Dissolution Of Bone Mineral Research Articles

Mechanism of osteoclast-mediated bone resorption

The osteoclast is the main bone resorbing cell. It has several structural features which are related to its function. In active resorption stage this multinucleated giant cell shows remarkable polarity which, with its basolateral and apical membrane surfaces, resembles a secreting epithelial cell. The third specialized membrane area, sealing zone, mediates the attachment of the osteoclast to the bone surface. The rate of bone resorption can be regulated either by changing the number or the activity of resorbing osteoclasts. These processes can be influenced by different systemic circulating factors as well as local, bone matrix or other bone cell-derived factors. After activation of stem cells, preosteoclasts are guided to the bone surface where they undergo fusion into multinucleated osteoclasts. A special type of cell attachment to mineralized bone surface precedes actual resorption process. During cell attachment cytoskeletal structures are organized into a typical belt-like structure. The actual attachment receptor as well as its counterpart in bone matrix are not yet known. Bone mineral dissolution is then initiated by active secretion of H+ through the ruffled border membrane area. Acidification of the resorption lacuna together with hydrolytic enzymes completes finally the degradation of organic matrix of bone.

The effect of cadmium on the formation and properties of hydroxyapatite in vitro and its relation to cadmium toxicity in the skeletal system.

In order to understand the biological action of cadmium (Cd) in inducing bone pathologies, the effect of Cd on the formation, structure, and properties of hydroxyapatite (HA) in vitro was investigated using three biologically relevant test systems: (1) direct precipitation of HA with no precursor phase; (2) transformation of amorphous calcium phosphate (ACP) to crystalline HA; and (3) growth of HA seed crystals. Cd-containing HA was prepared by transforming ACP to HA in the presence of Cd at a pH of 10; Cd/Ca ratios of 0.05, 0.10, and 0.20 were obtained. Infrared and x-ray diffraction analyses were performed on the Cd-HA samples, and measurements were made of Cd adsorption on HA and of the dissolution characteristics of Cd-containing HA. Cd incorporation in HA introduced little strain in the lattice but resulted in a decreasing C-axis spacing and a corresponding crystal size decrease in the C-axis direction. Cd incorporation had a nominal effect on HA dissolution. Cd had an inhibitory effect on HA formation kinetics in all three test systems. Infrared spectroscopy of Cd-HA showed a complex series of small changes in the spectra as a function of Cd concentration resulting from some distortion in the crystal perfection and symmetry. The interference of Cd with mineralization can be partially explained by its inhibitory effect on HA nucleation and growth in addition to any cellular involvement. Furthermore, Cd probably has little effect on bone mineral dissolution. Our results explain the Cd incorporation reported in bone.

Role of IL-1β and prostaglandins in β2-microglobulin-induced bone mineral dissolution

beta 2-microglobulin (beta 2m) induces an osteoclast-mediated net calcium efflux from neonatal mouse calvariae which occurs only after 48 hours of incubation, suggesting that beta 2m acts via other growth factors. To further test this hypothesis, calvariae were incubated with and without beta 2m in the presence of the prostaglandin inhibitor indomethacin, anti-interleukin-1 beta antibody (anti-IL-1 beta), or interleukin-1 beta receptor antagonist (IL-1 beta RA). The addition of beta 2m to the culture medium stimulated, whereas indomethacin inhibited basal calcium efflux following 48 hours. However, the difference (delta) between the calcium efflux induced in calvariae incubated with and without beta 2m in basal medium and that in calvariae incubated with and without beta 2m in indomethacin supplemented medium was similar, suggesting a prostaglandin independent mechanism. There was a time dependent increase in PGE2 in basal medium which was unaffected by beta 2m. In contrast, pre-incubating calvariae with either anti-IL-1 beta or IL-1 beta RA did not alter basal calcium efflux but completely blocked the beta 2m induced calcium efflux. Anti-IL-1 beta had no effect on the basal release of beta-glucuronidase but partially blocked the beta 2m induced release of beta-glucuronidase. Thus, the beta 2m-induced calcium efflux observed in neonatal mouse calvariae is dependent on interleukin-1 beta but not prostaglandins.

