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Biomedical subjects

G L Wong

Publications and source records attributed to G L Wong.

At least 19 recordsLinked to original sources
PubMed1992

Maturation-associated changes in the cellular composition of mouse calvariae and in the biochemical characteristics of calvarial cells separated into subclasses on Percoll density gradients.

The effects of tissue maturation on the cellular composition and biochemical characteristics of bone were studied in neonatal, young adult, and aging mice. Osteoblast subclasses were isolated on Percoll density gradients. Neonatal calvariae consisted almost exclusively of cells banding at low and intermediate buoyant density. High buoyant density cells constituted 5-10% of total cells at 10 days of age but increased to 50-60% by 5 weeks of age. These latter cells were released late during collagenase digestion. This indicates that they arise from the deeper layer of bone. For this reason, we consider them putative osteocytes. We established that constitutive secretion of IGF-I and TGF-beta and activities of cellular alkaline phosphatase paralleled those of the tissue of origin in all cell groups and was highest in cells of intermediate buoyant density. These activities declined rapidly after cessation of growth at 5 weeks of age in both bone and isolated cells. Between 5 and 8 weeks of age, the hormonal response to PTH also declined dramatically. The maximum cAMP induced by PTH declined by about 70% in highly responsive cells of intermediate buoyant density and fell to insignificant levels in cells of high buoyant density. We found that a cyclic AMP response to PTH was positively correlated with stimulated secretion of IGF-I by this hormone in cells from animals of all ages. Despite their inability to respond to PTH with increases in cAMP and IGF-I, adult bone cells of high buoyant density continued to respond to PTH with increases in the secretion of TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1991

Characterization of bone cells isolated on discontinuous Percoll gradients: distribution in sequentially derived populations.

Bone cell populations obtained by sequential digestion of newborn mouse calvariae remain morphologically heterogeneous despite well-documented biochemical differences. Fractionation of these populations on Percoll gradient reveal three major cell groups of low, intermediate, and high buoyant density (1.056, 1.070, and 1.095 g/ml) that are present in different ratios in early and late released populations. Cells of low and intermediate density dominate in early released populations. In contrast, late released populations contain mostly high-density cells. Basal levels of alkaline phosphatase are highest in cells of intermediate buoyant density. All cells respond to PTH with cAMP production and morphologic transformation, but biochemical responses to PTH, such as secretion of insulin-like growth factor I (IGF-I) and stimulation of alkaline phosphatase activity, occur mostly in cells of intermediate density. These data suggest that (1) subclasses of osteoblasts can be further separated by density and (2) PTH effects on alkaline phosphatase activity and IGF-I secretion are probably expressed by osteoblasts of a certain subclass and/or stage of development.

Alkaline Phosphatase↗
PubMed1990

IGF-I production by mouse osteoblasts.

Mouse osteoblasts contain and secrete insulinlike growth factor I (IGF-I), which can be measured by radioimmunoassay after separation from endogenous IGF-I binding activity. Our studies indicate that IGF-I is produced by all bone cell populations prepared by sequential digestion of mouse calvaria with collagenase and protease. Furthermore, relatively small amounts of IGF-I are cell associated, and IGF-I is recovered primarily in the cell medium after 24 h of culture. Basal IGF-I secretion is also density dependent, and secretion per cell is approximately 20-fold higher when cultures are inoculated at 0.125 versus 1.0 x 10(5) cells per cm2. Growth hormone increased the secretion of IGF-I only in cells released in the earlier stages of digestion. These growth hormone-responsive populations were previously shown to differ from late released cells in that they show a lower expression of the osteoblastic phenotype, harbor more EGF receptors per cell, and have a higher proliferative response to low doses of exogenous IGF-I and EGF. These data reaffirm the presence of different subclasses of bone cells in populations obtained by sequential digestion of bone and suggest that growth hormone stimulates IGF-I secretion by immature osteoblasts.

Animals↗
PubMed1989

Mitogenic responses to and binding of insulin-like growth factor 1 and/or epidermal growth factor by bone cells.

