Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development.

We examine clonal murine calvarial MC3T3-E1 cells to determine if they exhibit a developmental sequence similar to osteoblasts in bone tissue, namely, proliferation of undifferentiated osteoblast precursors followed by postmitotic expression of differentiated osteoblast phenotype. During the initial phase of developmental (days 1-9 of culture), MC3T3-E1 cells actively replicate, as evidenced by the high rates of DNA synthesis and progressive increase in cell number, but maintain a fusiform appearance, fail to express alkaline phosphatase, and do not accumulate mineralized extracellular collagenous matrix, consistent with immature osteoblasts. By day 9 the cultures display cuboidal morphology, attain confluence, and undergo growth arrest. Downregulation of replication is associated with expression of osteoblast functions, including production of alkaline phosphatase, processing of procollagens to collagens, and incremental deposition of a collagenous extracellular matrix. Mineralization of extracellular matrix, which begins approximately 16 days after culture, marks the final phase of osteoblast phenotypic development. Expression of alkaline phosphatase and mineralization is time but not density dependent. Type I collagen synthesis and collagen accumulation are uncoupled in the developing osteoblast. Although collagen synthesis and message expression peaks at day 3 in immature cells, extracellular matrix accumulation is minimal. Instead, matrix accumulates maximally after 7 days of culture as collagen biosynthesis is diminishing. Thus, extracellular matrix formation is a function of mature osteoblasts. Ascorbate and beta-glycerol phosphate are both essential for the expression of osteoblast phenotype as assessed by alkaline phosphatase and mineralization of extracellular matrix. Ascorbate does not stimulate type I collagen gene expression in MC3T3-E1 cells, but it is absolutely required for deposition of collagen in the extracellular matrix. Ascorbate also induces alkaline phosphatase activity in mature cells but not in immature cells. beta-glycerol phosphate displays synergistic actions with ascorbate to further stimulate collagen accumulation and alkaline phosphatase activity in postmitotic, differentiated osteoblast-like cells. Mineralization of mature cultures requires the presence of beta-glycerol phosphate. Thus, MC3T3-E1 cells display a time-dependent and sequential expression of osteoblast characteristics analogous to in vivo bone formation. The developmental sequence associated with MC3T3-E1 differentiation should provide a useful model to study the signals that mediate the switch between proliferation and differentiation in bone cells, as well as provide a renewable culture system to examine the molecular mechanism of osteoblast maturation and the formation of bone-like extracellular matrix.     

Three isolation techniques for primary culture of human osteoblast-like cells: A comparison

The culture of osteoblast-like cells of human origin has become an important experimental model in bone biology. We report here a comparison and evaluation of three of the most widely used systems available today: bone marrow stroma cell cultures (BMSC), human osteoblast explant cultures (hOB) and osteoblast explant cultures from collagenase-treated bone (hOB^col). Cultures from 16 bone specimens obtained from various donors were established and their expression of the osteoblast phenotype were then compared in secondary cultures by use of biochemical markers. BMSC had the highest basal and 1,25-dihy-droxyvitaminD₃ (1,25(OH)₂D.))-induced alkaline phosphatase activities in all cell isolations, with levels approximately twice those in explant cultures. Basal osteocalcin secretion was low-to-undetectable in all cell cultures but was detected in 1,25(OH)₂D₃-stimulated cultures. BMSC produced half of the amount of osteocalcin synthesized in explant cultures. The BMSC cultures also synthesized the lowest amounts of type I collagen, whereas collagen type III synthesis did not differ significantly among the various cultures. When secondary cultures were treated with 100 nM dexam-ethasone in the presence of ascorbic acid (50 u.g/mL) and B-glycerophosphate (10 mM), cultures deposited calcium mineral into the cell layer within 2-4 weeks. PTH-induced cAMP formation was detected in only 5 of 15 isolations and no consistent isolation-dependent response pattern was seen. We conclude that BMSC cultures differ significantly from explant cultures obtained from the same bone specimen. However, all cultures represent cells which can differentiate further and induce mineralization of the extracellular matrix in response to osteoinductive drugs.     

