Potassium,sodium, and the intracellular fluid space of cells from bone

Summary Cells enzymatically dispersed from fetal rat calvaria were analyzed for sodium and potassium content and intracellular fluid space (ICF). Even when obtained in comparatively high yield, the cells are damaged by the isolation procedure as evidenced by high sodium and low potassium content immediately after isolation. During a post-incubation period potassium is accumulated and sodium extruded to steady-state levels. Although electrolyte content of cells after recovery did not vary as a function of cell yield, ICF was increased in cells obtained in lower yield, suggesting cell swelling as a result of membrane damage. The weighted mean values obtained for the best cell preparations were 117 mM K⁺ and 27 mM Na⁺. Based on DNA assay of isolated cells and the whole tissue, 20- to 21-day calvaria were found to have an average of 8.1 × 10⁶ cells/calvarium. Combining cell data with analysis of total tissue sodium, potassium, and water, it was concluded that the tissue extracellular sodium is in equilibrium with blood but that the potassium concentration is approximately 5-fold higher than blood levels.     

In vitro bone resorption activity produced by a hypercalcemic adenocarcinoma tumor line (CAC-8) in nude mice

Summary Tumor extracts and conditioned tissue culture media from a canine adenocarcinoma tumor line (CAC-8) propagated in nude mice significantly increasedin vitro bone resorption in neonatal mouse calvaria as measured by release of previously incorporated⁴⁵Ca.In vitro bone resorption activity was induced in a dose-dependent manner, was not suppressible by indomethacin, and was heat- and acid-stable. Gel exclusion chromatography demonstrated peak bone resorbing activity at a relative molecular mass of approximately 28,000. The parathyroid hormone (PTH) antagonist (^8,18norleucine,³⁴tyrosine) bovine PTH (3–34) amide did not inhibit CAC 8-stimulated or (1–34) bPTH-induced bone resorption. There was an increased number of tartrate-resistant, acid phosphatase-positive cells in calvariae exposed to CAC-8 extract. Ultrastructural evaluation of calvaria revealed hypertrophy and maturation of osteoclasts in calvaria exposed to CAC-8 extract. The maturation effects included close contact to bone surfaces and the presence of clear zones and ruffled borders in osteoclasts. Similar structures were observed infrequently in osteoclasts of control calvaria. These data demonstrate that the tumor line (CAC-8) contained activity capable of stimulatingin vitro bone resorption by increasing osteoclast numbers and the activity of existing osteoclasts.     

Hormonal,pH, and Calcium Regulation of Connexin 43–Mediated Dye Transfer in Osteocytes in Chick Calvaria

Gap junctional intercellular communication among osteocytes in chick calvaria, their natural 3D environment, was examined using FRAP analysis. Cell–cell communication among osteocytes in chick calvaria was mediated by Cx43 and was regulated by extracellular pH, extracellular calcium ion concentration, and PTH. Introduction: The intercellular network of communication among osteocytes is mediated by gap junctions. Gap junctional intercellular communication (GJIC) is thought to play an important role in integration and synchronization of bone remodeling. We hypothesized that extracellular pH (pH_o) and extracellular calcium ion concentration ([Ca²⁺]_e), both of which are dynamically altered by osteoclasts during bone remodeling, affect GJIC among osteocytes. Using fluorescence replacement after photobleaching (FRAP) analysis, we examined the effect of changes in pH_o and [Ca²⁺]_e and addition of PTH on GJIC in osteocytes in chick calvaria. Additionally, we examined the role of intracellular calcium on the regulation of GJIC among osteocytes. Materials and Methods: Anti‐Connexin43 (Cx43) immunolabeling was used to localize gap junctions in chick calvaria. GJIC among osteocytes in chick calvariae was assessed using FRAP. Results: Cx43 immunoreactivity was detected in most of the osteocyte processes. FRAP analysis showed dye‐coupling among osteocytes in chick calvariae. In untreated osteocytes, fluorescence intensity recovered 43.7 ± 2.2% within 5 min after photobleaching. Pretreatment of osteocytes with 18 α‐GA, a reversible inhibitor of GJIC, significantly decreased fluorescence recovery to 10.7 ± 2.2%. When pH_o was decreased from 7.4 to 6.9, fluorescence recovery significantly decreased from 43.3 ± 2.9% to 19.7 ± 2.3%. Conversely, when pH_o was increased from 7.4 to 8.0, fluorescence recovery was significantly increased to 61.9 ± 4.5%. When [Ca²⁺]_e was increased from 1 to 25 mM, fluorescence recovery was significantly decreased from 47.0 ± 6.1% to 16.1 ± 2.1%. In bone fragments exposed to 1.0–10 nM rPTH for 3 h, replacement of fluorescence was significantly increased to 60.7 ± 7.2%. Chelating intracellular calcium ions affected GJIC regulation by [Ca²⁺]_e and PTH. Conclusions: Our study of cell–cell communication between osteocytes in chick calvaria showed for the first time that GJIC among osteocytes is regulated by the extracellular environment and by hormonal stimulation during bone remodeling. This method may be more biologically relevant to living bone than current methods.     