Response of genetic hypercalciuric rats to a low calcium diet

A fundamental mechanism for hypercalciuria in genetic hypercalciuric rats appears due to a primary increase in intestinal calcium absorption. However previous studies could not exclude additional mechanisms to account for the hypercalciuria. To determine if enhanced bone mineral dissolution either as a primary abnormality or secondary to a defect in renal tubule calcium reabsorption is responsible for a component of the augmented calcium excretion we studied rats continually inbred for hypercalciuria. Nineteenth generation adult female idiopathic hypercalciuric (IH) and non-inbred control (Ctl) rats were fed 13 g/day of a normal calcium diet (0.6% calcium, NCD) for 10 days. Urine calcium excretion over the last seven days was greater in IH (34 +/- 2 mg/7 day) than in Ctl (2.9 +/- 0.3, P < 0.01) rats. Some rats in each group were continued on the same diet while others were fed a low calcium diet (0.02% calcium, LCD) for an additional 10 days; balance measurements were made over the final seven days. With LCD, urine calcium excretion was approximately 8-fold higher in IH compared to Ctl (13 +/- 2 mg/7 day vs. 1.6 +/- 0.1, IH vs. Ctl, respectively, P < 0.01). In IH rats percent calcium absorption was greater (59 +/- 3% vs. 45 +/- 3, IH vs. Ctl, P < 0.01), however calcium retention was negative (-1.9 +/- 2.0 mg/7 day vs. 6.5 +/- 0.5, IH vs. Ctl, P < 0.01) compared to Ctl rats. The fall in urine calcium excretion when IH rats are fed LCD indicates that enhanced intestinal calcium absorption is a primary mechanism of the hypercalciuria.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterisation of cysteine proteinases from human osteoclastomas

The process of normal bone remodeling requires the continual resorption and replacement of bone mineral and bone matrix proteins. Excessive bone resorption is a feature of a number of pathological conditions. These include osteoporosis, rheumatoid arthritis and Paget's disease [l]. Bone resorption is accomplished by the osteoclast. Osteoclasts attached to bone are highly polarised cells [2]. The osteoclast attaches to bone through a peripheral attachment zone within which the surface (apical) membrane is thrown into complex ruffles. The space enclosed by the osteoclast apical surface and the bone surface is acidified, probably by the action of a proton pump, resulting in the dissolution of bone mineral. Lysosomal enzymes are released into the acidified subosteoclastic space where they are responsible for the degradation of bone matrix components. Bone matrix is largely composed of type I collagen with small amounts of types 111, V and VI collagens [31. In addition, bone contains a number of glycoproteins (e.g. osteopontin, osteonectin and bone sialoproteins) and proteoglycans [4]. In vitro studies using disaggregated osteoclasts have suggested that cysteine proteinases released by osteoclasts are responsible for the degradation of bone proteins 151. Investigations of the properties of osteoclast cysteine proteinases are hampered by the scarcity of osteoclasts in normal bone. As our starting material we have used osteoclastomas. These are primary bone tumours that contain large numbers of apparently normal osteoclasts. Using the synthetic peptide Z-PheArg-NHMec as substrate, we have separated six cysteine proteinase activities from osteoclastoma extracts by sequential chromatography on S-Sepharose, phenyl-Sepharose, heparin-Sepharose and Sephacryl S200HR. The proteinases have molecular weights ranging from 20,000 to 42,000 (determined by gel filtration). Z-Phe-Arg-NHMec was the preferred synthetic substrate. Four of the enzymes showed some activity towards Bz-Phe-Val-Arg-NHMec but only two demonstrated low activity against Z-Arg-Arg-NHMec. The kinetics of hydrolysis of these substrates gave values within the range expected for cathepsin B [61. All six activities were capable of degrading soluble and insoluble collagen. The pH optima for the hydrolysis of Z-Phe-Arg-NHMec and type I collagen by the six enzymes are shown in table 1. A variety of inhibitors of cysteine proteinases (eg. leupeptin, E-64, cystatin) were potent inhibitors of the osteoclastoma enzymes. The kinetics of inhibition by cystatin and the rate constants of inactivation by Z-PheTyr-(O-t-Bu)CHNz were similar to published values for cathepsin B [7,81. Immunolocalisation of cathepsin B in sections of osteoclastomas revealed that osteoclasts stained strongly with an antibody to cathepsin B whereas the neoplastic osteoblasts were only weakly stained. The Table 1 pH-Optima of osteoclastoma cysteiiie protciilases

