasted for 5 h and was followed by a pronounced decrease as noted at 24 h. A similar increase was observed with dexamethasone, whereas hydrocortisone and corticosterone led to a less significant effect. No significant changes took place with the use of deoxycorticosterone, progesterone, and estradiol. The effect of triamcinolone on the cellular content of exchangeable Ca²⁺ was completely blocked by both cycloheximide and puromycin when added shortly after the addition of the corticosteroid to the culture system. In order to determine the effect of steroid hormones on the initial rate of Ca²⁺ influx into cultured cells, cultures were first preincubated with the various hormones and thereafter⁴⁵Ca was added. Only fluorinated glucocorticoid analogs such as triamcinolone acetonide and dexamethasone induced significant rises in the initial rate of Ca²⁺ influx. Ultrastructural examinations showed that in 5-day-old control cultures osteoblast-like cells show multiple aggregates of calcium pyroantimonate along their plasma membrane. In contrast, similar cells cultured in the presence of triamcinolone (10⁻⁷M) for 3 h lacked such precipitates along their plasma membrane but instead contained aggregates of calcium pyroantimonate within enlarged mitochondria. Bone cells that were incubated with triamcinolone for a longer period of time (24 h) exhibited hypertrophied mitochondria that were devoid of such calcium precipitates. Hence this study indicates that potent synthetic analogs of glucocorticoids affect: (a) the rate of calcium influx into bone cells, (b) the intracellular concentration of calcium, and (c) the distribution of calcium within these cells.     

Comparative studies of intra- and extramitochondrial calcium phosphates from the hepatopancreas of the blue crab (Callinectes sapidus)

Subcellular fractions of cytoplasmic mineral granules and mineral-loaded mitochondria were isolated from whole homogenates of hepatopancreas of the blue crab (Callinectes sapidus). Chemical, physical, and morphologic studies were carried out on both subcellular fractions, with and without prior removal of organic components by hydrazine extraction. In contrast to cytoplasmic granules, whole mitochondria contained appreciable amounts of mineral ions not associated with a solid mineral phase. Quantitative analyses and infrared spectroscopy showed cytoplasmic and mitochondrial mineral phases to be calcium phosphates of similar but not identical composition. Both cytoplasmic and mitochondrial mineral phases, as in synthetic amorphous calcium phosphate, show noncrystalline patterns when examined by infrared spectroscopy and x-ray diffraction, and a common ultrastructure of clustered spheres of approximately 100 ? diameter. The findings suggest that amorphous calcium phosphate in biological systems may exhibit appreciable variation in Ca/P and in the content of foreign ions such as Mg²⁺, ADP, and ATP. A mitochondriogenic mechanism of calcification could not be confirmed nor refuted by this study.     

Influence of estrogens on bone resorption in organ culture

Estradiol benzoate, ethinyl estradiol, and human calcitonin were compared for their ability to inhibit the spontaneous or parathyroid extract-induced bone resorption in organ cultures of 19-day fetal rat fibulae. The criteria used for the assessment of bone resorption were: the release of⁴⁵Ca from paired prelabeled bone rudiments into the culture medium, the dry weight, and the number of osteoclasts per bone. Estradiol benzoate at concentrations of 1.3×10^–5 to 2.6×10^–4 M had no effect on the release of⁴⁵Ca from fetal fibulae or on the dry weight. Histologically, treated bones were well preserved and resembled the controls. Parathyroid extract (PTE) alone caused extensive bone resorption with numerous osteoclasts and enhanced⁴⁵Ca release without weight gain. The addition of estradiol benzoate to the culture medium did not prevent the resorptive action of PTE. Ethinyl estradiol alone provoked a dose-related inhibition of⁴⁵Ca release at concentrations of 3.4×10^–5 to 1.7×10^–4 M. However, this inhibition was accompanied by a decrease in bone dry weight and by various degrees of cellular damage. The same phenomenon was observed in PTE-treated fibulae. Human calcitonin, on the contrary, inhibited the release of⁴⁵Ca by decreasing the number of osteoclasts while the weight of the fibulae increased. The inhibitory action of ethinyl estradiol appears to be caused by a toxic effect on bone cells. It is concluded that estrogens have no direct physiological effect on bone resorption in vitro.     

Glutamate does not play a major role in controlling bone growth.

Bone cells express glutamate-gated Ca2+-permeable N-methyl-D-aspartate (NMDA) receptors and GLAST glutamate transporters. Blocking NMDA receptors has been reported to reduce the number of bone resorption pits produced by osteoclasts, and mechanical loading alters GLAST transporter expression, which should change the extracellular glutamate concentration and NMDA receptor activation. Thus, by analogy with the brain, glutamate is postulated to be an important intercellular messenger in bone, controlling bone formation and resorption. We found that activating or blocking NMDA receptors had no effect on bone formation by rat osteoblasts in culture. The number of resorption pits produced by osteoclasts was reduced by the NMDA receptor blocker MK-801 but not by another blocker AP-5, implying that this effect of MK-801 is unrelated to its glutamate-blocking action. By contrast, MK-801, AP-5, and NMDA had no consistent effect on the volume of pits. In mice with GLAST glutamate transporters knocked out, no differences were detected in mandible and long bone size, morphology, trabeculation, regions of muscle attachment, resorption lacunae, or areas of formation versus resorption of bone, compared with wild-type siblings. These data suggest that glutamate does not play a major role in controlling bone growth.     