Three isolation techniques for primary culture of human osteoblast-like cells: a comparison

The culture of osteoblast-like cells of human origin has become an important experimental model in bone biology. We report here a comparison and evaluation of three of the most widely used systems available today: bone marrow stroma cell cultures (BMSC), human osteoblast explant cultures (hOB) and osteoblast explant cultures from collagenase-treated bone (hOBcol). Cultures from 16 bone specimens obtained from various donors were established and their expression of the osteoblast phenotype were then compared in secondary cultures by use of biochemical markers. BMSC had the highest basal and 1,25-dihydroxyvitaminD3 (1,25(OH)2D3)-induced alkaline phosphatase activities in all cell isolations, with levels approximately twice those in explant cultures. Basal osteocalcin secretion was low-to-undetectable in all cell cultures but was detected in 1,25(OH)2D3-stimulated cultures. BMSC produced half of the amount of osteocalcin synthesized in explant cultures. The BMSC cultures also synthesized the lowest amounts of type I collagen, whereas collagen type III synthesis did not differ significantly among the various cultures. When secondary cultures were treated with 100 nM dexamethasone in the presence of ascorbic acid (50 microg/mL) and beta-glycerophosphate (10 mM), cultures deposited calcium mineral into the cell layer within 2-4 weeks. PTH-induced cAMP formation was detected in only 5 of 15 isolations and no consistent isolation-dependent response pattern was seen. We conclude that BMSC cultures differ significantly from explant cultures obtained from the same bone specimen. However, all cultures represent cells which can differentiate further and induce mineralization of the extracellular matrix in response to osteoinductive drugs.     

The dose-response effects of ethanol on the human fetal osteoblastic cell line.

Alcohol is a risk factor for the development of osteoporosis, especially in men. Chronic alcohol abuse decreases bone mass, which contributes to the increased incidence of fractures. To better understand the mechanism of action of ethanol on bone metabolism, we have studied the dose-response effects of ethanol on conditionally immortalized human fetal osteoblasts (hFOB) in culture. Ethanol treatment had no significant effects on osteoblast number after 1 day or 7 days. Ethanol treatment did not reduce type I collagen protein levels at either time point at any dose but slightly reduced alkaline phosphatase activity after 7 days. The messenger RNA (mRNA) levels for alkaline phosphatase, type I collagen, and osteonectin were unaltered by 24 h of ethanol treatment but a high dose (200 mM) reduced mRNA levels for the two bone matrix proteins after 7 days. Ethanol treatment led to dose-dependent increases in transforming growth factor beta1 (TGF-beta1) mRNA levels and decreases in TGF-beta2 mRNA levels. The concentration of ethanol in the medium decreased with time because of evaporation but there was little degradation caused by metabolism. These results, which show that cultured osteoblasts are less sensitive than osteoblasts in vivo, suggest that the pronounced inhibitory effects of ethanol on bone formation are not caused by direct cell toxicity.     

Human bone cellsin vitro

Summary Human bone cell cultures were established by maintaining collagenase-treated, bone fragments in low Ca⁺⁺ medium. The resulting cell cultures exhibited a high level of alkaline phosphatase activity and produced a significant increase in intracellular cAMP when exposed to the 1–34 fragment of human parathyroid hormone. With continued culture, the cells formed a thick, extracellular matrix that mineralized when cultures were provided daily with normal levels of calcium, fresh ascorbic acid (50 μg/ml) and 10 mM β-glycerol phosphate. Biosynthetically, these cells produced type I collagen (without any type III collagen), and the bone-specific protein, osteonectin. In addition, the cells produced sulfated macromolecules electrophoretically identical to those positively identified as the bone proteoglycan in parallel cultures of fetal bovine bone cells. This technique provides a useful system for the study of osteoblast metabolismin vitro     

Col3.6-HSD2 transgenic mice: A glucocorticoid loss-of-function model spanning early and late osteoblast differentiation

