The effects of sodium fluoride on the resorptive activity of isolated osteoclasts.

It is not clear to what extent the increased bone mass observed in vertebral trabecular bone of fluoride-treated mammals is a consequence of effects of fluoride on the number and activity of osteoclasts or of osteoblasts. In the present communication, we have analyzed the effects of NaF on the activity of isolated rabbit osteoclasts cultured on thin slices of devitalized compact bovine bone. Osteoclastic resorption was quantitated by counting the number of resorption lacunae and measuring their surface area and their depth using scanning electron microscopy. Our results show that NaF in concentrations of 0.5-1.0 mM decreased the number of resorption lacunae made by individual osteoclasts and decreased the resorbed area per osteoclast. We argue that the concentration of fluoride in these experiments may be within the range "seen" by osteoclasts in mammals treated for prolonged periods with approximately 1 mg of NaF/kg body weight (bw) per day.

Adhesion patterns and cytoskeleton of rabbit osteoclasts on bone slices and glass.

The ability of osteoclasts (OC) to migrate and resorb bone is thought to be dependent on cytoskeletal function and adhesion. Therefore, we investigated the cytoskeleton and the adhesion patterns of rabbit OC on glass and on devitalized bone slices, using specific antibodies to cytoskeletal elements and fluorescence and interference reflection microscopy. Microtubules (MT) were similar in OC on both substrata, and appeared in a pattern typical of that described for many cells. Multiple centriolar complexes were observed in most OC, either as one large aggregate in the center of the cell or dispersed singly or in small aggregates close to individual nuclei. Staining of microfilaments (MF) was similar on both substrata and appeared primarily as an F-actin network. MF distribution was different in OC associated with resorption lacunae with intense staining over those regions. In the OC on glass, high F-actin staining was detectable at the periphery in dots and rosette-like structures, which also stained for vinculin. The adhesion patterns indicated that OC on glass do not make large focal contacts, but appear to make a few tiny focal contacts that are not associated with the rosette-like structures. Most of the undersurface of the OC appeared either to be involved in close contacts or to be separated by distances of greater than 100 nm from the substratum. These studies indicate that the MF distribution and the adhesion patterns of rabbit OC are typical of motile cells, that the distribution of the cytoskeleton of rabbit OC on glass and on bone slices is similar, and that MF may be involved in the morphological changes associated with resorption.

Time course of "escape" from calcitonin-induced inhibition of motility and resorption of disaggregated osteoclasts.

The reversible calcitonin (CT)-induced inhibition of osteoclastic activity has been studied to clarify the mechanisms responsible for the so-called "escape phenomenon." Osteoclasts disaggregated from neonatal rabbits were cultured on glass coverslips or thin bovine bone slices. Resorption activity was evaluated by using time-lapse recording and scanning electron microscopy. Addition of CT to the cultures caused most osteoclasts on glass surfaces to be immotile and contracted. From 1.5 h onward, in cultures with CT, osteoclasts started to escape from CT-induced quiescence independently of other cells. CT also prevented osteoclasts on bone slices from excavating bone while concomitant cell immobility occurred. Inhibited osteoclasts were able to regain apparent bone-resorbing potency only after resumption of cytoplasmic immobility. The resumption of bone resorption could begin as early as 9.7 h after CT addition. The observations indicate that CT-induced inhibition of osteoclastic bone resorption is associated with inhibition of cytoplasmic motility and that the "escape" phenomenon reflects resumption of activity of osteoclasts that were previously inhibited by CT action rather than the resportive activity of newly formed osteoclasts.

Osteoclastic bone resorption: in vitro analysis of the rate of resorption and migration of individual osteoclasts.

Osteoclasts isolated from the long bones of newborn rabbits were cultured on translucent devitalized bone slices and observed by phase-contrast time-lapse cinemicrography and scanning electron microscopy (SEM). This has allowed us to measure the rate of resorption and the rate of migration of individual osteoclasts. Our films show that osteoclasts do not resorb while migrating. When the osteoclastic resorption areas, which are easily recognizable with phase-contrast microscopy as areas delineated by refractile lines, were observed by SEM, such areas appeared as excavated areas lined with a network of collagen fibrils. The rate of migration was calculated using time lapse recordings, and varied from 30 micron/hr to 248 micron/hr, with a mean +/- SEM of 105 +/- 10 micron/hr. The rate of resorption by individual osteoclasts was calculated using both time lapse and SEM data, and varied from 43 micron 3/hr to 1225 micron 3/hr with a mean +/- SEM of 390 +/- 109 micron 3/hr. Additional observations indicated not only that the same osteoclast can resorb at a different rate at different times without any definable alteration of the culture conditions, but also that the same osteoclast can simultaneously resorb two lacunae at different rates. These observations provide, for the first time, data on the rate of resorption and the rate of migration of individual osteoclasts on a bone substratum.

A band of F-actin containing podosomes is involved in bone resorption by osteoclasts.

