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J N Heersche

Publications and source records attributed to J N Heersche.

At least 19 recordsLinked to original sources
PubMed2001

Resorptive state and cell size influence intracellular pH regulation in rabbit osteoclasts cultured on collagen-hydroxyapatite films.

Diseases exhibiting excessive bone loss are often characterized by an increase in the size and number of osteoclasts in affected areas, suggesting that osteoclast size is associated with increased resorptive activity or efficiency. Because osteoclastic bone resorption depends on proton extrusion via a bafilomycin A1-sensitive vacuolar type H+ ATPase (V-ATPase), we investigated the relationship between osteoclast size and state of activity on the one hand, and proton-extruding mechanisms (bafilomycin A1-sensitive V-ATPase and amiloride-sensitive Na+/H+ exchange) on the other. In determining resorptive activities of individual osteoclasts, osteoclast-containing cell suspensions obtained from newborn rabbit long bones were cultured on apatite-collagen complex (ACC)-coated coverslips. Large osteoclasts resorbed 2.5 times more per cell than small osteoclasts, but the amount resorbed per nucleus was the same for the two categories. However, a much larger percentage of large osteoclasts was resorbing compared with small osteoclasts. To study pH regulatory mechanisms in individual large and small osteoclasts, the cells were loaded with the pH-sensitive indicator BCECF and analyzed by single-cell fluorescence. Small and large resorbing osteoclasts had significantly higher basal pH(i) than their nonresorbing counterparts. Also, small nonresorbing osteoclasts were insensitive to bafilomycin A1 addition or Na+ removal from the medium, large nonresorbing osteoclasts responded slightly, and all resorbing osteoclasts (small and large) responded strongly. Differences were also seen in the recovery from an acid load: both small and large nonresorbing osteoclasts were more sensitive to amiloride inhibition, while large resorbing cells were more sensitive to bafilomycin A1 inhibition. Small resorbing cells were inhibited equally by bafilomycin A1 and amiloride. These results clearly show that a greater proportion of large osteoclasts are active in resorption and that pH(i) regulation is associated with enhanced proton pump activity in actively resorbing osteoclasts. Thus, large and small osteoclasts differ in the proportion of cells that are resorbing, while pH regulatory mechanisms differ mainly between resorbing and nonresorbing cells.

Acid Phosphatase↗
PubMed2000

Insulin-like growth factor-1 and -2 stimulate osteoprogenitor proliferation and differentiation and adipocyte formation in cell populations derived from adult rat bone.

In the present study, we test the hypothesis that the stimulation of osteoprogenitor differentiation by the glucocorticoid dexamethasone (Dex) is mediated, at least in part, through components of the insulin-like growth factor (IGF) system. Because it has been suggested that osteoprogenitors and adipocyte progenitors originate from the same precursor cells, and that their differentiation in many systems is reciprocally regulated, the effects of Dex and IGF on adipocyte formation were also evaluated in the same cultures. In view of the presence of IGFs and their binding proteins in serum, we also evaluated to what degree the effects of IGF-1 and IGF-2 on differentiation of osteoblasts and adipocytes were affected by the serum concentration of the culture media. Bone cell populations were isolated from vertebrae of 3-month-old female Wistar rats using an explant culture technique. Osteoprogenitor differentiation was evaluated by a colony assay: Bone-forming osteoblastic colonies (bone nodules) derived from single osteoprogenitors were identified by alkaline phosphatase (ALP) staining and/or staining for mineralized matrix according to the von Kossa technique. Unmineralized nodules and osteoblastic colonies were subsequently identified by their distinctive color and morphology after methylene blue counterstaining. Differentiated adipocytes were identified by Sudan IV staining. IGF-1 and IGF-2 stimulated both osteoprogenitor and adipocyte progenitor differentiation in a dose-dependent pattern. The stimulation of osteoprogenitor differentiation by IGF was not dependent on Dex, but differentiation of adipocytes was. The stimulatory effects of IGF-1 and IGF-2 on osteoprogenitor differentiation were greater in media containing 2.5% fetal bovine serum (FBS) than in media containing 5% or 10% FBS, whereas stimulation of adipocyte formation was greater in media containing 10% FBS. Neutralizing antibody against the type 1 IGF receptor (IGF-1R) partially blocked IGF- and Dex-induced osteoprogenitor differentiation, but did not affect IGF-induced adipocyte formation. This suggests that IGF-stimulated osteoprogenitor differentiation is mediated through IGF-1R, that the stimulation of adipocyte formation by IGF is not, that the stimulatory effects of Dex on osteoprogenitor differentiation are partially mediated through IGF-1R, and that the effects on adipocyte differentiation appear to be mediated through signaling pathways other than the IGF-1R.