The goal of this study was to characterize the bone phenotype and molecular alterations in Col3.6-HSD2 mice in which a 3.6-kb Col1a1 promoter fragment drives 11ß-HSD2 expression broadly in the osteoblast lineage to reduce glucocorticoid signaling. Serum corticosterone was unchanged in transgenic females exluding a systemic effect of the transgene. Adult transgenic mice showed reduced vertebral trabecular bone volume and reduced femoral and tibial sub-periosteal and sub-endosteal areas as assessed by microCT. In adult female transgenic mice, histomorphometry showed that vertebral bone mass and trabecular number were reduced but that osteoblast and osteoclast numbers and the mineral apposition and bone formation rates were not changed, suggesting a possible developmental defect in the formation of trabeculae. In a small sample of male mice, osteoblast number and percent osteoid surface were increased but the mineral apposition bone formation rates were not changed, indicating subtle sex-specific phenotypic differences in Col3.6-HSD2 bone. Serum from transgenic mice had decreased levels of the C-terminal telopeptide of α1(I) collagen but increased levels of osteocalcin. Transgenic calvarial osteoblast and bone marrow stromal cultures showed decreased alkaline phosphatase and mineral staining, reduced levels of Col1a1, bone sialoprotein and osteocalcin mRNA expression, and decreased cell growth and proliferation. Transgenic bone marrow cultures treated with RANKL and M-CSF showed greater osteoclast formation; however, osteoclast activity as assessed by resorption of a calcium phosphate substrate was decreased in transgenic cultures. Gene profiling of cultured calvarial osteoblasts enriched in the Col3.6-HSD2 transgene showed modest but significant changes in gene expression, particularly in cell cycle and integrin genes. In summary, Col3.6-HSD2 mice showed a low bone mass phenotype, with decreased ex vivo osteogenesis. These data further strengthen the concept that endogenous glucocorticoid signaling is required for optimal bone mass acquisition and highlight the complexities of glucocorticoid signaling in bone cell lineages.     

Interleukin-1 beta is a potent inhibitor of bone formation in vitro

The effect of interleukin-1 beta, the major component of osteoclast-activating factor (OAF), on bone formation by fetal rat osteoblast-rich cells was investigated. An in vitro culture system developed by Ecarot-Charrier et al. (1983) and Bellows et al. (1986) was utilized in which osteoblasts form mineralized nodules which closely resemble woven bone. Continuous exposure of cultures to homogenous IL-1 beta resulted in potent inhibition of mineralized nodule formation, which was half maximal at 0.1 U/ml (7.5 X 10(-13) M). Bone formation may thus be considerably more sensitive to IL-1 beta than is bone resorption (half maximal at 3.8 X 10(-11) M). Inhibition of bone formation occurred when cultures were exposed to IL-1 beta at both early and late time periods and was unaffected by the presence of indomethacin or by the osteoclast inhibitors calcitonin and gamma-interferon. Instead, IL-1 beta exerted multiple inhibitory effects on osteoblast functions, including inhibition of collagen and noncollagen protein synthesis, alkaline phosphatase expression, and cell replication. On a dose response basis, the inhibition of protein synthesis correlated most closely with inhibition of bone formation. IL-1 beta is clearly inhibitory rather than stimulatory for bone formation as assessed in this system and is therefore unlikely to function as a coupling factor linking the processes of bone resorption and bone formation.     

Osteoblastic cells from rat long bone. I. Characterization of their differentiation in culture