Quantitative analyses of osteoclast changes in resorbing bone organ cultures

Summary The stimulation of bone resorption, assessed by the release of⁴⁵Ca from prelabeled bones, was associated with an increase in number of osteoclasts per bone section in parathyroid hormone (PTH)-treated bones, but not in lipopolysaccharide (LPS)-treated bones. By contrast the number of nuclei per osteoclast increased following LPS treatment, but was not affected by PTH. LPS-treated bones had more multinucleated cells, some having as many as 27 nuclei per osteoclast. More osteoclasts were adjacent to the bone collar in bones treated with LPS or PTH than in control bones. In LPS-treated bones this area also contained the largest osteoclasts, as determined by the greatest number of nuclei per osteoclast. The results suggest that LPS and PTH stimulate osteoclastic resorption by different mechanisms.     

Comparison in localization between cystatin C and cathepsin K in osteoclasts and other cells in mouse tibia epiphysis by immunolight and immunoelectron microscopy

We compared the distribution of a cysteine proteinase inhibitor, cystatin C, with that of cathepsin K in osteoclasts of the mouse tibia by immunolight and immunoelectron microscopy. Light microscopically, strong immunoreactivity for cystatin C was found extracellularly along the resorption lacuna and intracellularly in the organelles of osteoclasts. In serial sections, various patterns of cystatin C and cathepsin K localization were seen, specifically: (1) some resorption lacuna were positive for both cystatin C and cathepsin K; (2) others were positive for either cystatin C or cathepsin K, but not both; and (3) some lacuna were negative for both. In osteoclasts, the localization of cystatin C was similar to that of cathepsin K. Furthermore, cystatin C immunoreactivity was detected in preosteoclasts and osteoblasts, whereas cathepsin K was seen only in preosteoclasts. Electron microscopically, cystatin C immunoreactive products were found in the rough endoplasmic reticulum (ER), Golgi apparatus, vesicles, granules, and vacuoles of osteoclasts. These cystatin C-positive vesicles had fused or were in the process of fusion with the ampullar vacuoles (extracellular spaces) containing cystatin C-positive, fragmented, fibril-like structures. The extracellular cystatin C was deposited on and between the cytoplasmic processes of ruffled borders, and on and between type I collagen fibrils. In the basolateral region of osteoclasts, cystatin C-positive vesicles and granules also fused with vacuoles that contained cystatin C-positive or negative fibril-like structures. These results indicate that osteoclasts not only synthesize and secrete cathepsin K from the ruffled border into the bone resorption lacunae, but also a cysteine proteinase inhibitor, cystatin C. Therefore, it is suggested that cystatin C regulates the degradation of bone matrix by cathepsin K, both extracellularly and intracellularly.     

Alendronate Disturbs Vesicular Trafficking in Osteoclasts

The nitrogen-containing bisphosphonate alendronate inhibits osteoclast-mediated bone resorption through inhibition of the mevalonate pathway. This results in impaired protein prenylation and may affect the function of small GTPases in osteoclasts. Since these proteins are important regulators of vesicle transport in cells, we investigated the possible interference of alendronate with these processes in isolated rat osteoclasts. We show here that alendronate-induced inhibition of bone resorption coincides with accumulation of tartrate-resistant acid phosphatase- and electron dense material-containing tubular vesicles in osteoclasts. Alendronate-induced changes in osteoclasts also included widening of the sealing zone areas and incomplete organization of tight attachments and ruffled borders. Osteoclasts also appeared partially detached from the bone surface, and organic matrix was typically dissolved only at the edges of the resorption pits on alendronate-coated bone slices. In contrast, resorption pits on the control and clodronate-coated bone slices were thoroughly resorbed. Inhibition of bone resorption by alendronate was not, however, related to a decrease in osteoclast number. In conclusion, our findings suggest that alendronate inactivates osteoclasts by mechanisms that impair their intracellular vesicle transport, apoptosis being only a secondary phenomenon to this.     

Effects of ethane-1-hydroxy-1, 1-diphosphonate on cell differentiation, and proteoglycan and calcium metabolism, in the proximal tibia of young rats

The effects of ethane-1-hydroxy-1, 1-diphosphonate(EHDP) on cell differentiation, and on the metabolism of proteoglycan and calcium in the epiphyseal plate and metaphysis of rats were investigated through histology and autoradiograms of [35S]-sulfate, 45Ca, and [3H]-thymidine. Suppression of bone resorption in the metaphysis due to low dose EHDP administration was associated with a proliferation of osteoclasts with an increased number of nuclei. High dose EHDP induced enlargement of the hypertrophic zone of the epiphyseal plate and suppression of calcification of the cartilage matrix. This change had a significant association not only with the suppression of chondroitin sulfate synthesis and the degradation in the cartilage matrix, but also with the suppression of growth and differentiation of chondrocytes. Calcification was also inhibited in the metaphysis, and growth and differentiation from undifferentiated mesenchymal cells to osteoblasts, osteocytes, and osteoclasts were also suppressed by high dose EHDP.     