Adipocytes↗
PubMed2000

Differences in regulation of pH(i) in large (>/=10 nuclei) and small (</=5 nuclei) osteoclasts.

Osteoclasts are multinucleated cells that resorb bone by extrusion of protons and proteolytic enzymes. They display marked heterogeneity in cell size, shape, and resorptive activity. Because high resorptive activity in vivo is associated with an increase in the average size of osteoclasts in areas of greater resorption and because of the importance of proton extrusion in resorption, we investigated whether the activity of the bafilomycin A(1)-sensitive vacuolar-type H(+)-ATPase (V-ATPase) and amiloride-sensitive Na(+)/H(+) exchanger differed between large and small osteoclasts. Osteoclasts were obtained from newborn rabbit bones, cultured on glass coverslips, and loaded with the pH-sensitive indicator 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Intracellular pH (pH(i)) was recorded in single osteoclasts by monitoring fluorescence. Large (>/=10 nuclei) and small (</=5 nuclei) osteoclasts differed in that large osteoclasts had a higher basal pH(i), their pH(i) was decreased by bafilomycin A(1) addition or removal of extracellular Na(+), and the realkalinization upon readdition of Na(+) was bafilomycin A(1) sensitive. After acid loading, a subpopulation of large osteoclasts (40%) recovered by V-ATPase activity alone, whereas all small osteoclasts recovered by Na(+)/H(+) exchanger activity. Interestingly, in 60% of the large osteoclasts, pH(i) recovery was mediated by both the Na(+)/H(+) exchanger and V-ATPase activity. Our results show a striking difference between pH(i) regulatory mechanisms of large and small osteoclasts that we hypothesize may be associated with differences in the potential resorptive activity of these cells.

Acids↗
PubMed1999

Aluminum accelerates osteoblastic differentiation but is cytotoxic in long-term rat calvaria cell cultures.

We have examined the effects of aluminum (Al) on osteoprogenitor proliferation and differentiation, cell survival, and bone formation in long-term rat calvaria (RC) cell cultures. RC cells were grown in alpha minimal essential medium containing 10% fetal bovine serum, 50 microg/ml ascorbic acid, and 10 mM beta-glycerophosphate with or without Al added to final concentrations of 1 microM-1 mM. Al caused a dose-dependent increase in the number of bone nodules present at early times (day 11) but had no significant effect on nodule numbers at later times (day 17). Time course experiments showed that Al increased nodule number beginning from day 7. Alkaline phosphatase activity, assessed at four stages during the differentiation sequence of RC cell cultures (from 4 to 13 days) was stimulated by Al at all times. However, Al decreased colony formation, inhibited cell growth in late log phase, and decreased saturation density of the treated cultures. Al concentrations of 30 microM and above resulted in degeneration of the cell layer and an increasing fibrillar appearance of the matrix present in between or adjacent to nodules when cultures were maintained for more than 15 days. The presence of Al significantly decreased the viability of cells obtained from 13-17 days cultures, as determined by plating efficiency and trypan blue exclusion. We frequently observed cellular toxicity (in 8 of 10 experiments) in cultures containing 300 microM Al, and by days 17-19, cells, nodules, and matrix were disintegrating in these cultures. We conclude that Al accelerates the rate of osteoprogenitor cell differentiation and the formation of bone nodules while concomitantly inhibiting nodule mineralization. However, concentrations that accelerate differentiation appear to be cytotoxic in long-term cultures.