We here report the characterization for osteogenic markers alkaline phosphate (AP), osteocalcin, and mineral deposition of osteoblast cultures derived from cells migrating out from seven-day-old rat tibia fragments. The cells outgrown from bone fragments responded to stimulation with PTH with cAMP increase. We show in these cultures a high level of biosynthesis of type III collagen and osteonectin, and report the stimulatory effect that conditioned serum-free media (CM) from these cultures exert on the migration of endothelial cells (EA by 926). The cultures were kept for the initial seven days in a Coon's modified F12 medium, then were switched to a medium containing ascorbic acid and β-glycerophosphate (BGP), and cultured for another 41 days. They showed constitutive and timely restricted osteoblast markers and mineralization. Maximal AP activity occurred in concomitance with cell doubling, then fell to low levels by the time cultures were stationary. ⁴⁵Ca incorporation in the monolayer increased after four weeks of culture, in concomitance with the appearance of unmineralized nodules, and remained high throughout the phase of mineral deposition. Biosynthesis of collagens type I, type III, and type V was detected at all times; secreted newly synthesized collagens decreased overall, relative to total secreted newly synthesized proteins, and on a per cell basis, with progression of the culture, while the ratio of collagen type III/collagen type I increased. Osteonectin was detected by immunohistochemistry and high amounts of osteonectin were synthesized constitutively. Osteocalcin was detected on virtually all cells tested at 21 and 28 days. A preliminary step in neoangiogenesis is the migration of endothelial cells. CMs collected from osteoblast cultures at different times were potent inducers of endothelial cell migration and acted as such in a dose dependent fashion.     

Inhibition of bone matrix apposition by (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP) in the mouse

To elucidate the mechanism of action of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP, formerly APD) on bone metabolism, we have studied the influence of low doses of AHPrBP on bone resorption and formation in the mouse. Thirty-five-day-old mice were given daily injections of 0.16, 1.6, or 16 mumol/kg BW per day of AHPrBP for 10 days. At sacrifice biochemical parameters were measured in serum and bone ash, and histomorphometric parameters of bone formation and resorption were determined on undecalcified sections of caudal vertebrae after double 3H-proline and double tetracycline labelings. Serum calcium and 1,25-dihydroxyvitamin D levels remained normal at all dosage levels. Compared to controls, AHPrBP at doses of 1.6 and 16 mumol/kg per day increased the number of osteoclasts and the number of nuclei per osteoclast but markedly decreased the number of acid phosphatase-stained osteoclasts. Thus, AHPrBP appears to inhibit osteoclastic activity in vivo in part through reduction of acid phosphatase activity. At doses of 1.6 and 16 mumol/kg per day AHPrBP reduced serum alkaline phosphatase and the osteoblastic surface and decreased the endosteal osteoid surface and thickness. Both the matrix apposition rate and the mineral apposition rate were progressively reduced at the endosteal level, although they were not significantly changed at the periosteal level. Greater inhibition of bone resorption than bone formation resulted in increased endosteal bone density and bone mineral content. AHPrBP at a dose of 0.16 mumol/kg per day did not alter either the osteoclastic bone resorption or the mineral and matrix apposition rates.     

Expression of Bone Microsomal Casein Kinase II, Bone Sialoprotein, and Osteopontin During the Repair of Calvarial Defects

The temporal expression of bone microsomal casein kinase II, osteopontin, bone sialoprotein, alkaline phosphatase, and the accumulation of a solid calcium–inorganic orthophosphate mineral phase, have been charted from day 2 to day 21 during the repair of calvarial defects in rats induced by the implantation of decalcified rat bone matrix. Unlike the sequence of events that occur when the same decalcified bone matrix is implanted subcutaneously or intramuscularly, in which cases the first tissue to form in response to the implant is cartilage that subsequently calcifies and is later resorbed and replaced by bone, the repair of cranial defects is quite different. In the latter case, the first cells induced are undifferentiated mesenchymal cells and early fibroblasts followed by osteoblastic direct bone formation. Somewhat later a few small islands of cartilage are formed, widely separated and spatially distinct from the newly formed bone matrix. All of the cartilage and most of the implanted decalcified bone matrix are later resorbed and replaced by new bone by day 21. This in vivo model of the repair of a bone defect by direct bone formation has provided an excellent system to follow specific biochemical and physicochemical events. The total accumulation and rate of accumulation of the mineral and the two noncollagenous phosphoproteins (bone sialoprotein and osteopontin), as well as the activities of alkaline phosphatase, and for the first time either in vivo or in cell culture, the activity of microsomal casein kinase II, the major enzyme that phosphorylates the bone phosphoproteins, have been determined as a function of healing time in vivo. The overall general pattern of accumulation of the phosphoproteins and calcium-phosphate mineral phase and their relationships are similar to those reported in osteoblast cell cultures also monitored as a function of time.     