Isolated rabbit osteoclasts cultured on devitalized thin bone slices excavate resorption lacunae that can be visualized with brightfield or phase-contrast microscopy. Superimposition of the brightfield images of such resorption lacunae and the fluorescence images of the corresponding osteoclasts after fixation and staining with rhodamine-conjugated phalloidin revealed that a bright fluorescent band of F-actin-containing podosomes precisely outlined the resorption lacunae in stationary osteoclasts. When the resorption lacunae were being extended laterally, the clearly delineated band of podosomes corresponded to the advancing edge of the resorbing osteoclast and the most recently excavated part of the lacunae. Reshaping and reorganization of the bright bands preceded development of the lateral boundary of the lacunae. Podosomes forming these bands were highly dynamic, changed in size and location, and appeared and disappeared continuously. Their lifespan varied between 2 and 12 min. Similar bands were also seen in vivo in bone-resorbing osteoclasts on the endocranial surface of growing calvariae. Podosomes disappeared in osteoclasts treated with calcitonin, resulting in the disruption of the fluorescent bands. Our results suggest that podosomes are an essential part of the resorption apparatus of osteoclasts.

Lectin-binding glycoconjugates and immunoreactive proteoglycans in resorption pits freshly excavated by isolated rabbit osteoclasts.

Staining with FITC-conjugated concanavalin A, wheatgerm agglutinin and peanut agglutinin demonstrated that abundant sugar residues are present in resorption pits produced in vitro and over the cell surface of rabbit osteoclasts resorbing bovine femoral bone slices. After chondroitinase ABC digestion the stubs of chondroitin 4-sulphate and dermatan sulphate could be detected in the resorption pits by monoclonal antibodies.

Salivary fibronectin in man: an immunoblotting, radioimmunoassay and immunohistochemical study.

An antiserum and monoclonal antibody against plasma fibronectin recognized a 230-kDa, intact fibronectin molecule when 1000-fold diluted human plasma was subjected to immunoblot analysis. Immunoblot analysis of the parotid saliva demonstrated that the rabbit antiserum to human plasma fibronectin bound to five molecules (200, 110, 85, 75 and 65 kDa), other than the 230-kDa, intact fibronectin molecule, while the mouse monoclonal antibody recognized only the 230-kDa molecule. The 230-kDa type was not found in whole saliva with either the antiserum or monoclonal antibody. The antiserum reacted with 85-, 75- and 33-kDa molecules, and the monoclonal antibody recognized 75-, 33- and 20-kDa molecules in the whole saliva. Radioimmunoassay revealed that the mean +/- SD of fibronectin concentration in parotid saliva was 2.5 +/- 1.4 ng/ml (n = 20) and 149.8 +/- 46.2 ng/ml (n = 30) for whole saliva. Immunoperoxidase staining with rabbit antiserum and the mouse monoclonal antibody showed positive cytoplasmic staining of cells in the intralobular and interlobular ducts in parotid, submandibular, and sublingual glands. No acinar cells were stained.

Electrophysiological responses of osteoclasts to hormones.

Electrophysiological measurements were carried out on osteoclasts in vitro. Such isolated osteoclasts are able to resorb bone in vitro and contract in response to calcitonin (CT). Our measurements show that individual osteoclasts respond to CT with a significant transient hyperpolarization of membrane potential. Application of parathyroid hormone (PTH) and dibutyryl cAMP produced a transient hyperpolarization in some osteoclasts. Measurements on an osteoblastlike line (ROS 17/2.8) showed a sustained hyperpolarizing response to CT, which is similar to but smaller than the hyperpolarizing response to PTH and dibutyryl cAMP in this and some other osteoblastlike lines. In contrast to osteoblastlike cells, the osteoclasts have no long term membrane potential response to CT, to PTH, or to dibutyryl cAMP. These results show that there are distinct differences between osteoclasts and osteoblasts in their ion transport responses to hormones.

Osteoclasts and osteoblasts migrate in opposite directions in response to a constant electrical field.

We have investigated in vitro the effects of the electrical field produced by constant current on freshly isolated rabbit osteoclasts and on well characterized clonal rat osteoblastlike cells. At field strengths of 0.1 and 1 V/mm, the osteoclasts migrated rapidly toward the positive electrode, whereas the osteoblastlike cells migrated in the opposite direction, toward the negative electrode. Thus, different cell types from the same tissue can respond differently to the same electrical signal. These results have important implications for hypotheses concerning the cellular mechanism of galvanotaxis, and may also clarify the cellular basis of the clinical application of electrical stimulation of bone healing.

In vitro bone resorption by isolated multinucleated giant cells from giant cell tumour of bone: light and electron microscopic study.