Alkaline Phosphatase↗
PubMed1999

Expression of mRNAs for type-I collagen, bone sialoprotein, osteocalcin, and osteopontin at different stages of osteoblastic differentiation and their regulation by 1,25 dihydroxyvitamin D3.

We have used in situ hybridization to evaluate the effects of 1,25 dihydroxyvitamin D3 (1,25 (OH)2 D3) on the expression of mRNA for bone-matrix proteins and to determine whether mature osteoblasts respond differently to 1,25 (OH)2 D3 than younger, newly differentiated osteoblasts. Rat calvaria cells were cultured for 7, 12, 15, and 19 days to obtain a range of nodules from very young to very mature. At each time point, some cultures were treated with 10 nM 1,25 (OH)2 D3 for 24 h prior to fixation. In control cultures, type-I collagen mRNA was detectable in osteoblastic cells in very young nodules and increased with increasing maturity of the nodules and the osteoblasts lining them. The bone sialoprotein mRNA signal was weak in young osteoblasts, increased in older osteoblasts, and decreased in mature osteoblasts. Weak osteocalcin and osteopontin signals were seen only in osteoblasts of intermediate and mature nodules. 1,25 (OH)2 D3 treatment markedly upregulated osteocalcin and osteopontin mRNAs and downregulated mRNA levels of bone sialoprotein and, to a lesser extent, type-I collagen in both young and mature osteoblasts. However, a marked diversity of signal levels for bone sialoprotein, osteocalcin, and osteopontin existed between neighboring mature osteoblasts, particularly after 1,25 (OH)2 D3 treatment, which may therefore selectively affect mature osteoblasts, depending on their differentiation status or functional stage of activity.

Animals↗
PubMed1999

Progesterone- and dexamethasone-dependent osteoprogenitors in bone cell populations derived from rat vertebrae are different and distinct.

Previous experiments have demonstrated that bone cell populations derived from explants of lumbar vertebral bone of adult female rats contain osteoprogenitors that require dexamethasone (Dex) or progesterone (Prog) to proliferate and differentiate into fully differentiated bone-forming osteoblasts. We now show that the Prog-dependent population cannot be detected in male rats after sexual maturation, but is present in prepubertal rats of both sexes and can be induced in adult male-derived populations by culturing the explants in medium containing 17beta-estradiol (10(-9)-10(-8) M). This suggested that the Prog- and Dex-dependent osteoprogenitors in adult female-derived populations were probably distinct populations and that the survival of the Prog-dependent osteoprogenitors and/or their ability to proliferate are dependent on the presence of estrogen. We then proceeded to prove this by using replica plating. When one of the paired colonies duplicated was cultured in medium containing Dex (10(-8) M) and the other in medium containing Prog (10(-5) M), 5.0% of duplicates formed bone in Prog only, 11.1% formed bone in Dex only, and 3.4% formed bone in both Prog and Dex. In all cases the size of the bone-forming colonies in Dex-treated cultures was larger than that in Prog-treated cultures, indicating that the effects of Dex on osteoprogenitor proliferation are greater than those of Prog. The results demonstrate the existence of three classes ofosteoprogenitors in adult female rat-derived bone cell populations: a class responding to Dex only, a class responding to Prog only, and a class responding to both Dex and Prog. The results also indicate that the effects of Prog are not mediated by Prog binding to the glucocorticoid receptor and imply that Prog plays an important role in maintaining bone mass through regulating the class of osteoprogenitors responsive to Prog.

Aging↗
PubMed1999

Macrophage colony stimulating factor increases bone resorption in dispersed osteoclast cultures by increasing osteoclast size.