Influence of irradiation on the osteoinductive potential of demineralized bone matrix

Summary Samples of demineralized bone matrix (DBM) were exposed to graduated doses of radiation (1–15 Megarad) (Mrad) utilizing a linear accelerator and then implanted into the thoracic region of Long-Evans rats. Subcutaneous implantation of DBM into allogenic rats induces endochondral bone. In response to matrix implantation, a cascade of events ensues; mesenchymal cell proliferation on day 3 postimplantation, chondrogenesis on day 7, calcification of the cartilagenous matrix and chondrolysis on day 9, and osteogenesis on day 11 resulting in formation of an ossicle containing active hemopoietic tissue. Bone formation was assessed by measuring alkaline phosphatase activity, the rate of mineralization was determined by measuring⁴⁵Ca incorporation to bone mineral, and⁴⁰Ca content measured the extent of mineralization; acid phosphatase activity was used as a parameter for bone resorption. The dose of radiation (2.5 Mrad) currently used by bone banks for sterilization of bone tissue did not destroy the bone induction properties of DBM. Furthermore, radiation of 3–5 Mrad even enhanced bone induction, insofar as it produced more bone at the same interval of time than was obtained from unirradiated control samples. None of the radiation doses used in these experiments abolished bone induction, although the response induced by matrix irradiated with doses higher than 5 Mrad was delayed.     

Low dose phenytoin is an osteogenic agent in the rat

Long-term use of phenytoin for the treatment of epilepsy has been associated with increased thickness of craniofacial bones. The aim of the present study was to evaluate the possibility that low doses of phenytoin are osteogenic in vivo by measuring the effects of phenytoin administration on serum and bone histomorphometric parameters of bone formation in two rat experiments. In the first experiment, four groups of adult male Sprague-Dawley rats received daily I.P. injections of 0, 5, 50, or 150 mg/kg/day of phenytoin, respectively, for 47 days. Serum alkaline phosphatase (ALP) and osteocalcin were increased by 5 and 50 mg/kg/day phenytoin. The increases in osteocalcin and ALP occurred by day 7 and day 21, respectively. The tibial diaphyseal mineral apposition rate (MAR) at sacrifice (day 48) was significantly increased in rats receiving 5 mg/kg/day phenytoin. At a dose of 150 mg/kg/day, the increase in serum ALP, osteocalcin and MAR was reversed. No significant differences in serum calcium, phosphorus, or 1,25(OH)₂D₃ levels were seen. In a second experiment, three groups of rats received daily I.P. injection of lower doses of phenytoin (i.e., 0, 1, or 5 mg/kg/day, respectively) for 42 days. Phenytoin also did not affect the growth rate or serum calcium, phosphorus, and 25(OH)D₃ levels. Daily injection of 5 mg/kg/day phenytoin significantly increased several measures of bone formation, i.e., serum ALP and osteocalcin, bone ALP, periosteal MAR, and trabecular bone volume. However, rats receiving lower doses of phenytoin (i.e., 1 mg/kg/day) did not show significant increases in the serum bone formation parameters. In contrast, metaphyseal osteoblast surface, osteoblast number, osteoid thickness, surface, and volume were all significantly increased in rats treated in 1 mg/kg/day but not with 5 mg/kg/day phenytoin, suggesting that the tibial diaphysis and metaphysis bone formation parameters might have different dose-dependent responses to phenytoin treatment. Administration of the test doses of phenytoin did not significantly affect the histomorphometric bone resorption parameters. In conclusion, these findings represent the first in vivo evidence that phenytoin at low doses (i.e., between 1 and 5 mg/kg/day) is an osteogenic agent in the rat.     