True hyponatremia secondary to intravenous immunoglobulin

Hyponatremia is characterized as either “true hyponatremia,” which represents a decrease in the Na⁺ concentration in the water phase of plasma, or “pseudohyponatremia,” which is due to an increased percentage of protein or lipid in plasma, with a normal plasma water Na⁺ concentration ([Na⁺]). Pseudohyponatremia is a known complication of intravenous immunoglobulin (IVIG). Because IVIG has been reported to result in post-infusional hyperproteinemia, IVIG-induced hyponatremia has been attributed to pseudohyponatremia. In this case report, we demonstrate that IVIG therapy can result in true hyponatremia, resulting from sucrose-induced translocation of water from the intracellular compartment (ICF) to the extracellular compartment (ECF), as well as the infusion of a large volume of dilute fluid, in patients with an underlying defect in urinary free water excretion.     

Exposure of macrophage-like cells to titanium particles does not affect bone resorption, but inhibits bone formation

We examined the capacity of culture supernatants of macrophage-like cells exposed to titanium particles to influence bone formation and bone resorption, our aim being to elucidate the mechanism of implant loosening. A mouse macrophage-like cell line, J774, was exposed to titanium particles and the concentrations of prostaglandin E₂, tumor necrosis factor-α, interleukin-1α, and interleukin-6 in the supernatants were measured. Titanium particles stimulated the J774 cells to release tumor necrosis factor-α, whereas prostaglandin E₂, interleukin-1α and interleukin-6 concentrations remained low. The bone resorptive activity of the supernatants was measured by determining ⁴⁵Ca release from cultured pre-labeled newborn mouse calvariae. The culture supernatants of J774 cells exposed to titanium particles showed no significant difference in bone resorptive activity in mouse calvariae from that of culture supernatants of J774 cells not exposed to titanium particles. The bone-forming activity of the supernatant was evaluated by determining bone nodule formation and alkaline phosphatase activity in cultured mouse calvaria cells. The bone-forming activity of the supernatants exposed to titanium particles was significantly decreased compared with the supernatants of unexposed J774 cells. This inhibition was reversed by the addition of anti-tumor necrosis factor-α neutralizing antibody. We conclude that tumor necrosis factor-α released from J774 cells exposed to titanium particles played an important role in the inhibition of bone formation rather than in the stimulation of bone resorption. Received for publication on May 20, 1998; accepted on Aug. 28, 1998     

Omeprazole,a specific inhibitor of H+−K+-ATPase,inhibits bone resorptionin vitro

Summary Omeprazole has been previously shown to block gastric acid secretion by specific inhibition of gastric parietal cell membrane H⁺−K⁺-ATPase. It is now demonstrated that similar concentrations of omeprazole will inhibit PGE₂- and PTH-stimulated Ca⁺⁺ release from prelabelled neonatal mouse calvariae without affecting the viability of cultured calvaria explants. This study was supported by grants from Aaltonen Foundation and Paulo Foundation, Finland.     

