Cell-cell interactions in the osteogenic compartment of bone

The interactions between two different cell populations within the osteogenic compartment have been examined. The proliferation of periosteal fibroblasts (PF) in the presence or absence of osteoblast-like cells (OB), whose proliferative capacity was inhibited by irradiation, was measured. OB stimulated [3H]thymidine incorporation in PF and parathyroid hormone (PTH) enhanced the stimulation. In the reverse situation, however, PF inhibited OB. Irradiated OB also stimulated 3H-thymidine incorporation in OB, and irradiated PF in PF, but both to a lesser extent. Co-culture experiments showed that direct cell-cell contact was a prerequisite for stimulation of PF. Medium mediated contact between physically separated OB and PF did not stimulate, but rather inhibited PF proliferation. These results demonstrate that OB regulate the proliferation of cells in the PF population and can transmit the proliferation stimulating message of PTH to PF. This implies that in vivo the mature osteoblast may play a pivotal role in the (hormonal) regulation of osteoprogenitor cell proliferation and therefore bone formation.     

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

Summary 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.     

Fluid shear stress induces less calcium response in a single primary osteocyte than in a single osteoblast: implication of different focal adhesion formation.

The immediate calcium response to fluid shear stress was compared between osteocytes and osteoblasts on glass using real-time calcium imaging. The osteoblasts were responsive to fluid shear stress of up to 2.4 Pa, whereas the osteocytes were not. The difference in flow-induced calcium may be related to differences in focal adhesion formation. INTRODUCTION: To explore the immediate response to mechanical stress in a bone cell population, we examined flow-induced calcium transients. In addition, the involvement of focal adhesion-related calcium transients in response to fluid flow in the cells was studied. MATERIALS AND METHODS: Bone cells were isolated from 16-day-old embryonic chicken calvaria by serial treatment with EDTA and collagenase. Single cells on glass without intercellular connections were subjected to fluid flow, and intracellular calcium concentration was measured using imaging with fluo-3. The identification of cell populations in the same field was performed with a chick osteocyte-specific antibody, OB7.3, and an alkaline phosphatase substrate, ELF-97, for osteoblast identification afterward. Immunofluorescence staining of vinculin was performed to visualize focal adhesions. RESULTS: The percentage of cells responding to fluid shear stress at 1.2 Pa was 5.5% in osteocytes, 32.4% in osteoblasts, and 45.6% in OB7.3/ELF-97-negative cells. Furthermore, osteoblasts and OB7.3/ELF-97-negative cells were more responsive to 2.4 Pa than 1.2 Pa, whereas osteocytes were less responsive. The elevation of calcium transients over baseline did not show any significant differences in the populations. To elucidate the mechanism accounting for the fact that single osteocytes are less sensitive to fluid shear stress of up to 2.4 Pa than osteoblasts, we studied focal adhesion-related calcium transients. First, we compared focal adhesion formation between osteocytes and osteoblasts and found a larger number of focal adhesions in osteoblasts than in osteocytes. Next, when the cells were pretreated with GRGDS (0.5 mM) before flow treatment, a significant reduction of calcium transients in osteoblasts (18%) was observed, whereas calcium transients in osteocytes were not changed by GRGDS. Control peptide GRGES did not reduce the calcium transients in either cell type. Furthermore, we confirmed that osteoblasts in calvaria showed a marked formation of vinculin plaques in the periphery of the cells. However, osteocytes in calvaria showed faint vinculin plaques only at the base of the processes. CONCLUSIONS: On glass, single osteocytes are less sensitive to fluid shear stress up to 2.4 Pa than osteoblasts. The difference in calcium transients might be related to differences in focal adhesion formation. Shear stress of a higher magnitude or direct deformation may be responsible for the mechanical response of osteocytes in bone.     

Osteoblastlike cells in a serum-free methylcellulose medium form colonies: Effects of insulin and insulinlike growth factor I