The differences in calcium distribution pattern between preodontoblasts and preameloblasts in developing rat molar tooth germs

Summary Ultrastructural localization of calcium in preodontoblasts and preameloblasts was investigated using the potassium pyroantimonate technique, and it was confirmed that there were clear differences in calcium distribution pattern between preodontoblasts and preameloblasts. In preodontoblasts, pyroantimonate reaction products were mainly observed in the Golgi region, lateral intercellular spaces, and secretory granules, especially in the distal portion of cell body; however, few were found in mitochondria and on the plasma membrane. In preameloblasts, on the other hand, the precipitates were located in mitochondria, nuclei, and on the inner face of the plasma membrane; however, few reaction products were observed in the intercellular spaces, lysosomelike granules, secretory granules, and stippled materials. Granular reaction product approximately 20–40nm in diameter adhered preferentially to the growing end of needlelike crystals in the initial enamel matrix.     

Electrophysiological characterization of ion channels in osteoclasts isolated from human deciduous teeth

Ion channels contribute to several important processes in osteoclasts, including proton transport and volume regulation. Although ion channels have been described in osteoclasts from several species, little is known about their properties in human osteoclasts. We devised a method for isolation of authentic human osteoclasts from deciduous teeth undergoing root resorption, and characterized currents in these cells using patch-clamp techniques. Three types of K(+) current were identified. Hyperpolarization elicited an inwardly rectifying K(+) current in most osteoclasts, which was inhibited by Ba(2+) in a voltage- and time-dependent manner. Depolarization elicited an outwardly rectifying and tetraethylammonium-sensitive current, consistent with a large-conductance Ca(2+)-dependent K(+) channel. In addition to these basal currents, extracellular adenosine 5'-triphosphate (ATP) elicited a linear current that was identified as a Ca(2+)-dependent K(+) current, based on its reversal potential close to that predicted for K(+), its blockade by quinine, and its activation by Ca(2+) ionophore. Last, an outwardly rectifying current was observed to activate spontaneously or in response to ATP, with properties of a swelling-activated Cl(-) current. This current reversed direction close to the Cl(-) equilibrium potential and was blocked by the anion channel blocker, niflumic acid, identifying it as a Cl(-) current. In summary, we have developed a novel method for isolation of authentic human osteoclasts and have characterized K(+) and Cl(-) currents. Cl(-) current mediates charge compensation during electrogenic H(+) transport, so activation of Cl(-) current may contribute to the stimulatory effects of extracellular ATP on bone resorption.     

Calcium absorption and osseous organ-, tissue-, and envelope-specific changes following ovariectomy in rats

Because cancellous bone loss occurs following ovariectomy (OVX) in rats, this has become a popular model to explore therapeutic modalities for postmenopausal bone loss in humans. The purpose of this study was to determine intestinal calcium absorption in situ and organ-, tissue-, envelope-, and site-specific changes in osseous tissues at six weeks after OVX in rats using chemical, biochemical, absorptiometric, microradiographic, and morphometric methods. There were no changes in intestinal absorption of calcium, but duodenal weight per length was significantly increased in the OVX animals compared with age-matched, sham-operated controls. There was an increase in wet bone weight, but decreases in ash/dry bone weight, total bone Ca, and Ca per ash weight in the OVX animals. There were significant decreases in the OVX animals in metaphyseal bone mineral content, as determined by photon absorptiometry and metaphyseal cancellous bone volume. The perimeter to area ratio of the metaphyseal cancellous bone in the OVX animals was increased compared with controls. Endochondral growth rates were increased in the OVX animals, attributable to an increased growth plate hypertrophic cell size and rate of chondrocyte proliferation. In the OVX animals there was an increase in modeling in the formation mode of the periosteal surface at the tibio-fibular junction. Increased periosteal modeling in the formation mode was also observed in the body of the mandible, suggesting that the changes in periosteal bone formation are not strictly coupled with changes in endochondral growth. There was an increase in modeling in the resorption mode of the endocortical surface at the tibio-fibular junction in the OVX animals. There was increased bone turnover in the OVX animals compared with controls on the endosteal surface, as indicated by increases in both formation and resorption indices, including an increase in the osteoclast population. In the long bones, OVX results in larger bones due to increases in endochondral growth and modeling in the formation mode at the periosteal surfaces, with a loss of cancellous bone and total bone calcium due to increased resorption on the endocortical surfaces and turnover (increased formation and resorption) on endosteal surfaces. This study emphasizes that osseous tissue changes following OVX in rats are tissue-specific, envelop-specific, and site-specific.