Parathyroid hormone and calcitonin interactions in bone: Irradiation-induced inhibition of escape in vitro

Summary Calcitonin (CT) inhibits hormonally stimulated bone resorption only transiently in vitro. This phenomenon has been termed “escape,” but the mechanism for the effect is not understood. One possible explanation is that bone cell differentiation and recruitment of specific precursor cells, in response to stimulators of resorption, lead to the appearance of osteoclasts that are unresponsive to CT. To test this hypothesis, cell proliferation in neonatal mouse calvaria in organ culture was inhibited by irradiation from a cobalt-60 source. At a dose of 6000 R, [³H]thymidine incorporation into intact calvaria was inhibited approximately 90%. Irradiation had no effect on the resorptive response to 0.1 U/ml parathyroid hormone (PTH). However, irradiation induced a dose-dependent inhibition of the escape response which was maximal at 6000 R. A dose of 6000 R did not affect the binding of¹²⁵I-salmon CT to calvaria and decreased PTH stimulation of cyclic AMP release from bone without affecting the cyclic AMP response to CT. Although irradiation caused a dose-dependent inhibition of DNA synthesis, the dose-response curves for that effect and inhibition of escape were not superimposable. A morphologic study of hormonally treated calvaria demonstrated that irradiation prevented the early increase in number of osteoclasts in PTH-treated calvaria that had been observed previously in unirradiated bones. Autoradiography showed that irradiation also prevented the PTH-stimulated recruitment of newly divided mononuclear cell precursors into osteoclasts. This may be correlated with the effect of irradiation to prevent the loss of responsiveness to CT in the presence of PTH.     

The effects of stable strontium on calcium metabolism.: II. Effects of 1α-hydroxyvitamin D3 in strontium-fed rats,and inhibitory effect of strontium on bone resorptionin vitro

It has been postulated that the effect of strontium on bone metabolism due to the reduced plasma 1,25-dihydroxyvitamin D₃ level following the inhibition of 1α-hydroxylation by strontium. The effects of strontium were examined on intestinal calcium absorption when rats were received synthetic 1α-hydroxyvitamin D₃. Four groups of rats at the age of 36 days were fed a semi-synthetic vitamin D-deficient diet for 4 weeks containing 1% strontium and vitamin D₃ (Sr-D group), 1% strontium and 1α-hydroxyvitamin D₃ (Sr-α group), vitamin D₃ (Co-D group), or 1α-hydroxyvitamin D₃ (Co- α group), respectively. At the age of 60 days, calcium and strontium balance studies were conducted to determine intestinal calcium absorption over a 3-day period, and 1,25-dihydroxyvitamin D level was then measured. Serum 1,25-dihydroxyvitamin D in Sr-D group was undetectable, and intestinal calcium absorption significantly decreased. Replacement of vitamin D₃ with 1α-hydroxyvitamin D₃ recovered serum 1,25-dihydroxyvitamin D to the level in Co-D group. However, this substitution in Sr-α group failed to increase intestinal calcium absorption. We also examined the direct of strontium on bone resorption using⁴⁵Ca pre-labeled mouse calvaria. Strontium was injected every day until sacrifice, and percent⁴⁵Ca release from cultured calvariae was measured. Bone resorption was inhibited by strontium dose-dependently in groups which had and had not received parathyroid hormone in culture. These results suggest that strontium inhibits intestinal calcium absorption and has a direct inhibitory effect on bone resorption.     

Passive accumulation of magnesium,sodium, and potassium by chick calvaria

Summary Four-day-old chick calvaria were used to determine the passive concentrations of magnesium, sodium, and potassium in metabolically poisoned bone. When incubated in buffers containing the blood levels of sodium and magnesium, these calvaria contained sodium and magnesium at the identical concentrations found in freshly dissected calvaria. Calvarial sodium and magnesium levels could be varied by altering the buffer concentrations of these cations. The potassium content of metabolically poisoned calvaria incubated in buffers containing 4 mM potassium was less than 20% of the content of freshly dissected calvaria. When the buffer concentrations of sodium and potassium were systematically varied, ouabain-poisoned calvaria concentrated these cations in the bone extracellular fluid by a factor of approximately two above buffer cation levels. Presumably, the hydroxyapatite crystal zeta potential is responsible for this concentrative phenomenon. These results are discussed in terms of the control of the ionic content of the bone extracellular fluid by the postulated “bone membrane.”     