Summary Osteoblastlike (OB) cells obtained from a heterogeneous primary cell population by enzymatic cell digestion of calvaria of newborn rats are grown in a serum-free viscous α-MEM/F-12 medium containing 0.8% methylcellulose. In contrast to cell monolayers in conventional tissue cultures OB cells proliferate into colonies of rounded-up cells to form morulalike spherical cell clusters containing up to 100 cells. These colonies, with different cell numbers, are clearly not fibroblastlike since fibroblasts from the same rats always grow as a cell monolayer. Alkaline phosphatase activity and cAMP responsivness to PTH are expressed more markedly (70% and 250% respectively) by OB cells in the described culture system than in conventional tissue cultures. Rounded-up OB cells sediment and colonies stick to the dish; proliferation of OB cells is favored and starts 3–4 days after inoculation. Increasing concentrations of insulinlike growth factor (IGF) I (0.4–35 nM) and insulin (20–660 nM), as well as increasing initial cell density, enhances mitogenic activity of these cells in a dose-dependent way. On a molar ratio IGF I (physiological concentrations) is 10 times as potent as insulin (pharmacological concentrations) with respect to proliferation. If less than 10⁵ cells/ml are inoculated, there exists an apparent relationship between initial cell density and major onset of replication, indicating the presence and accumulation of local growth factors. For OB cells, the described culture system (1) comes closer to thein vivo situation (2) leads to a clear morphological difference between OB cells and fibroblasts (3) provides an excellent system to study hormone actions on OB cell proliferation, and (4) allows inoculation at a wide variety of initial cell densities.     

Cell autonomous requirement of connexin 43 for osteocyte survival: consequences for endocortical resorption and periosteal bone formation

Connexin 43 (Cx43) mediates osteocyte communication with other cells and with the extracellular milieu and regulates osteoblastic cell signaling and gene expression. We now report that mice lacking Cx43 in osteoblasts/osteocytes or only in osteocytes (Cx43(ΔOt) mice) exhibit increased osteocyte apoptosis, endocortical resorption, and periosteal bone formation, resulting in higher marrow cavity and total tissue areas measured at the femoral mid-diaphysis. Blockade of resorption reversed the increased marrow cavity but not total tissue area, demonstrating that endocortical resorption and periosteal apposition are independently regulated. Anatomical mapping of apoptotic osteocytes, osteocytic protein expression, and resorption and formation suggests that Cx43 controls osteoclast and osteoblast activity by regulating osteoprotegerin and sclerostin levels, respectively, in osteocytes located in specific areas of the cortex. Whereas empty lacunae and living osteocytes lacking osteoprotegerin were distributed throughout cortical bone in Cx43(ΔOt) mice, apoptotic osteocytes were preferentially located in areas containing osteoclasts, suggesting that osteoclast recruitment requires active signaling from dying osteocytes. Furthermore, Cx43 deletion in cultured osteocytic cells resulted in increased apoptosis and decreased osteoprotegerin expression. Thus, Cx43 is essential in a cell-autonomous fashion in vivo and in vitro for osteocyte survival and for controlling the expression of osteocytic genes that affect osteoclast and osteoblast function.     

Intermittent PTH treatment can delay the transformation of mature osteoblasts into lining cells on the periosteal surfaces

Mature osteoblasts have three fates: as osteocytes, quiescent lining cells, or osteoblasts that undergo apoptosis. However, whether intermittent parathyroid hormone (PTH) can modulate the fate of mature osteoblasts in vivo is uncertain. We performed a lineage-tracing study using an inducible gene system. Dmp1-CreERt2 mice were crossed with Rosa26R reporter mice to obtain targeted mature osteoblasts and their descendants, lining cells or osteocytes, which were detected using X-gal staining. Rosa26R:Dmp1-CreERt2(+) mice were injected with 0.25 mg 4-OH-tamoxifen (4-OHTam) on postnatal days 5, 7, 9, 16, and 23. In a previous study, at 22 days after the last 4-OHTam, most LacZ+ cells on the periosteal surface were inactive lining cells. On day 25 (D25), the mice were challenged with an injection of human PTH (1–34, 80 μg/kg) or vehicle daily for 10 (D36) or 20 days (D46). We evaluated the number and thickness of LacZ+ osteoblast descendants in the calvaria and tibia. In the vehicle group, the number and thickness of LacZ+ osteoblast descendants at both D36 and D46 significantly decreased compared to D25, which was attenuated in the PTH group. In line with these results, PTH inhibited the decrease in the number of LacZ+/osteocalcin-positive cells compared to vehicle at both D36 and D46. As well, the serum levels of sclerostin decreased, as did the protein expression of sclerostin in the cortical bone. These results suggest that intermittent PTH treatment can increase the number of periosteal osteoblasts by preventing mature osteoblasts from transforming into lining cells in vivo.     

Differential serum dependence of cultured osteoclastic and osteoblastic bone cells

Summary Sequential collagenase digestion of mice calvariae provides populations of bone cells that express either osteoclastic (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.     