Several reports indicate that macrophage colony stimulating factor (MCSF) is one of the major factors required for osteoclast proliferation and differentiation. Paradoxically, it has also been reported that MCSF inhibits osteoclastic activity. We therefore decided to investigate in detail the effects of MCSF on resorption and osteoclast formation to try and clarify this issue. Osteoclast-containing cultures were obtained from rabbit long bones and cultured on plastic culture dishes or devitalized bovine bone slices. MCSF (4-400 ng/ml) stimulated osteoclastic bone resorption in a time-dependent manner and at all doses examined. After 48 h of culture in the presence of MCSF, we observed a 2-fold increase in the total area of bone resorbed, as well as a significant increase in the area of bone resorbed per osteoclast and the number of resorption pits per osteoclast. This effect was paralleled by an increase in the number of larger osteoclasts (as determined by the number of nuclei per cell) and an increase in the size and depth of the resorption pits. Since the total number of osteoclasts remained the same, the MCSF-induced increase in resorptive activity appeared to be related to an increase in the average size of the osteoclasts. When resorption was expressed as the amount of bone resorbed per osteoclast nucleus, larger osteoclasts resorbed more per nucleus, suggesting that large osteoclasts, as a population, are more effective resorbers than small osteoclasts. Interestingly, when osteoclasts were plated at one-fifth the standard density, the amount of bone resorbed per osteoclast decreased considerably, indicating that resorptive activity is also affected by cell density of osteoclasts and/or of other cells present. However, at this lower density MCSF still increased osteoclast size and resorption by the same fold increase over control, suggesting that the effect of MCSF was independent of factors related to cell density.

Animals↗
PubMed1998

Retinoic acid responsiveness of cells and tissues in developing fetal limbs evaluated in a RAREhsplacZ transgenic mouse model.

Limb morphogenesis is a complex phenomenon in which retinoids play an important role. Abnormal maternal retinoid levels from high oral doses cause fetal malformations, including abnormalities of the musculoskeletal system. Our purpose was to identify the retinoid-responsive cells in bone and cartilage during limb development by using a transgenic line of mice containing a reporter gene insert consisting of a retinoic acid response element linked to an Escherichia coli beta-galactosidase gene. Transgenic fetuses from day 11.5 after conception to birth (day 20) were analyzed histologically. Retinoid-responsive cells and tissues were first seen in the limb bud at 12.5 days in the webs between the forming digits. The webs stained maximally at 14.5 days, after which staining intensity subsided. Staining in the muscles was detectable at 13.5 days, at a stage coinciding with myoblast fusion. Specific regions of perichondrium and periosteum also stained at this stage. Occasional staining was observed in individual chondroblasts in all chondrogenic regions, including hypertrophic chondroblasts and certain articular surfaces of developing joints. Staining of these tissues decreased in intensity in subsequent stages. Osteoclasts started to express beta-galactosidase at 15.5 days and continued to stain into maturity. Our results indicate that specific subsets of cells respond to retinoids at specific stages in the course of normal limb development. In hypertrophic chondrocytes and cells in the webs and joints that display such a response, retinoid-induced effects may be linked to cell death that occurs in these regions. Staining in muscle, perichondrium, and periosteum may reflect retinoid-induced effects associated with cell differentiation and growth. These results suggest that retinoids play a role in a variety of tissues, including bone and cartilage, at specific stages during morphogenesis.

Animals↗
PubMed1998

The decrease in bone mass associated with aging and menopause.

The human skeleton accumulates bone up to approximately age 30, after which bone is gradually lost. Although estrogen replacement therapy prevents postmenopausal bone loss, it is not certain that estrogen deficiency alone is responsible for the decrease in bone mass. Progesterone deficiency could also be a factor, and progesterone replacement therapy has been shown to prevent postmenopausal bone loss associated with ovarian dysfunction. This article reviews what is known about bone remodeling and bone loss as a function of age and gender, discusses evidence from studies in rats that progesterone plays an important role in regulating bone formation, and suggests directions for future studies in predicting the success or failure of implant therapy based on the number and kinds of osteoprogenitor cells present.

Adult↗
PubMed1998

Osteoprogenitor cells in cell populations derived from mouse and rat calvaria differ in their response to corticosterone, cortisol, and cortisone.