Mechanism of aluminum-induced calcium efflux from cultured neonatal mouse calvariae

Aluminum has been shown to increase unidirectional 45Ca efflux from prelabeled bones in vitro; whether aluminum affects net calcium efflux and, if so, by what mechanism has not been studied. To examine the effects of aluminum on net calcium flux from bone we cultured live and dead neonatal mouse calvariae with and without graded concentrations of aluminum (10(-8) to 10(-5) M). Aluminum induced a dose-dependent net calcium efflux from live bone after 24 h, but not 3 h, which was similar in magnitude to that produced by 10(-8) M parathyroid hormone. The normal calcium influx into dead bone was not altered by aluminum. Release of beta-glucuronidase, a lysosomal enzyme released by osteoclasts, increased after a 24-h incubation in aluminum-containing medium and was correlated with net calcium efflux. Calcitonin, an inhibitor of osteoclastic bone mineral dissolution, abolished the increase in beta-glucuronidase release and nullified the aluminum-induced net calcium efflux. Thus aluminum induces cell-mediated net calcium efflux from bone and increases beta-glucuronidase release. Calcitonin inhibits the increase in both calcium efflux and beta-glucuronidase release, suggesting that aluminum stimulates osteoclasts to release bone mineral.

Net calcium efflux from live bone during chronic metabolic, but not respiratory, acidosis.

In vivo chronic metabolic acidosis induces bone mineral dissolution. Whether the dissolution is due to alterations in physicochemical factors alone, as in acute metabolic acidosis, or requires participation of bone cells is not clear. The effect of chronic respiratory acidosis on bone has also not been established. To determine the effects of chronic metabolic and respiratory acidosis on net calcium flux from bone, we cultured live and dead neonatal mouse calvariae for 99 h in control medium or in medium acidified (pH approximately equal to 7.1) either by lowering the bicarbonate concentration (Met) or by increasing the PCO2 (Resp). We measured net calcium flux (JCa) over 0-48, 48-96, and 96-99 h. Over the first 48 h, there was greater net calcium efflux from live and dead Met than from both Resp groups. All four acidic groups had greater net calcium efflux than controls. Over the last 51 h of the chronic 99 h culture, there was net calcium efflux only from live Met (JCa = 285 +/- 129 nmol.bone-1.3 h-1) and not from any of the other groups (live control, JCa = -183 +/- 24; live Resp, JCa = -110 +/- 22; dead control, JCa = -256 +/- 12; dead Met, JCa = 11 +/- 78; dead Resp, JCa = -27 +/- 47; each P less than 0.02 vs. live Met). There is net calcium efflux from live cultured neonatal mouse calvariae during chronic metabolic, but not respiratory, acidosis. During chronic acidosis, decreased medium bicarbonate, and not just a fall in pH, is necessary to enhance net calcium efflux from live bone.

Bisphosphonates and extrarenal acid buffering capacity

Both the intracellular compartment and bone mineral are supposed to play a role in acid-base balance by contributing to the extrarenal acid buffering capacity. Bisphosphonates could affect extrarenal acid buffering capacity by interfering with the formation and/or dissolution of bone mineral. In the present study, rats were pretreated with either 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP, 10 mg/kg.day sc), with prevailing inhibitory action on bone mineral formation, or dichloromethylene bisphosphonate (Cl2MBP, 10 mg p/kg.day sc) with prevailing action on bone resorption, or NaCl injections (controls) for 7 days. In intact rats, blood acid-base variables were influenced by neither HEBP, nor Cl2MBP. Two hours after nephrectomy and before acute acid loading, HEBP-but not Cl2MBP-pretreated rats displayed a significant increase in both blood HCO3- and PCO2. After HCl infusion (2.5 mEq/kg), the relative decrement in blood HCO3- (difference in blood HCO3- before and after acid loading) was transiently more important in the two bisphosphonate pretreated groups than in controls. After a 24 hour fasting period, nephrectomized animals pretreated with Cl2MBP displayed significantly lower blood HCO3- and pH values than controls or HEBP-pretreated rats. These results suggest that bisphosphonates influence extrarenal buffering capacity according to their prevailing inhibitory action on either bone mineral formation and/or dissolution. These compounds could interfere with the release rate of bone proton buffers. However, in the presence of normal renal function, this effect does not disturb the blood acid-base equilibrium.