Populations of cells with different biochemical characteristics can be obtained following enzymatic digestion of newborn mouse calvaria. Previous studies had suggested an enrichment for periosteal fibroblasts in early released cells, based on their proliferative response to PGE2 and for osteoblasts in late released cells due to their high cAMP response to PTH. Intermediate cells were presumed to be a mixture of those two phenotypes. We have continued our characterization of these populations by studying their mitogenic responses to IGF-1 and EGF since these polypeptide growth factors have been reported to stimulate proliferation of immature bone cells in organ cultures of bone. [3H]Thymidine incorporation into acid insoluble radioactivity was stimulated by IGF-1 in all populations but the greatest increase was in intermediate populations. These latter cells were also the most highly responsive to EGF, which had a small growth stimulating effect on early released cells and no significant effect on late released cells. In bone cells both low and high affinity EGF binding sites were identified (Kd approximately 4 nM and 0.5 nM) and the total EGF binding sites/cell were approximately two-fold higher in intermediate than in early and late released populations. These data suggest that among sequentially released populations of bone cells the intermediate populations may be enriched for immature bone cells from the proliferative zone of bone.

Animals↗
PubMed1989

A comparison of three methods to determine the radiochemical purity of 99Tcm-hexamethylpropylene amine oxime (99Tcm-HMPAO).

Determining the radiochemical purity of 99Tcm-HMPAO using the standard method suggested by the manufacturer of the HMPAO kit is slow, consuming much of the 30 min useful shelf-life of the radiopharmaceutical. We have compared two new methods (a solvent extraction technique and a method involving a disposable, pre-packed reverse phase chromatography cartridge) with the standard method for determining the radiochemical purity of 99Tcm-HMPAO. There were no significant differences (F test, p less than 0.05) in the results obtained by all three methods. However, the reversed phase chromatography method gave better agreement (correlation coefficient of 0.877) with results obtained using the standard method than did the solvent extraction technique (correlation coefficient of 0.693). The solvent extraction technique took about 10 min to perform whereas the reversed phase chromatography method took only 5 min. Both of the new methods did not achieve complete separation of the secondary, less lipophilic 99Tcm-HMPAO complex from the primary, lipophilic 99Tcm-HMPAO complex but the error introduced was small (typically only 3-5%). The new methods offer the capability of determining the radiochemical purity of 99Tcm-HMPAO quickly, reliably and accurately, prior to administration of the radiopharmaceutical to the patient.

Organotechnetium Compounds↗
PubMed1987

Production of and response to growth-stimulating activity in isolated bone cells.

Autologous growth-promoting activity has been shown to be secreted by bone organ cultures. To identify the cellular source of these growth factors, we have studied the activity present in cell extracts prepared from isolated bone cells released early or late from mouse calvariae following collagenase digestion. Previous studies have established that early released cell populations reside on the bone surface and consist of a heterogeneous mixture of cells that are less osteoblastic than late released cells. We find that soluble extracts of the latter cells contain more growth-promoting activity/mg of cellular protein than those of the former. By this criterion late released cells appear to be the major source of endogenous growth factors. On the other hand, upon exposure to bone cell-derived cell extracts, early released cells express a greater-fold increase in [3H]thymidine incorporation into acid insoluble radioactivity than late released osteoblasts because of the high basal mitogenic activity of the latter which may be related to their production of autologous growth factor. These data suggest that both early and later released cells may be major targets for bone growth factors produced in situ by late released osteoblasts.

Animals↗
PubMed1986

Isolation and characterization of highly serum-dependent cells released early from collagenase digested calvaria.

A subclass of highly serum-dependent bone cells has been identified among the cells released early from calvaria following digestion in collagenase. Partial purification for these cells has been carried out based on the observation that they require serum for attachment to polystyrene culture flasks. This subclass of bone cells differs from adherent cells and late released osteoblasts, in that they express almost no cAMP response to PTH, require high levels of serum (10%) for initial growth and proliferation, and do not increase DNA synthesis in response to PTH. In common with adherent cells and late released osteoblasts, their proliferation is decreased by 1,25(OH)2D3 at doses above 0.2 ng/ml and they respond to PGE2 with increased DNA synthesis. These similarities suggest an ontogenic relationship with osteoblasts. Based on their differences, however, provisional identification of these cells as relatively undifferentiated mesenchymal cells is suggested.