Osteoprogenitors present in cell populations derived from fetal or newborn rat and mouse calvaria differentiate in long term culture and form osteoblastic bone-forming colonies (bone nodules). Previous reports have indicated considerable differences between bone cell populations derived from these two species with regard to their proliferation in response to glucocorticoids. In the present investigation, we have focused on proliferation and differentiation of osteoprogenitor cells in these bone cell populations and evaluated the effect of corticosterone, the principal glucocorticoid of both mouse and rat. Cells were isolated by sequential collagenase digestion from calvaria of newborn (2-5 days) CD-1 mice [mouse calvariae (MC) cells] and term fetal Wistar rats [rat calvaria (RC) cells] and cultured for up to 25 days in alpha-minimal essential medium containing 10% fetal bovine serum (FBS), antibiotics, 50 microg/mL ascorbic acid, and 8-10 mmol/L beta-glycerophosphate. In agreement with previous observations by us and others, corticosterone increased cell growth in RC cell cultures, but inhibited cell growth in MC cultures. In RC cell cultures, corticosterone (1-1000 nmol/L) increased the nodule number in a dose-dependent manner (p < 0.001 for all concentrations above 3 nmol/L) with a maximal effect at 300 and 1000 nmol/L (threefold increase over control). In MC cells, on the other hand, corticosterone (0.3-1000 nmol/L) increased the nodule number only at 30 nmol/L (50%, p < 0.01) but inhibited nodule formation by 33% (p < 0.001) at 1000 nmol/L. In both RC and MC cultures a linear relationship was found between the number of cells plated and number of nodules formed. When cultures were treated with cortisol (30-300 nmol/L), similar effects were observed; the number of nodules dose dependently increased in RC cell cultures and dose dependently decreased in MC cell cultures. Significantly, however, the inactive glucocorticoid cortisone also increased bone nodule formation in RC cell cultures and decreased bone nodule formation in MC cell cultures. Carbenoxolone, which blocks 11 beta hydroxysteroid dehydrogenase and thus prevents conversion of cortisone to cortisol, partially inhibited the cortisone-induced effects on bone nodule formation in both RC and MC cell cultures, indicating that both RC and MC cells can convert inactive glucocorticoids to active metabolites. In conclusion, our results show that the glucocorticoids corticosterone and cortisol inhibit proliferation and differentiation of osteoprogenitors in MC cell cultures but stimulate these processes in rat-derived osteoprogenitors.

Animals↗
PubMed1997

Chronic extracellular acidosis induces plasmalemmal vacuolar type H+ ATPase activity in osteoclasts.

Proton extrusion into an extracellular resorption compartment is an essential component of bone degradation by osteoclasts. Chronic metabolic acidosis is known to induce negative calcium balance and bone loss by stimulating osteoclastic bone resorption, but the underlying mechanism is not known. The present studies were undertaken to evaluate whether chronic acidosis affects proton extrusion mechanisms in osteoclasts cultured on glass coverslips. Acidosis, mimicked experimentally by maintaining the cells at extracellular pH 6.5, rapidly lowered intracellular pH to 6.8. However, after 2 hours, a proportion of cells demonstrated the capacity to restore intracellular pH to near normal levels. To define the mechanism responsible for this recovery, the activity of individual H+ transport pathways was analyzed. We found that chronic acid treatment for up to 6 h did not significantly affect the cellular buffering power or Na+/H+ antiport activity. In contrast, chronic acidosis activated vacuolar H+ pumps in the osteoclasts. Although only approximately 5% of the control cells displayed proton pump activity, about 40% of cells kept at extracellular pH 6. 5 for 4-6 h were able to recover from the acute acid load by means of bafilomycin A1-sensitive proton extrusion. Conversely, the H+-selective conductance recently described in the plasma membrane of osteoclasts was clearly inhibited in the cells exposed to chronic acidosis. Following acid treatment, the activation threshold of the H+ conductance was shifted to more positive potentials, and the current density was significantly reduced. Considered together, these results suggest that induction of plasmalemmal vacuolar type ATPase activity by chronic acidosis, generated either systemically due to metabolic disease or locally at sites of inflammation, is likely to stimulate osteoclastic bone resorption and thus to promote bone loss.

Acidosis↗
PubMed1997

Progesterone-mediated stimulation of osteoprogenitor proliferation and differentiation in cell populations derived from adult or fetal rat bone tissue depends on the serum component of the culture media.