Net proton influx into bone during metabolic, but not respiratory, acidosis.

During acute metabolic acidosis there is a net influx of protons into bone, decreasing the elevated proton concentration. Whether there is an influx of protons into bone during acute respiratory acidosis is not known. To determine the effect of respiratory acidosis on net proton flux (JH) relative to bone, we compared JH from neonatal mouse calvariae incubated for 3 h in medium acidified by an increase in PCO2 (respiratory acidosis) with that from calvariae incubated in medium acidified to the same extent by a decrease in bicarbonate concentration (metabolic acidosis). The initial medium pH with respiratory acidosis was not different from that with metabolic acidosis (7.108 +/- 0.005 vs. 7.091 +/- 0.007, respectively, P = NS). During respiratory acidosis there was no JH from bone relative to the medium (JH = 236 +/- 93 neq.bone-1.3h-1, P = NS vs. 0); however, during metabolic acidosis there was net proton influx from the medium into bone (JH = -703 +/- 108, P less than 0.05 vs. 0, P less than 0.001 vs. respiratory acidosis). There was less calcium efflux from bone during respiratory than during metabolic acidosis (JCa = 68 +/- 6 nmol.bone-1.3 h-1 vs. 100 +/- 9, respectively, P less than 0.001). There is a net influx of protons into bone in vitro during acute metabolic, but not during acute respiratory, acidosis. The smaller calcium efflux during respiratory acidosis may indicate less net bone mineral dissolution and thus less buffer release into the medium.

Ion microprobe determination of bone surface elements: effects of reduced medium pH.

When neonatal mouse calvariae are cultured in a medium having a low pH they release calcium (Ca) while buffering protons (H). However, for 1 neq Ca released, 16-21 neq H enter the calvariae, arguing strongly against simple dissolution of bone mineral as the mechanism of H buffering. To determine if H for sodium (Na) exchange could explain the lack of stoichiometry between H and Ca, we cultured calvariae for 3 h in control (pH = 7.40 +/- 0.01) or reduced-pH (pH = 7.21 +/- 0.01) medium and then examined the surface Na, K, and Ca using a high-resolution scanning ion microprobe. The calvarial surface was rich in Na and K relative to Ca (Na/Ca, 52 +/- 17; K/Ca, 61 +/- 17; all values are the ratios of counts per second of detected secondary ions, means +/- SE). Compared with the surface the calvarial cross section contained far less Na and K relative to Ca (Na/Ca, 2 +/- 1; K/Ca, 1 +/- 1; both P less than 0.01 vs. surface). Compared with the control surface, culture in a reduced-pH medium reduced the surface Na and K relative to Ca (Na/Ca, 5 +/- 1; K/Ca, 7 +/- 1; both P less than 0.025 vs. surface) to values still greater than the cross section (P less than 0.05 for both). Neonatal mouse calvariae have a surface that is rich in Na and K relative to Ca. Reduced medium pH depletes surface Na and K of cultured calvariae with respect to Ca.

Diphosphonates inhibit bone resorption by macrophages in vitro

The diphosphonates are a group of synthetic compounds which are adsorbed onto hydroxyapatite crystal surfaces and inhibit both the growth and dissolution of these crystals. They also inhibit mineralisation and resorption of bone in vivo. The effects of diphosphonates were tested on the attachment of macrophages to bone and on the dissolution of bone mineral by macrophages. Attachment was unaffected but resorption was inhibited. Diphosphonates were found to be cytotoxic at much lower concentrations when bone mineral was present, and the most likely explanation for the effects of diphosphonate on bone resorption is that the drug is adsorbed onto the mineral surface where it reaches cytotoxic concentrations.