The effects of prostaglandin E2, parathyroid hormone, 1,25 dihydroxycholecalciferol,and cyclic nucleotide analogs on alkaline phosphatase activity in osteoblastic cells

Summary Clone MC3T3-E1 cells isolated from newborn mouse calvaria had the same type of alkaline phosphatase (ALP) as that found in adult mouse calvaria (the liver-bone-kidney type), as judged by polyacrylamide gel electrophoresis as well as by heat lability and amino acid inactivation. The effects of prostaglandin E₂ (PGE₂), parathyroid hormone (PTH), 1,25 dihydroxycholecalciferol [1,25(OH)₂D₃], and adenosine-3′, 5′-cyclic monophosphate (cAMP) analogs on ALP were investigated. PGE₂ and 1,25(OH)₂D₃ increased ALP activity in dose-related manner with a maximal effect at concentrations of 10 ng/ml and 40 pg/ml, respectively. N⁶,O²-dibutyryl adenosine-3′, 5′-cyclic monophosphate (DBcAMP) also induced an increase in ALP activity in a dose-related fashion with a maximal effect at a concentration of 0.5 mM which was 2.2-fold over that of the controls. Induced ALP was of the “liver-bone-kidney” type. Antinomycin D and cycloheximide inhibited the increase in ALP activity induced by DBcAMP. The level of ALP was elevated by 8-bromo-adenosine-3′,5′-cyclic monophosphate and theophylline, but not by N⁶,O²-dibutyryl guanosine-3′,5′-cyclic monophosphate and sodium butyrate. Moreover, PGE₂ dramatically increased the level of cAMP in the cells with a maximal effect at a concentration of 10 ng/ml, indicating that PGE₂ and DBcAMP induce an increase of ALP activity in clone MC3T3-E1 cells by increasing the cAMP level; PTH did not affect enzyme activity and cAMP, level in the cells. These results suggest that PGE₂, DBcAMP, and 1,25(OH)₂D₃ are involved in bone formationin vivo as well.     

Analysis of hydroxyproline by high performance liquid chromatography and its application to collagen turnover studies in bone cultures

Summary We describe a high performance liquid chromatography (HPLC) technique for separating and quantitating hydroxyproline in calvarial cultures. Using a reverse-phase Nova-Pak C₁₈ column and a 140 mM sodium acetate, 0.05% triethylamine (TEA), 6% acetonitrile solvent system, we obtained a complete separation of hydroxyproline. Recovery of added standards ranged from 89 to 103% and intraassay variability was <8%. [³H]hydroxyproline measurements were used to examine changes in collagen turnover in rat calvariae labeled with [³H]proline and “chased” in the presence of 10 mM unlabeled proline. The addition of parathyroid hormone (PTH) during a 24–48 hour “chase” period increased the release of acid-soluble [³H]hydroxyproline into the culture medium, indicating an increase of fully degraded collagen. This method offers a sensitive and reproducible technique for monitoring changes in bone matrix degradation and in studying agents that modify this process.     

Biological characterization of interleukin-1-like cytokine produced by cultured bone cells from newborn mouse calvaria

Summary We have investigated the role of interleukin-1 (IL-1) and IL-1-like factor in the regulatory mechanisms of a bone remodeling system. To determine whether the bone cell itself produces IL-1-like cytokine, we examined bone cells cultured from newborn mouse calvaria. Bone cells migrating from fragments of newborn mouse calvaria were used in this study. We also used bone cells obtained by consecutive digestion of the calvaria with enzymes. These bone cells were cultured in fetal calf serum-containing alpha-MEM. IL-1-like cytokine activity was measured by incorporation of [³H]thymidine into C3H/HeJ thymocytes stimulated with PHA. When treated with lipopolysaccharide (LPS) fromEscherichia coli 0111 B4, the cultured bone cells produced a significant amount of IL-1-like cytokine. The maximum concentration of IL-1-like cytokine was observed in culture supernatants of the bone cells cultured for 24 horus with the LPS in serum-free medium. The IL-1-like cytokine closely resembles IL-1 in some of its biological characteristics: (1) stimulation of mitogen-induced thymocyte proliferation, (2) stimulation of fibroblast proliferation, (3) pyrogenicity, and (4) molecular weight. These results show that cultured bone cells from newborn mouse calvariae produce an IL-1-like cytokine that closely resembles IL-1.     