We have shown previously that progesterone (Prog) and dexamethasone (Dex) stimulate osteoprogenitor proliferation and differentiation in cell populations derived from adult rat vertebrae and in primary cultures of fetal rat calvariae. In these two in vitro systems, osteoprogenitors can be identified by the appearance of colonies of differentiated osteoblasts producing bone (bone nodule formation). Culture conditions supporting proliferation and differentiation of osteoprogenitors include a requirement for the presence of serum in the culture media. Our major interest in the present study was to investigate whether Prog- and Dex-mediated osteoprogenitor proliferation and differentiation was observed to the same degree in different lots of fetal bovine serum (FBS). In addition, we wanted to investigate whether osteoprogenitors present in cell populations derived from fetal calvarial bone and those present in populations derived from adult vertebral bone would respond similarly under the different culture conditions. We found that, in populations derived from adult rat vertebrae, the effects of the serum component of the culture medium on the number of bone nodules induced by Prog and on the dose-dependency of the Prog effect were striking: in culture media containing the most effective serum the number of bone nodules was 22-fold higher than that in the least effective serum. In addition, Prog responses were detectable at 10(-5) M only in some sera but were significant at 10(-7) M in others. The effect of Dex in the adult rat vertebrae-derived populations was much less dependent on the serum used: the number of bone nodules in culture media containing the most effective serum was only 1.3 times greater than that in media containing the least effective serum. In cell populations derived from fetal calvariae, the serum dependence of the Prog response was less pronounced: a 4.3-fold increase over control was observed in the most effective serum, and a 2.4-fold increase in the least effective serum. No effects of the serum component of the culture medium on the Dex response were detectable. Thus, Prog-induced bone nodule formation appears to be strongly dependent on the particular type of FBS used for osteoprogenitors present in bone cell populations derived from adult rat vertebrae but much less so in populations obtained from fetal rat calvariae. Preliminary experiments suggest that the estrogen content of the culture media may be one of the determinants regulating Prog responsiveness of the osteoprogenitors. Dex-induced proliferation and differentiation of osteoprogenitors in bone cell populations derived from both adult rat vertebrae and fetal rat calvariae, on the other hand, did not appear to be strongly dependent on factor(s) present in the FBS component of the culture medium.

Animals↗
PubMed1997

Progesterone stimulates proliferation and differentiation of osteoprogenitor cells in bone cell populations derived from adult female but not from adult male rats.

We examined the effects of the synthetic glucocorticoid dexamethasone (Dex) and of the sex steroids progesterone (Prog) and testosterone (Testo) on proliferation and differentiation of progenitors for osteoblasts, adipocytes, and macrophages in cell populations derived from lumbar vertebrae of adult male and female rats. To assay for these progenitors, we used a previously described colony assay, where progenitors are identified by the appearance of colonies of the differentiated phenotype in long term cultures of cell populations containing these progenitors. In cell populations derived from both males and females, Dex (10(-9)-10(-6) mol/L) induced a similar dose-dependent increase in the number of osteoblast colonies (bone nodules), colonies of alkaline phosphatase (AP)-positive cells, adipocyte colonies and macrophage colonies (ED2-positive cells). Prog (10(-8)-10(-5) mol/L), on the other hand, increased bone nodule formation in female-derived populations but not in male-derived populations. Maximal stimulation was seen at 10(-5) mol/L. 17 beta-Estradiol (E2) enhanced the Prog-induced increase in the number of bone nodules in a dose-related fashion. Maximal stimulation was seen at 10(-8) mol/L E2. E2 (10(-9)-10(-6) mol/L) had no effect on Dex-induced bone nodule formation, indicating that the effect is specific for Prog-induced stimulation of bone nodule formation. Prog also caused a dose-dependent increase in the number of colonies of AP-positive cells. Interestingly, the effect of Prog on the number of AP-positive colonies was the same in populations derived from both sexes. Prog-induced stimulation of adipocyte and macrophage development in female-derived populations was significantly greater than that in male-derived populations. Testo (10(-9)-10(-5) mol/L) had no effect on any of the parameters evaluated in populations derived from either males or females. These observations demonstrate that the effect of Prog on proliferation and differentiation of progenitors for osteoblasts, adipocytes, and macrophages in cell populations derived from lumbar vertebrae of adult male or female rats is sex-dependent and is seen either only (bone nodule formation) or more pronounced (adipocyte and macrophage development) in female-derived populations.