The reactions of H + and Zn 2+ with the surfaces of bone and bone mineral

1. 1. Although the greater uptake by bone and bone mineral of 65Zn 2+ than of 47Ca 2+ as previously reported 1 was confirmed, it was also found that the fraction of bone Ca 2+ exchanged, due to the differences between isoionic and heteroionic exchange, was in some cases the same for the two radioisotopes. 2. 2. 65Zn 2+ was taken up by bone and bone mineral much more rapidly than 47Ca 2+ in the initial stages of reaction. However, as uptake continued, diffusion through bone appeared to become the rate-limiting process. Apparently this differed little for the two radioisotopes. 3. 3. The amount of Ca 2+ that entered the solution in these exchange experiments was equal to the amount of Zn 2+ that entered bone powder at pH 7.3 and anorganic bone at pH 6.0. Ca 2+ in solution was greater than Zn 2+ entering bone powder at pH 6.0, and less than Zn 2+ entering anorganic bone at pH 7.3. 4. 4. H + was shown to be a powerful competitor of Ca 2+ in exchange reactions, with a superiority of about 1000: 1. A mechanism was proposed to explain its behavior. 5. 5. Zn 2+ replaced Ca 2+ on the surface of bone mineral so rapidly that it protected the surface from attack by H + at pH 6.0. It thus blocked the mechanism of action of H +, suppressed much of the rise in pH that was found in its absence, and reduced to a considerable extent the amount of phosphate that entered the solution. These effects were interesting from the theoretical point of view, and are of potential practice importance. 6. 6. After bone powder or anorganic bone was shaken with solutions containing Zn 2+, stable Mg 2+ was found in the solutions in amounts up to 0.23 mmole/l. In some cases it was as much as 20% of the Zn 2+ that entered bone by heteroionic exchange. 7. 7. Ca 2+/P ratios of solutions which had been shaken with bone and bone mineral were either significantly greater than or significantly less than the ratios in the original bone samples and in theoretical hydroxyapatite, depending on conditions. These, and other data, suggest that the increase in Ca 2+ and P concentration in solution had little to do with solubility proper but was the result of chemical reaction. 8. 8. Bone powder and anorganic bone differed in quantitative aspects of their relations with 47Ca 2+, 65Zn 2+, H +, and stable Ca 2+ and P. Differences with regard to pH and rate of dissolution of bone mineral in the two forms were especially striking, and suggested that anorganic bone, as usually prepared, differed from bone powder not only in its lack of organic matrix, but in relative content of amorphous phosphate and crystalline hydroxyapatite as well.

Regulation of bone resorption and formation: Influences of thyrocalcitonin, parathyroid hormone, neutral phosphate and vitamin D 3

The destruction of bone collagen and dissolution of bone mineral induced by parathyroid hormone administration is accompanied by a concomitant renal action of the hormone, causing decreased tubular reabsorption of phosphorus and increased reabsorption of calcium and magnesium. The hormone also acts on soft tissue cells, resulting in a shift of phosphorus, potassium and magnesium from cells. Thyrocalcitonin inhibits bone resorption and the action of parathyroid hormone on bone resorption and may also stimulate osteoblastic activity. The effect of thyrocalcitonin on phosphorus excretion is influenced by the ratio of calcium to magnesium cations perfusing the kidney. Minimal amounts of parathyroid hormone are required for the action of vitamin D 3 upon bone, and bone resorption induced by the vitamin is suppressed by thyrocalcitonin. Since the hormone is required for the action of the vitamin on bone, cases of hypoparathyroidism resistant to vitamin D may respond to treatment with the combination of vitamin plus hormone. Although thyrocalcitonin protects against hypercalcemia the physiologic significance of this is unknown. Another important function of the hormone may be in the regulation of bone remodeling. Administration of neutral phosphate stimulates bone formation and bone mineralization. A combination of neutral phosphate with thyrocalcitonin may prove to be useful in the treatment of osteoporosis.