Animals↗
PubMed1984

Paracrine interactions in bone-secreted products of osteoblasts permit osteoclasts to respond to parathyroid hormone.

During bone remodeling, activation of resorption is followed by a cycle of formation and this ordered sequence of events has long suggested that local interactions between osteoclasts and osteoblasts are an important regulatory mechanism in bone metabolism. To study this phenomenon, we have prepared bone cells containing primarily osteoclasts by brief digestion of mice calvariae in collagenase, overnight attachment to polystyrene tissue culture flasks in serumless medium supplemented with OB (osteoblast) cell conditioned medium and subsequent growth in low serum. These OC (osteoclast) cells were found to be highly enriched in acid phosphatase activity and expressed cAMP responses to PTH (parathyroid hormone) and prostaglandin E2 but exhibited no PTH-stimulated hyaluronate synthesis in contrast to prostaglandin E2. PTH effects on hyaluronate, however, could be restored upon coculture of OC cells with OB cells (noncontact) or with OB cell conditioned medium, thereby suggesting that OB cells regulate OC cell PTH responsiveness and/or differentiation by soluble cell products secreted into the medium.

Acid Phosphatase↗
PubMed1984

A comparison of the PTH-dependent cAMP responses in osteoclastic and osteoblastic bone cells.

Isolated bone cells enriched for either osteoclastic (OC) or osteoblastic (OB) characteristics have been previously described. Only OC cells respond to calcitonin (sCT), but both types of cell populations demonstrate adenosine 3':5'-cyclic phosphate (cAMP) increases with parathyroid hormone (PTH), suggesting a common PTH-responsive cell in both. In view of this, the cAMP responses to PTH were compared in terms of turnover time, induction of dose-dependent desensitization, recovery from desensitization and inhibition by trifluoperazine (TFP). In the absence of new protein synthesis, OC cell cAMP response to PTH declined more slowly than that of OB cells (t/2 of approximately 24 h vs. 8 h for OB cells). The relative stability of PTH responsiveness in OC cells was unique in that the same was not seen for sCT or prostaglandin E1 (PGE1) responsiveness which declined with a t/2 of 8 and 6 h, respectively. In comparison to OB cells, OC cells also demonstrated (1) a more rapid recovery from autologous desensitization with PTH, regaining responsiveness with a t/2 of approximately 6 h vs. 14 h for OB cells; (2) greater resistance to desensitization by submaximal doses of PTH (4 X 10(-10) -4 X 10(-9) M), and (3) resistance to inhibition by low doses of TFP (less than 5 microM). These results suggest that the PTH-responsive cells in OC populations are metabolically different from those in OB populations.

Alprostadil↗
PubMed1983

Differential sensitivity of osteoclasts and osteoblasts suggests that prostaglandin E1 effects on bone may be mediated primarily through the osteoclasts.

Prostaglandin E (PGE) stimulates resorption in bone. Since osteoblast-like osteosarcoma cells secrete PGE2, the possibility that osteoclasts were the major target for PGE was considered. To study this question, it was first established that in isolated bone cells enriched for either osteoclastic (OC) or osteoblastic (OB) characteristics, PGE1 can induce biochemical effects similar to those seen with bovine parathyroid hormone 1-84 (PTH), another potent stimulator of bone resorption. These changes include increased cAMP and hyaluronate synthesis in OC cells, and increased cAMP but decreased citrate decarboxylation in OB cells. By following these markers, it is demonstrated that PGE1 can activate OC cells at doses as low as 1 nM, whereas OB cells require 250 nM. Bone cell responses to various doses of PTH and PGE1 were also compared. In OC cells the lowest effective dose of PGE1 and PTH was similar (1 nM), but increasing response to PGE1 was seen up to 1000 nM in contrast to PTH response which peaked at 20 nM. In addition, the magnitude of PGE1-induced OC cell hyaluronate was two to four times greater than that of PTH at all doses tested. In OB cells, PTH induced significant decreases in citrate decarboxylation at 0.1 nM, compared to 250 nM for PGE1. Half-maximal inhibition of citrate decarboxylation (19% of control) by PTH occurred at 0.5 nM, whereas 500 nM of PGE1 was required for an equivalent effect. Thus, (i) OC cells responded to PGE1 doses that were approximately 200 times lower than the minimum required by OB cells, and (ii) OB cells responded to 100 times lower doses of PTH than PGE1.