Adipocytes↗
PubMed1997

Experimentally induced periodontitis in beagle dogs causes rapid increases in osteoclastic resorption of alveolar bone.

This study was undertaken to observe osteoclast differentiation related to inflammatory progression in aggressive periodontitis induced in beagle dogs by ligature of the gingival sulcus. To monitor osteoclastic activity, we used histochemical methods (staining for tartrate-resistant acid phosphatase [TRAP]) to visualize osteoclasts and their TRAP-positive precursors and biochemical methods (ELISA assay of pyridinium crosslinks) to detect bone matrix degradation products in gingival crevicular fluid (GCF), serum, and urine. For histochemical study, tissue specimens were prepared from 3 adult female beagle dogs induced with experimental periodontitis by silk ligature placement below the gingival margin of mandibular molars ligated for 3, 7, and 21 days. For biochemical study for pyridinoline measurement, the 24 mandibular molars of 4 male beagle dogs were ligated. GCF, urine, and serum were collected at day 0 and at 3, 7, 14, and 21 days after ligation. In the early inflammatory phase of ligature-induced periodontitis (day 3), TRAP+ mononuclear and TRAP+ multinucleated cells were present in the gingival connective tissue, and active bone-resorbing cells were found in excavated lacunae at the alveolar crest, but osteoclasts were not infiltrating the periodontal ligament during this early phase. During later stages of the inflammatory process (7 and 21 days), osteoclasts appeared at both the gingival and ligament side of the alveolar bone. Osteoclastic bone resorption appeared to be more severe on the bone surface at the gingival side than on the bone surface of the periodontal ligament side. Measurement of pyridinoline significantly increased in GCF and urine 3 days after ligation. The results suggested that bone at the crest of the alveolar bone is rapidly resorbed within 3 days of inducing experimental periodontitis.

Acid Phosphatase↗
PubMed1996

Progesterone and dexamethasone stimulate proliferation and differentiation of osteoprogenitors and progenitors for adipocytes and macrophages in cell populations derived from adult rat vertebrae.

We investigated the effects of the sex hormone progesterone (Prog) and the synthetic glucocorticoid dexamethasone (Dex) on proliferation and differentiation of progenitor cells of osteogenic, adipocytic, and hemopoietic lineages in cell populations derived from explants of adult female rat lumbar vertebrae. The cell populations were obtained by culturing bone explants in plasma clots immersed in alpha-minimum essential medium plus 10% fetal calf serum (standard medium) and then subculturing the outgrowth cells in standard medium plus 50 micrograms/ml of ascorbic acid, 5 mM beta-glycerophosphate, and with or without Prog or Dex. On day 6 of culture, these populations were analyzed for cAMP responses to parathyroid hormone (PTH), prostaglandin E2 (PGE2), and isoproterenol (IPT). Increases in intracellular cAMP were seen in response to PTH, PGE2, and IPT, and culturing in medium containing Prog increased these responses. At various time periods between days 4-27 of culture, the cultures were evaluated for the presence of bone nodules, alkaline phosphatase (AP)-positive colonies, adipocytes, monocytes, and macrophages. Prog and Dex increased the number of bone nodules and AP-positive colonies. The effect of Prog on bone nodule formation was smaller than that of Dex. In addition, the effect of Dex on bone nodule formation was evident after 10 days of culture, while the Prog-induced effects became significant at days 16-20 of culture. Both hormones also increased the number of Sudan IV-positive colonies (adipocytes), certain types of alpha-naphthyl butyrate esterase (alpha-NBE)-positive colonies (monocytes, macrophages, and T-lymphocytes), and ED2-positive colonies (macrophages). Prog-treated cultures contained more colonies of small spindle-shaped alpha-NBE-positive cells and fewer colonies of small round alpha-NBE-positive cells when compared with Dex-treated cultures. These data indicate that cell populations derived from adult rat lumbar vertebrae contain, among others, osteoprogenitors and progenitors for adipocytes and macrophages that are stimulated to proliferate and differentiate by Prog and Dex. The data also suggest that the effects of Prog and Dex differ qualitatively and quantitatively.