Mineralization and the Expression of Matrix Proteins During In Vivo Bone Development

The in vivo expression of fibronectin, type I collagen, and several non-collagenous proteins was correlated with the development of bone in fetal and early neonatal rat calvariae. Fibronectin was the earliest matrix protein expressed in calvariae, with a peak expression in fetal 16- and 17-day (d) bones. Fibronectin expression coincided with the condensation of preosteoblasts prior to calcification and decreased once bone mineralization commenced. The expression of type I collagen, osteonectin, bone sialoprotein, and alkaline phosphatase mRNAs was found at 17 d. The increase in type I collagen mRNA levels was correlated with a 3.5-fold increase in calcium deposition at 19–20 d. Bone sialoprotein and alkaline phosphatase peaked on fetal 21 d while osteonectin remained at a low level and was localized to the osteoblast layer and the osteocyte lacunae. Osteopontin mRNA levels increased rapidly in neonatal calvariae. After birth, osteonectin and fibronectin were mainly associated with blood vessels. Thus, fibronectin is one of the earliest matrix proteins expressed in calvariae and is rapidly followed by type I collagen, bone sialoprotein, and alkaline phosphatase. Osteocalcin, osteonectin, and osteopontin mRNAs have similar patterns of expression in the developing fetal calvaria, and their synthesis coincided with mineralization. Received: 31 December 1996 / Accepted: 5 June 1997     

Potassium, sodium, and the intracellular fluid space of cells from bone.

Cells enzymatically dispersed from fetal rat calvaria were analyzed for sodium and potassium content and intracellular fluid space (ICF). Even when obtained in comparatively high yield, the cells are damaged by the isolation procedure as evidenced by high sodium and low potassium content immediately after isolation. During a post-incubation period potassium is accumulated and sodium extruded to steady-state levels. Although electrolyte content of cells after recovery did not vary as a function of cell yield, ICF was increased in cells obtained in lower yield, suggesting cell swelling as a result of membrane damage. The weighted mean values obtained for the best cell preparations were 117 mM K+ and 27 mM Na+. Based on DNA assay of isolated cells and the whole tissue, 20- to 21-day calvaria were found to have an average of 8.1 x 10(6) cells/calvarium. Combining cell data with analysis of total tissue sodium, potassium, and water, it was concluded that the tissue extracellular sodium is in equilibrium with blood but that the potassium concentraiton is approximately 5-fold higher than blood levels.     

Inhibition of parathyroid hormone-stimulated bone resorption by monovalent cation ionophores

Summary The actions of divalent cation ionophores on bone resorption in vitro are complex; both enhancement of resorption and inhibition of stimulated resorption have been observed with the ionophore A23187. We have found in neonatal mouse calvaria, in which divalent ionophores were only inhibitory, that monovalent cation ionophores were even more potent inhibitors of stimulated bone resorption. Nigericin, monensin, and X206 each inhibited the release of calcium (Ca) from calvaria that were stimulated to resorb by 0.1 U/ml parathyroid hormone (PTH). Actions of the three ionophores were dose dependent and were maximal at 10⁻⁷M, 3×10⁻⁷M, and 1.2×10⁻⁷M, respectively, compared to A23187, which was maximally inhibitory at 2×10⁻⁶M. After pretreatment with nigericin alone or together with PTH for 24 h, inhibition of stimulated resorption was partially reversible. Prolonged (48 h) treatment, either with ionophore alone or together with PTH, caused irreversible inhibition of stimulated Ca release. However, the ionophore was only partially inhibitory if it was added to calvaria stimulated by pretreatment with PTH alone for 24 or 48 h. Resorption stimulated by prostaglandin E₂, 1,25-dihydroxyvitamin D₃, and epidermal growth factor was also inhibited by monovalent ionophores, indicating that the inhibition was not at the level of the PTH receptor. In addition, ionophores did not lower basal or PTH-stimulated production of cyclic AMP by calvaria. Submaximal doses of nigericin were synergistic with calcitonin or ouabain in inhibiting PTH-stimulated resorption. These results are consistent with the hypothesis that stimulated release of Ca from bone occurs by a Na/Ca exchange mechanism. Thus monovalent cation ionophores would increase intracellular Na⁺, thereby decreasing the Na gradient across bone cell membranes, leading to conditions unfavorable for Ca efflux coupled to further Na influx.     