Intermittent parathyroid hormone administration converts quiescent lining cells to active osteoblasts

Intermittent administration of parathyroid hormone (PTH) increases bone mass, at least in part, by increasing the number of osteoblasts. One possible source of osteoblasts might be conversion of inactive lining cells to osteoblasts, and indirect evidence is consistent with this hypothesis. To better understand the possible effect of PTH on lining cell activation, a lineage tracing study was conducted using an inducible gene system. Dmp1‐CreERt2 mice were crossed with ROSA26R reporter mice to render targeted mature osteoblasts and their descendents, lining cells and osteocytes, detectable by 5‐bromo‐4‐chloro‐3‐indolyl‐β‐d‐galactopyranoside (X‐gal) staining. Dmp1‐CreERt2(+):ROSA26R mice were injected with 0.25 mg 4‐OH‐tamoxifen (4‐OHTam) on postnatal days 3, 5, 7, 14, and 21. The animals were euthanized on postnatal day 23, 33, or 43 (2, 12, or 22 days after the last 4‐OHTam injection). On day 43, mice were challenged with a subcutaneous injection of human PTH (1–34, 80 µg/kg) or vehicle once daily for 3 days. By 22 days after the last 4‐OHTam injection, most X‐gal (+) cells on the periosteal surfaces of the calvaria and the tibia were flat. Moreover, bone formation rate and collagen I(α1) mRNA expression were decreased at day 43 compared to day 23. After 3 days of PTH injections, the thickness of X‐gal (+) cells increased, as did their expression of osteocalcin and collagen I(α1) mRNA. Electron microscopy revealed X‐gal–associated chromogen particles in thin cells prior to PTH administration and in cuboidal cells following PTH administration. These data support the hypothesis that intermittent PTH treatment can increase osteoblast number by converting lining cells to mature osteoblasts in vivo. © 2012 American Society for Bone and Mineral Research.     

Mineralization and metabolic response in serially passaged adult rat bone cells

Summary Cell populations derived from adult rat bone were grown in cell culture and characterized with respect to their morphology and response to hormones. The cells were isolated from adult rat calvaria by mechanical rather than enzymatic methods. Cultures were initiated in modified BGJb medium supplemented with fetal bovine serum. These cultures and several cloned populations derived from them retained the ability to mineralize in vitro even after extended serial passage. Cultures derived from an osteoblast-enriched population showed an initial positive cAMP response to PTH and PGE₂, but not to TCT. The PTH and PGE₂ responses diminished with serial passage. The PTH response was no longer measurable at passage 6, and the PGE₂ response was not evident in passage 11. In one clone, the PGE₂ response persisted through passage 16. Adult rat skin fibroblasts cultured similarly did not respond to PTH or TCT, but still had a significant PGE₂ response through passage 21. The cultured cells formed multiple layers with localized areas of higher cell density. Mineral plaques with major diameters as great as 0.75 mm were evident in the areas of greater cell density. Less extensive mineral deposits were present throughout the culture. The mineral plaques consisted of apatite-like crystals deposited on an organic matrix. Matrix vesicles and mineralized spherules appeared to be associated with initial mineral deposition. The spherules apparently coalesced to form more complex mineralized structures. A limited amount of mineralization also was observed in rat skin fibroblast cultures.     

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

Summary 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.     

Effects of PTH on osteocyte function

Osteocytes are ideally positioned to detect and respond to mechanical and hormonal stimuli and to coordinate the function of osteoblasts and osteoclasts. However, evidence supporting the involvement of osteocytes in specific aspects of skeletal biology has been limited mainly due to the lack of suitable experimental approaches. Few crucial advances in the field in the past several years have markedly increased our understanding of the function of osteocytes. The development of osteocytic cell lines initiated a plethora of in vitro studies that have provided insights into the unique biology of osteocytes and continue to generate novel hypotheses. Genetic approaches using promoter fragments that direct gene expression to osteocytes allowed the generation of mice with gain or loss of function of particular genes revealing their role in osteocyte function. Furthermore, evidence that Sost/sclerostin is expressed primarily in osteocytes and inhibits bone formation by osteoblasts, fueled research attempting to identify regulators of this gene as well as other osteocyte products that impact the function of osteoblasts and osteoclasts. The discovery that parathyroid hormone (PTH), a central regulator of bone homeostasis, inhibits sclerostin expression generated a cascade of studies that revealed that osteocytes are crucial target cells of the actions of PTH. This review highlights these investigations and discusses their significance for advancing our understanding of the mechanisms by which osteocytes regulate bone homeostasis and for developing therapies for bone diseases targeting osteocytes. This article is part of a Special Issue entitled "The Osteocyte".     