Animals↗
PubMed1983

Actions of parathyroid hormone and 1,25-dihydroxycholecalciferol on citrate decarboxylation in osteoblast-like bone cells differ in calcium requirement and in sensitivity to trifluoperazine.

The actions of PTH in OB bone cells appear to involve both calcium and cAMP. At present little information exists regarding the relationship, if any, between these two putative second messengers of hormone action in bone cells. In this report the molecular role of calcium in the actions of PTH and 1,25(OH)2D3 has been compared, since like PTH, the steroid 1,25(OH)2D3 is a potent bone resorbing hormone that exerts inhibition of citrate decarboxylation in OB cells, but unlike PTH does not activate adenylate cyclase. It was found that 1,25(OH)2D3 could initiate near maximum inhibition of citrate decarboxylation at extracellular calcium levels as low as 0.05 mM, whereas PTH effects began to be apparent only at 0.1 mM calcium, and maximum inhibition of citrate decarboxylation by PTH required 0.5 mM Ca. In addition, PTH-induced decrease in citrate decarboxylation was inhibited by low doses of TFP, an inhibitor of calmodulin and calcium-dependent, phospholipid-sensitive protein kinases, in contrast to 1,25(OH)2D3, whose effects were not reduced by this agent. These results suggest that: (a) the actions of 1,25(OH)2D3 may not be directly dependent on calcium influx; (b) in OB cell response to PTH a relationship probably exists between cAMP and calcium; and (c) this relationship may involve calmodulin, or calcium-dependent protein kinases that can be inhibited by TFP.

Animals↗
PubMed1983

Differential serum dependence of cultured osteoclastic and osteoblastic bone cells.

Sequential collagenase digestion of mice calvariae provides populations of bone cells that express either osteoclasts (OC) or osteoblastic (OB) activities after growth for 6 days in similar culture conditions consisting of minimal essential medium supplemented with 10% fetal calf serum (FCS). The OC characteristics (acid phosphatase activity and hyaluronate synthesis, and their stimulation by PTH) were recovered in the cell populations released early from calvariae, but these also contained OB cells and numerous spindle-shaped alkaline phosphatase positive cells that resembled fibroblasts. We have attempted to select for growth of OC cells in these early populations by exploiting differences in growth requirements of OC, OB, and fibroblastic cells. We find that after growth for 6 days in low serum (2% FCS), OC cell populations demonstrated a threefold increase in OC activity/cell, and cell yield was reduced to one-third of that obtained in 10% FCS. Spindle-shaped cells were absent in 2% FCS and OB marker activities (alkaline phosphatase and citrate decarboxylation) were reduced threefold. In contrast to OC cells, high serum (10% FCS) favored the growth and phenotypic expression of OB cells (late populations). Cell yield and OB marker activities/cell were twofold higher in OB cells grown in 10% FCS vs 2% FCS, whereas growth but not phenotypic expression was retained at 5% FCS. These data suggest that differential serum dependence of OC and OB cells may provide a basis for further enrichment for each cell type following sequential digestion.

Acid Phosphatase↗
PubMed1982

Characterization of subpopulations of OC and OB bone cells obtained by sedimentation at unit gravity.

Immediately after isolation from calvaria, OC and OB bone cells populations 1-6 were individually characterized by sedimentation at unit gravity. This procedure was used to generate from each population 4 fractions that contained cells of different sizes. Sedimentation results suggested that freshly isolated OC cell populations consisted of cells that were generally smaller and demonstrated less size heterogeneity than OB cells. After sedimentation the cells in each fraction were cultured for 6 days and then characterized with regard to cell separation based on basal biochemical characteristics and hormonal responses to PTH and CT. The largest cells in the later released OC cells appeared to be a mixture of OC and OB cells (approx. 15% of populations 2 + 3). All OB cell fractions appeared to be free of OC cells. The highest basal OC activities and hormonal responses occurred in the larger cells of population 2, whereas in OB cells (populations 5 and 6), this occurred in the cells of small to intermediate size. Finally, although the absolute size of the cells in each fraction increased during culture, the size differential within the fractions and between OC and OB cells was maintained even after 6 days.