Differential effects of nicotine and smoke condensate on bone cell metabolic activity

OBJECTIVE: Delayed or impaired healing of skeletal trauma in patients who smoke has been attributed to vascular responses of nicotine absorption and/or a direct effect of nicotine or other smoke components on bone cells. In vivo studies indicate variability in osteosynthetic response to nicotine versus smoke inhalation. We tested the hypothesis that components of cigarette smoke other than nicotine may be responsible for the adverse skeletal effects of smoking. DESIGN: In vitro cultures of MC3T3-E1 osteoblastlike cells were exposed to varying doses of nicotine or condensates of cigarette smoke. Metabolic assays included alkaline phosphatase activity, collagen synthesis, and total protein synthesis as well as cell proliferation. RESULTS: Variations in the degree of response were noted between bone cell preparations. Nicotine elicited a significant dose-dependent stimulation of bone cell metabolism in all studies. This was detected as increases in alkaline phosphatase activity and increases in total protein and collagen synthesis. Responses were noted with nicotine doses as low as 12.5 ng/mL (half the nicotine level circulating in smokers). In one study, maximum stimulation occurred at 250 ng/mL with levels reaching 74% (total protein) and 104% (collagen) greater than control cultures. In a second study, 222% and 627% stimulation of protein and collagen synthesis over controls was noted using 100 ng/mL. Addition of the nicotine receptor antagonist mecamylamine reduced the nicotine stimulation. Preparations of smoke condensate with equivalent nicotine concentrations reduced all indices of metabolic activity. Cell proliferation was stimulated by both nicotine (20-25%) and smoke condensate (38-46%). CONCLUSION: The data suggest that nicotine acts as a direct stimulant of bone cell metabolic activity. Smoke condensate containing equivalent levels of nicotine elicits an inhibitory effect. A probable speculation is that the delay in clinical healing of skeletal trauma in smoking patients may in part be a result of absorption of components of smoke other than nicotine.     

Osteoblast Gene Expression in Rat Long Bones: Effects of Ovariectomy and Dihydrotestosterone on mRNA Levels

The steroid sex hormones exert major effects on bone formation although the molecular events associated with their activity remain unclear. We have investigated the effects of ovariectomy and dihydrotestosterone (DHT) administration to both sham-operated and ovariectomized (ovx) rats on the bone mRNA levels of osteoblast genes. Rats were randomly allocated to either sham or ovariectomy operations and were administered either vehicle or 40 mg/kg body weight DHT by silastic tube implants at the time of operation for 8 weeks, at which time they were killed and total RNA was extracted from the long bones. Northern blot analysis indicated that the mRNA levels of the bone cell genes α1(I) collagen, alkaline phosphatase, osteocalcin, and osteopontin were markedly increased in ovx rats between 6- and 30-fold. DHT administration to ovary-intact, estrogen-sufficient rats increased the mRNA levels of α1(I) collagen, alkaline phosphatase, osteopontin, and osteocalcin between 3- and 9-fold. In contrast, DHT did not alter levels of these mRNA species in ovx rats. The data demonstrate that estrogen deficiency increased mRNA levels of genes expressed during osteoblast development and suggest an interplay between estrogen and androgen action in regulating the expression of a number of bone cell genes. Received: 20 May 1999 / Accepted: 21 January 2000     

Alendronate administration and skeletal response during chronic alcohol intake in the adolescent male rat.