Cell Surface Expression of Stem Cell Antigen-1 (Sca-1) Distinguishes Osteo-, Chondro-, and Adipoprogenitors in Fetal Mouse Calvaria

The flat bones of the skull (calvaria) develop by balanced cell proliferation and differentiation in the calvarial sutures and the bone tips. As the brain grows and the calvaria expand, cells within the sutures must remain undifferentiated to maintain suture patency, but osteoprogenitors also need to be recruited into the osteogenic fronts. The exact identity of calvarial osteoprogenitors is currently not known. We used immunomagnetic cell sorting to isolate Sca-1⁺ and Sca-1⁻ cells from fetal mouse calvaria and determined their differentiation potential in in vitro differentiation asssays and in vivo subcutaneous transplantations. Cells within the Sca-1⁺ cell fraction have a higher adipogenic potential, whereas cells within the Sca-1⁻ cell fraction have a higher osteogenic and chondrogenic potential. The Sca-1⁻ fraction retains its chondrogenic potential after in vitro expansion but not its osteogenic potential. The Sca-1⁺ fraction does not retain its adipogenic potential after in vitro expansion. Subcutaneous transplantation resulted in islands of bone and cartilage in implants that had been seeded with Sca-1⁻ cells. In conclusion, immunomagnetic cell sorting with Sca-1 antibodies can be used to separate a Sca-1⁺ cell fraction with adipogenic potential from a Sca-1⁻ cell fraction with osteogenic and chondrogenic potential. Isolation of pure populations of calvarial adipoprogenitors, osteoprogenitors, and chondroprogenitors will be beneficial for cellular studies of calvarial development, adipogenesis, osteogenesis, and chondrogenesis. Calvaria-derived osteogenic cell populations may be useful in craniofacial tissue regeneration and repair.     

The effects of hydrocortisone,parathyroid hormone and the bisphosphonate,APD, on bone resorption in neonatal mouse calvaria

Summary The effects of hydrocortisone and parathyroid hormone (PTH) upon bone resorption rates in neonatal mouse calvaria have been studied. Bone resorption (measured as⁴⁵Ca release) was significantly increased by hydrocortisone (10⁻⁷ M and 10⁻⁶ M) and there was a dose-dependent rise with PTH (0.3–0.9 μg/liter). When both PTH 0.3 μg/liter and hydrocortisone 10⁻⁸ M were present in the incubating medium, bone resorption did not differ from control, but increasing the hydrocortisone concentration to 10⁻⁷ M augmented⁴⁵Ca release by 25% (P<0.02) and doubling of the PTH level was associated with a 10% increase (nonsignificant). When both PTH and hydrocortisone were present in the higher concentrations (0.6 μg/liter and 10⁻⁷ M, respectively)⁴⁵Ca release increased by 39% (P<0.005) above that resulting from the lower levels of both hormones (0.3 μg/l and 10⁻⁸ M, respectively). (3-Amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD) in concentrations of 3×10⁻⁵ M and 10⁻⁴ M, produced inhibition of basal and hydrocortisone/PTH-stimulated bone resorption without evidence of toxicity. These results indicate that hydrocortisone stimulates bone resorption in neonatal mouse calvariain vitro, in contrast to the result found in fetal rat bone culture systems. PTH has a similar effect, which is additive to that of hydrocortisone and the combined stimulation can be overcome by APD. The possible relevance of these results to the development and prevention of glucocorticoid-induced osteoporosis is discussed.     