Acid Phosphatase↗
PubMed1980

Glucocorticoids increase osteoblast-like bone cell response to 1,25(OH)2D3.

Recent reports indicate that some hormones may regulate the binding of, and subsequent response to, other hormones by their target tissue. The adrenal glucocorticoids are prominent among these modulating hormones. Glucocorticoids have been shown to enhance bone cell sensitivity to parathyroid hormone (PTH) in vitro and this in turn has permitted PTH-induced effects to be measured at physiological doses of PTH for the first time in isolated osteoblast-like (OB) and osteoclast-like (OC) cells. It is unknown whether these findings represent a specific interaction between glucocorticoids and PTH or indicate a general role for glucocorticoids in the development and/or maintenance of bone cell differentiation, of which hormonal responsiveness would be one expression. In the event of a general glucocorticoid effect on cell differentiation, increased responsiveness to other bone resorbing hormones should also be observed. We have therefore examined whether glucocorticoids enhance the sensivity of bone cells to a steroid hormone, 1,25 dihydroxycholecalciferol (1,25(OH)2,D3), and we report here that they do.

Biological Transport↗
PubMed1980

Bone cell cultures as an experimental model.

The isolation and separation of bone cells with populations enriched for osteoclastic or osteoblastic phenotypes are described. Such systems offer the opportunity to compare and contrast the controls exerted by hormones, ions, and other agents on the functions of the individual bone cell types and may in the future provide explanation for the changes seen in bone tissue in various diseased states.

Bone and Bones↗
PubMed1979

Basal activities and hormone responsiveness of osteoclast-like and osteoblast-like bone cells are regulated by glucocorticoids.

Isolated bone cells demonstrate cell-type specific responses to glucocorticoids. Osteoclast-like (OC) cells exhibit a large decrease in basal hyaluronate synthesis at physiological doses of glucocorticoids and resistance to further inhibition by pharmacological doses up to 10(-4) M. This effect is not accompanied by decreases in protein synthesis. In contrast, osteoblast-like (OB) cell metabolism is not inhibited by physiological doses of glucocorticoids. However, in OB cells both citrate decarboxylation and collagen synthesis are decreased at pharmacological doses of glucocorticoids and these effects are accompanied by a decrease in general protein synthesis. In addition to these effects on basal and general cell activities, physiological doses of glucocorticoids modulate the hormonal sensitivity of OC and OB bone cells such that lower concentrations of bovine parathyroid hormone (PTH) are necessary to elicit measurable biochemical changes. As a result, the presence of glucocorticoids permits significant responses to PTH to be detected at doses as low as 2 x 10(-13) M in OC and OB bone cells.

Animals↗
PubMed1979

Induction of metabolic changes and down regulation of bovine parathyroid hormone-responsive adenylate cyclase are dissociable in isolated osteoclastic and osteoblastic bone cells.

Bovine parathyroid hormone (PTH), dibutyryl cAMP, and calcium each induce similar metabolic changes in isolated bone cells. PTH and calcium, but not dibutyryl cAMP, result in desensitization of osteoclastic and osteoblastic bone cells to PTH. In osteoblastic cells, calcium effects are specific for PTH receptor.adenylate cyclase complexes and responsiveness to other hormones is not reduced while in osteoclastic cells, small effects of high calcium on prostaglandin E1- and epinephrine-inducible cAMP accompany the large decreases seen in cAMP response to PTH. The membrane effects of calcium and of PTH appear to be independently regulated as PTH-induced desensitization can be initiated in the absence of calcium. In addition, calcium effects on PTH-sensitive adenylate cyclase follow a different calcium dose-response than PTH-like metabolic changes. These results suggest that the effect of calcium on the membrane is not directly related to its induction of PTH-like metabolic changes. A possible role of calcium as an in vivo regulator of bone cell sensitivity to PTH is discussed.

Adenylyl Cyclases↗