Adipocytes↗
PubMed1996

Analysis of chondroprogenitor frequency and cartilage differentiation in a novel family of clonal chondrogenic rat cell lines.

We have isolated through sequential steps of subcloning a series of normal clonal cell lines enriched for chondroprogenitors that undergo differentiation in vitro from progenitors to mature chondroblasts and chondrocytes forming three-dimensional cartilage nodules. In the parental chondroblast clone RCJ 3.1C5 (C5), differentiation and cartilage formation occurred without added hormones or growth factors, but chondrogenesis could be stimulated markedly in the presence of the glucocorticoid steroid Dexamethasone (Dex). Limiting dilution analysis indicated that greater than one in ten C5 cells plated was a chondroprogenitor capable of differentiating and forming a cartilage nodule in low density cultures, but chondrogenesis was down-regulated in higher density cultures. Dex elicited a greater stimulatory effect on cartilage nodule formation when C5 cells were plated at higher rather than lower densities. Since Dex also maintained the chondrogenic potential of C5 cells passaged repeatedly, we subcloned C5 in the presence of Dex. Eight of eleven subclones were chondrogenic and the frequency of chondroprogenitors capable of cartilage formation in isolated subclones ranged from lower to much higher than in the parental C5 clone. Both Dex-independent as well as Dex-dependent clones were identified, although long-term maintenance of the chondrocyte phenotype in all subclones required Dex. These data suggest that there are Dex-dependent and Dex-independent chondroprogenitor cells, that cell-cell interactions and/or local factors can modulate cartilage nodule formation and that Dex-responsive steps are involved in long-term maintenance of chondroprogenitors in vitro. Thus, this unique family of non-transformed, clonal chondrogenic cell lines provides a quantifiable, readily manipulatable system in which cartilage differentiation and metabolism can be assessed.

Animals↗
PubMed1995

Regulation of cytoplasmic pH in osteoclasts. Contribution of proton pumps and a proton-selective conductance.

Osteoclasts resorb bone by secreting protons into an extracellular resorption zone through vacuolar-type proton pumps located in the ruffled border. The present study was undertaken to evaluate whether proton pumps also contribute to intracellular pH (pHi) regulation. Fluorescence imaging and photometry, and electrophysiological methods were used to characterize the mechanisms of pH regulation in isolated rabbit osteoclasts. The fluorescence of single osteoclasts cultured on glass coverslips and loaded with a pH-sensitive indicator was measured in nominally HCO(3-)-free solutions. When suspended in Na(+)-rich medium, the cells recovered from an acute acid load primarily by means of an amiloride-sensitive Na+/H+ antiporter. However, rapid recovery was also observed in Na(+)-free medium when K+ was used as the substitute. Bafilomycin-sensitive, vacuolar-type pumps were found to contribute marginally to pH regulation and no evidence was found for K+/H+ exchange. In contrast, pHi recovery in high K+ medium was largely attributed to a Zn(2+)-sensitive proton conductive pathway. The properties of this conductance were analyzed by patch-clamping osteoclasts in the whole-cell configuration. Depolarizing pulses induced a slowly developing outward current and a concomitant cytosolic alkalinization. Determination of the reversal potential during ion substitution experiments indicated that the current was due to H+ (equivalent) translocation across the membrane. The H+ current was greatly stimulated by reducing pHi, consistent with a homeostatic role of the conductive pathway during intracellular acidosis. These results suggest that vacuolar-type proton pumps contribute minimally to the recovery of cytoplasmic pH from intracellular acid loads. Instead, the data indicate the presence of a pH- and membrane potential-sensitive H+ conductance in the plasma membrane of osteoclasts. This conductance may contribute to translocation of charges and acid equivalents during bone resorption and/or generation of reactive oxygen intermediates by osteoclasts.

Animals↗