The behaviour of multinucleated giant cells (GCs), obtained from a giant cell tumour of the tibia and cultured on glass coverslips or on devitalized bone slices, was studied using light and electron microscopy. Monitoring the GCs on bone slices by phase-contrast microscopy revealed that they had removed calcified bone matrix resulting in excavation of lacunae, with subsequent lateral extension and perforation of the bone slices. Electron microscopy demonstrated for the first time that the GCs responsible for exavating lacunae had two specific membrane modifications, ruffled border and clear zone, and showed basically similar cytoplasmic fine structures to those of osteoclasts. Fluorescence images of the GCs on glass and on bone after rhodamine-conjugated phalloidin staining revealed that most of the GCs had an intensely fluorescent peripheral band composed of a number of F-actin dots called podosomes. Some GCs showed unusual arrangements of podosomes suggesting abortive attempts at GC formation. We have demonstrated that the band structure of the GCs cultured on bone is intimately involved in bone resorption. Two stromal cell types could be recognized. The predominant type, which seemed to be the only neoplastic element because of its proliferative capability, showed quite different fine structural and cytoskeletal features from the GCs. The other type, which was much less frequent and seemed not to proliferate, had morphological similarities to the GCs, and seemed to be their precursor. Importantly GCs cultured on bone and the osteoclasts share common structures for adhesion to and resorption of bone, strongly supporting the view that the GCs of the giant cell tumour of bone are potentially active bone resorbers and can be regarded as osteoclasts.

[Marginal periodontitis and the immune system. III. Differences in peripheral blood lymphocyte subsets before and after treatment in adult periodontitis patients].

The lymphocyte subsets in the peripheral blood of 8 individuals, aged 25 to 57 yr, with moderate to advanced adult periodontitis were analyzed before and after treatment by means of flow cytometry, and then compared with the findings in 20 normal adults (aged 30 to 45 yr). The results were as follows: the percentage of OKT-8+ (CD 8) in the patients (20.25 +/- 7.15%) was lower than that in normal individuals (31.50 +/- 6.03%, P less than 0.01) while the OKT-4/OKT-8 ratio (2.34 +/- 1.18) was higher than the corresponding ratio in the controls (1.23 +/- 0.37); however, there was no significant difference in the percentages of OKT-3+ (CD 3), OKT-4+ (CD 4) and Leu-12+ (CD 19) between patients and normal subjects. The percentages of OKT-3+, OKT-4+, OKT-8+, Leu-12+ and OKT-4/OKT-8 after the periodontal therapy did not show a significant difference compared with the data obtained before treatment. These results indicate that the decrease in suppressor/cytotoxic T-cell population may be a kind of pathological disposition of the initiation and/or course of adult periodontitis.

[Regulation of osteoclastic bone resorption. 1. Limitation of osteoclast motile activity by fibroblast-like cells].

The cell-to-cell interaction between osteoclasts and fibroblast-like cells, which were mechanically disaggregated from long bones of neonatal rabbits, was investigated to clarify the cellular mechanisms responsible for the regulation of osteoclastic bone resorption by using phase-contrast time lapse recording and fluorescent microscopy for visualization of F-actin cytoskeleton. Osteoclasts showed rapid extension and retraction of pseudopodia with intense ruffling activity and active translocation over a glass surface when there was enough open space around them, while fibroblast-like cells seemed much more stationary. Osteoclasts were not able to cross over cell bodies of fibroblast-like cells which interfered with migration of the osteoclasts. As fibroblast-like cells proliferated, they gradually took over the open glass surface and finally tightly surrounded the osteoclasts. Such osteoclasts usually became contracted and pseudopodial and migratory activities decreased. Fluorescent microscopy of well-spread osteoclaststs stained with rhodamine-labeled phalloidin revealed development of a band-like structure composed of a number of brightly fluorescent F-actin dots at the cell periphery and fine F-actin bundles which run through the cell bodies. On the other hand, contracted osteoclasts tightly surrounded by fibroblast-like cells showed conglomerates composed of accumulated F-actin dots instead of the band-like structure. An F-actin pattern of fibroblast-like cells, which was very different from that of osteoclasts, displayed thick and straight bright bundles with in the cell bodies. The present observation suggests that ongoing osteoclastic bone resorption may be regulated by fibroblast-like cells through limitation of osteoclastic motile activity, and alteration of cytoskeleton and cell-substratum interaction which occurred in osteoclasts may be involved in the mechanism.

Case report of chondroma of the tongue.

A 20-year-old Japanese woman was referred to the hospital for examination and treatment of an asymptomatic swelling of the left dorsum of the tongue. Intraoral examination revealed that the tumor was covered with normal mucosa and oval, firm on palpation and measured 1.5 cm in diameter. The lesion was movable suggesting benignity in nature. No cervical lymph node was palpated. The cut surface of the extirpated tumor after fixation showed a chalky white "core region" surrounded with a yellowish white adipose tissue. Histopathological examination revealed the lesion to be a benign cartilaginous tumor. We report this case of chondroma which arose from the tongue because of its rarity, and reviewed related literature.