Effect of cortisol on periosteal and nonperiosteal collagen and DNA synthesis in cultured rat calvariae

Summary The effects of cortisol on bone formation are complex and may be modulated by the presence of periosteal cells or by factors released by the periosteal tissue. To test these possibilities, cortisol was examined for its effects on the incorporation of³H-proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP), on DNA synthesis and on alkaline phosphatase activity in intact and in the periosteum and nonperiosteal bone of dissected calvariae from 21-day-old fetal rats. After 24 h of treatment, cortisol increased the incorporation of³H-proline into CDP in intact bones and in the nonperiosteal bone of calvariae dissected after the culture. Cortisol inhibited the incorporation of³H-thymidine into calvarial DNA but it caused a small increase in nonperiosteal DNA content. Cortisol did not affect the incorporation of³H-proline into CDP in calvariae dissected prior to the culture if the periosteum and nonperiosteal central bone were incubated separately; the stimulatory effect was observed only if the two tissues were cultured in the same vial and were in contact. In contrast, cortisol stimulated alkaline phosphatase activity in the central nonperiosteal bone of calvariae dissected before or after the culture. After 72–96 h of treatment, cortisol inhibited the labeling of CDP, NCP, and DNA and the DNA content in intact bones and in both periosteal and nonperiosteal central bone of calvariae dissected after the culture. In contrast, when the periosteum was removed before the incubation, these inhibitory effects were observed in the periosteum and not in the nonperiosteal bone. Cortisol inhibited alkaline phosphatase activity in intact bones treated for 96 h, but removal of the periosteum resulted in a stimulatory effect in the nonperiosteal central bone. These studies indicate that 24 h treatment with cortisol stimulates collagen synthesis in nonperiosteal bone, an effect that requires the presence of periosteal tissue. Exposure to cortisol for 72–96 h inhibits collagen, noncollagen protein, and DNA synthesis, an effect that is secondary to an inhibition of periosteal cell replication. Cortisol does not cause a direct inhibition of osteoblastic function.     

Comparison of the mechanisms of bone resorption induced by 1α,25-dihydroxyvitamin D3 and lipopolysaccharides

Summary The mechanisms of increase in bone resorption induced by 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] and bacterial lipopolysaccharides (LPS) were compared in anin vitro dead bone assay and a living bone assay. 1α,25(OH)₂D₃ at concentrations of 0.05–5 ng/ml dose-dependently enhanced the ability of alveolar macrophages to release⁴⁵Ca from prelabeled dead bone particles (dead bone assay). In addition, the vitamin promoted fusion of the macrophages to form multinucleated cells and also enhanced glucose consumption, a marker of activation of macrophages. LPS at 0.05–5 μg/ml similarly enhanced the release of⁴⁵Ca from the dead bone particles and glucose consumption by alveolar macrophages, but it did not induce fusion of the cells at any concentration. Both 1α,25(OH)₂D₃ and LPS dose-dependently stimulated the release of⁴⁵Ca from fetal mouse calvaria prelabeled with⁴⁵Ca (living bone assay). Compared to control bone, there were several times as many osteoclasts per given length of trabecular bone surface in calvaria treated for 5 days with either 5 ng/ml of 1α,25(OH)₂D₃ or 5 μg/ml of LPS. Indomethacin (10⁻⁵ M) completely inhibited the LPS-induced increase of osteoclasts, but not the 1α,25(OH)₂D₃-induced increase. These results suggest that 1α,25(OH)₂D₃ and LPS similarly stimulate bone resorption by activating macrophages as well as by promoting fusion of precursor cells to form multinucleated cells. 1α,25(OH)₂D₃ induced formation of multinucleated cells with bone-resorbing activity directly, whereas LPS appeared to induce multinucleated cells through prostaglandin synthesis by some other types of cells present in living bone tissues.     

Morphological evidence of gap junctions between bone cells

Summary Cell membrane specializations occur at contact sites between adjacent osteoblasts and osteoblasts and osteocytes. These junctions have been described by other investigators as being important in preventing the extracellular movement of material around bone cells. Previously we described how certain small proteins circumvented the osteoblast population and rapidly penetrated the canalicular-osteocyte system. In the present study we used lanthanum colloid as an extracellular marker; the lanthanum readily penetrated the bone cell junctions and the extracellular space of bone. Morphologically, these junctions were not “tight” or “occluding” structures, but resembled “gap” junctions. These gap junctions contained elements which formed intercellular bridges between adjacent cells but also maintained a 2 nm space between cells that contained extracellular fluid. These gap junctions may have an important function in the control or coordination of bone cell activity throughout a given volume of bone.