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.     

Modulation of PTH-stimulated cyclic AMP in cultured rodent bone cells: The effects of 1,25(OH)2 vitamin D3 and its interaction with glucocorticoids

Summary Parathyroid hormone (PTH)-stimulated cyclic adenosine monophosphate (cAMP) in rat osteoblastlike (OB) cells has been shown to be modulated by steroid hormones; glucocorticoids are known to increase the level, while the effects of 1,25(OH)₂D₃ are inhibitory. In the present study, we found that the PTH-stimulated cAMP responses are similar in neonatal mouse and fetal rat OB cells. Dexamethasone (0.13–13nM) augmented PTH-stimulated cAMP in both species. Mouse cells showed a higher maximal response to dexamethasone (100% increment) than rat cells (60–70% increment) with similar sensitivity to dexamethasone (ED₅₀ ∼ 1.0 nm). On the other hand, 1,25(OH)₂D₃ decreased PTH-stimulated cAMP, but the effect required pharmacological levels of hormone; mouse cells responded at a lower dose (1.3 nM) and were more sensitive than rat cells (responded at 13 nM) to 1,25(OH)₂D₃ treatment. Introduction of physiological concentrations of 1,25(OH)₂D₃ (0.013–1.3 nm) in addition to dexamethasone (13 nM) resulted in a synergistic enhancement of PTH-stimulated cAMP in rat cells. In contrast, a dose-dependent antagonistic effect was observed in mouse cells. In summary, our findings demonstrate species and concentration-dependent differences in hormonal responses to 1,25(OH)₂D₃ and a complex interplay among PTH, dexamethasone, and 1,25(OH)₂D₃.     

The cell biology of parathyroid hormone in osteoblasts

Continuous exposure to parathyroid hormone (PTH) is associated with catabolic effects, whereas intermittent exposure to low doses of PTH is associated with anabolic effects. By controlling osteoblast function, PTH increases bone formation on cancellous, endocortical, and periosteal bone surfaces. In general, PTH does not affect the replication of uncommitted osteoblast progenitors but suppresses proliferation of committed osteoprogenitors. Intermittent PTH promotes osteoblast differentiation, in part, by its ability to promote exit from the cell cycle, to activate Wnt signaling in osteoblasts, and to inhibit the Wnt antagonist sclerostin in osteocytes. Insulin-like growth factor-1 is also required for the actions of PTH to increase osteoblast numbers. Intermittent PTH prolongs osteoblast survival in rodents by mechanisms that involve activation and proteolytic degradation of Runx2. PTH’s ability to orchestrate a dynamic range of signaling cascades that determine osteoblast fate may explain both its catabolic and beneficial actions on the skeleton.     

Expression of parathyroid hormone-related peptide and insulin-like growth factor I during rat fracture healing.

Parathyroid hormone-related peptide (PTHrP) and insulin-like growth factor I (IGF-I) are both involved in the regulation of bone and cartilage metabolisms and their interaction has been reported in osteoblasts. To investigate the interaction of PTHrP and IGF-I during fracture healing, the expression of mRNA for PTHrP and IGF-I, and receptors for PTH/PTHrP and IGF were examined during rat femoral fracture healing using an in situ hybridization method and an immunohistochemistry method, respectively. During intramembranous ossification, PTHrP mRNA, IGF-I mRNA and IGF receptors were detected in preosteoblasts, differentiated osteoblasts and osteocytes in the newly formed trabecular bone. PTH/PTHrP receptors were markedly detected in osteoblasts and osteocytes, but only barely so in preosteoblasts. During cartilaginous callus formation, PTHrP mRNA was expressed by mesenchymal cells and proliferating chondrocytes. PTH/PTHrP receptors were detected in proliferating chondrocytes and early hypertrophic chondrocytes. IGF-I mRNA and IGF receptor were co-expressed by mesenchymal cells, proliferating chondrocytes, and early hypertrophic chondrocytes. At the endochondral ossification front, osteoblasts were positive for PTHrP and IGF-I mRNA as well as their receptors. These results suggest that IGF-I is involved in cell proliferation or differentiation in mesenchymal cells, periosteal cells, osteoblasts and chondrocytes in an autocrine and/or paracrine fashion. Furthermore, PTHrP may be involved in primary callus formation presumably co-operating with IGF-I in osteoblasts and osteocytes, and by regulating chondrocyte differentiation in endochondral ossification.     