Alendronate is an aminobisphosphonate that inhibits bone resorption in osteoporotic humans and rats but does not induce osteomalacia. Several bisphosphonates, including alendronate, also have direct positive actions on osteoblasts, bone formation, and mineralization. We studied the effects of alendronate on skeletal development in adolescent male rats during chronic alcohol intake. Four groups of age- and weight-matched male Sprague-Dawley rats (35 days of age) were fed the Lieber-DeCarli diet containing 36% of calories as EtOH (E), the EtOH diet plus 60 mg/kg alendronate (EA) every other day intraperitoneally (ip), an isocaloric diet (I), or the isocaloric diet plus 60 mg/kg alendronate (IA) every other day ip. Body weight, femur length, serum levels of osteocalcin (OC), insulin-like growth factor 1 (IGF-1), testosterone, and luteinizing hormone (LH); femur distal metaphyseal and middiaphyseal bone mineral density (BMD) and tibial metaphyseal gene expression for alpha-1-type I collagen (Col I), OC, and bone alkaline phosphatase (AP); and femur strength by four-point bending to failure were measured after 28 days of feeding and alendronate injections. Serum alcohol levels at death were 156 +/- 13 mg/dl (E) and 203 +/- 40 mg/dl (EA). Alendronate given to alcohol-fed rats increased metaphyseal BMD by more than 3-fold over rats fed alcohol alone. Alendronate given to isocaloric pair-fed rats increased metaphyseal BMD by more than 2.5-fold over rats fed the isocaloric diet alone. Cortical BMD was reduced by alcohol but was increased by alendronate. Alcohol consumption reduced serum IGF-1 levels, and alendronate increased IGF-1 levels in alcohol-fed rats. Serum OC, testosterone, and LH were unaffected by alcohol and alendronate. Quantitative dot blot hybridization using rat complementary DNA (cDNA) probes and normalization against 18S subunit ribosomal RNA (rRNA) levels revealed no changes in tibial metaphyseal gene expression for type I collagen, osteocalcin, or alkaline phosphatase. Alcohol significantly reduced the biomechanical properties of the femurs that were partially compensated by alendronate. Chronic alcohol consumption uncouples formation from ongoing resorption, and resorption is inhibited by alendronate. However, alendronate's positive effects on osteoblast-mediated mineralization during chronic alcohol consumption point to the potential use of bisphosphonates in the treatment of decreased bone formation secondary to alcohol-induced diminished osteoblast function.     

ATP-mediated mineralization of MC3T3-E1 osteoblast cultures

While bone is hypomineralized in hypophosphatemia patients and in tissue-nonspecific alkaline phosphatase (Tnsalp)-deficient mice, the extensive mineralization that nevertheless occurs suggests involvement of other phosphatases in providing phosphate ions for mineral deposition. Although the source of phosphate liberated by these phosphatases is unknown, pyrophosphate, ATP, pyridoxal-5'-phosphate (PLP) and phoshoethanolamine (PEA) are likely candidates. In this study, we have induced mineralization of MC3T3-E1 osteoblast cultures using ATP, and have investigated potential phosphatases involved in this mineralization process. MC3T3-E1 osteoblasts were cultured for 12 days and treated either with beta-glycerophosphate (betaGP) or ATP. Matrix and mineral deposition was examined by biochemical, cytochemical, ultrastructural and X-ray microanalytical methods. ATP added at levels of 4-5 mM resulted in mineral deposition similar to that following conventional treatment with betaGP. Collagen levels were similarly normal in ATP-mineralized cultures and transmission electron microscopy and X-ray microanalysis confirmed hydroxyapatite mineral deposition along the collagen fibrils in the ECM. Phosphate release from 4 mM ATP into the medium was rapid and resulted in approximately twice the phosphate levels than after release from 10 mM betaGP. ATP treatment did not affect mineralization by altering the expression of mineral-regulating genes such as Enpp1, Ank, and Mgp, nor phosphatase genes indicating that ATP induces mineralization by serving as a phosphate source for mineral deposition. Levamisole, an inhibitor of TNSALP, completely blocked mineralization in betaGP-treated cultures, but had minor effects on ATP-mediated mineralization, indicating that other phosphatases such as plasma membrane Ca2+ transport ATPase 1 (PMCA1) and transglutaminase 2 (TG2) are contributing to ATP hydrolysis. To examine their involvement in ATP-mediated mineralization, the inhibitors cystamine (TG2 inhibitor) and ortho-vanadate (PMCA inhibitor) were added to the cultures - both inhibitors significantly reduced mineralization whereas suppression of the phosphate release by ortho-vanadate was minor comparing to other two inhibitors. The contribution of PMCA1 to mineralization may occur through pumping of calcium towards calcification sites and TG2 can likely act as an ATPase in the ECM. Unlike the GTPase activity of TG2, its ATPase function was resistant to calcium, demonstrating the potential for participation in ATP hydrolysis and mineral deposition within the ECM at elevated calcium concentrations.