Osteocyte and bone structure

The osteocyte is the most abundant cell type of bone. There are approximately 10 times as many osteocytes as osteoblasts in adult human bone, and the number of osteoclasts is only a fraction of the number of osteoblasts. Our current knowledge of the role of osteocytes in bone metabolism is far behind our insight into the properties and functions of the osteoblasts and osteoclasts. However, the striking structural design of bone predicts an important role for osteocytes in determining bone structure. Over the past several years, the role of osteocytes as the professional mechanosensory cells of bone, and the lacunocanalicular porosity as the structure that mediates mechanosensing have become clear. Strain-derived flow of interstitial fluid through this porosity seems to mechanically activate the osteocytes, as well as ensure transport of cell signaling molecules, nutrients, and waste products. This concept explains local bone gain and loss—as well as remodeling in response to fatigue damage—as processes supervised by mechanosensitive osteocytes. Alignment during remodeling seems to occur as a result of the osteocyte’s sensing different canalicular flow patterns around the cutting cone and reversal zone during loading, therefore determining the bone’s structure.     

Effects of vitamin D and parathyroid hormone on cyclic AMP production by bone cells isolated from rat calvariae

Summary Studies presented here were designed to investigate further the basis for an impaired cAMP response to parathyroid hormone (PTH) in osteoblastlike calvarial bone cells isolated from vitamin D-deficient rat pups. The goal was to perturb Ca, PTH, and vitamin Din vivo in order to see which factors might be responsible for the impairedin vitro bone cell cAMP response. Pups either were parathyroidectomized (PTX) 3–5 days, implanted with osmotic minipumps delivering high doses of PTH, given repeated, high doses of 1,25(OH)₂D₃, or were D-deficient (-D, i.e., born and suckled by D-deficient mothers). Osteoblastlike bone cells, isolated by sequential enzyme digestion and centrifugation, were exposed to PTH for 5 min in the presence of a phosphodiesterase inhibitor. In bone cells isolated from -D rat pups, both basal and PTH-induced cAMP accumulation were significantly lower than in +D bone cells. Earlier, we had shown that two daily injections of -D pups with 50 ng 1,25(OH)₂D₃ restores this reduced bone cAMP response of -D pups toward normal. In the present study, neither basal nor PTH-induced bone cell cAMP accumulation was affected by subjecting D-replete pups to PTX, PTH infusion, or repeated high doses of 1,25(OH)₂D₃ despite the fact that each treatment markedly changed serum Ca or serum immunoreactive PTH. The results indicate that the impaired bone cell cAMP response seen in -D pups is not a direct result of chronic hypocalcemia and that the “heterologous desensitization” seenin vitro with added 1,25(OH)₂D₃ could not be duplicated byin vivo treatment of +D pups with supraphysiologic doses of 1,25(OH)₂D₃. Finally the lack of alteration in the bone cell cAMP response to PTHin vitro after chronic PTH infusionin vivo fails to support the notion that the impaired response in -D bone cells can be explained entirely by “homologous desensitization” induced by high circulating levels of PTH in the hypocalcemic, -D rat pup.     

Differential response of bone cells isolated by sequential digestion to dichloromethylenebisphonate in culture

Summary Dichloromethylenebisphosphonate (Cl₂MBP), a compound structurally related to inorganic pyrophosphate but resistant to hydrolysis of endogenous phosphatase to yield inorganic phosphate, inhibits bone resorption and soft tissue mineralizationin vivo. Previously, we have shown that bone cells isolated from rat calvaria respond profoundly to the exposure of Cl₂MBP. To determine whether the cellular effects evoked by Cl₂MBP are confined to a particular bone cell type, calvaria from 1 day postnatal rats were subjected to a sequential time-dependent enzyme digestion, yielding five bone cell populations marked by differences in PTH response, alkaline phosphatase activity and collagen, as well as hyaluronic acid synthesis. Culturing these bone cell populations with Cl₂MBP revealed that previously observed results found with mixed bone cells (inhibition of cell proliferation, diminution of hyaluronic acid synthesis, and increase in alkaline phosphatase) were limited to cell populations which, according to the isolation scheme, stem from the outer tissue layer(s) of the calvaria. Collagen synthesis, however, was found to be equally increased regardless of cell type. These present results indicate that the action of Cl₂MBP on bone may be cell specific.