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Biomedical subjects

I M Shapiro

Publications and source records attributed to I M Shapiro.

At least 19 recordsLinked to original sources
PubMed2007

Expression of HIF prolyl hydroxylase isozymes in growth plate chondrocytes: relationship between maturation and apoptotic sensitivity.

The overall goal of the current study was to examine the functional activity of the prolyl hydroxylases (PHDs) in maturing chondrocytes. Herein, we show for the first time that the PHDs are expressed in the maturing zone of the growth plate, and by a chondrocytic cell line. We determined if this protein and its substrate, hypoxia inducible factor (HIF)-1alpha, modulated the induction of apoptosis. Using a chondrocyte cell line that matured in culture, we inhibited HIF-1alpha expression using siRNA technology and pharmacologically blocked PHD activity. We noted that PHD suppression sensitized the cells to an apoptotic challenge with H(2)O(2). We next examined the interplay between the PHDs and HIF-1alpha by suppressing HIF-1alpha and blocking PHD activity. We noted reduced killing when the mature HIF-silenced cells were challenged with H(2)O(2). In contrast, there was limited change in the viability of immature cells. Based on these differences in chondrocyte susceptibility, it is concluded that HIF-1alpha sensitizes maturing cells to H(2)O(2)-mediated killing. We next determined if this change in the viability of the PHD-inhibited cells was linked to changes in activation of caspase-3. It was noted that there was a minimal change in enzyme activity of the PHD-inhibited HIF-1alpha suppressed cells. Finally, we found that as the chondrocytes mature, the activities of catalase and SOD were significantly reduced and that there was a decrease in the levels of Bcl-2 and Bcl(XL). This loss of protective activity together with the changes mediated by HIF would be expected to generate conditions that would favor the induction of chondrocyte apoptosis.

Amino Acids, Dicarboxylic↗
PubMed2005

Nucleation and growth of calcium phosphate on amine-, carboxyl- and hydroxyl-silane self-assembled monolayers.

Upon implantation, calcium phosphate (Ca-P) surfaces form on materials that are bone bioactive. In this study, the evolving surface characteristics associated with calcium phosphate precipitation are modeled using self-assembled monolayers (SAMs), in a one-step nucleation process. SAMs were used to create amine (-NH2), carboxyl (-COOH) and hydroxyl (-OH) functionalized surfaces by grafting 3-aminopropyltriethoxysilane, 3-triethoxysilylpropyl succinic anhydride and glycidoxypropyl tri-methoxysilane, respectively, onto oxidized silicon wafers. The SAM surfaces were characterized using ellipsometry to establish the presence of grafted molecules. On the surfaces incubated in simulated physiological fluids for 7 days, the thickness of Ca-P layer grew slowly over the first few hours, increasing strongly between 1 and 5 days and then slowed down again. FTIR showed the dependence of calcium phosphate morphology on the type of surface groups, with stronger P-O bands seen on the OH-terminated surface. SEM analysis showed dispersed Ca-P precipitates on the -COOH and -OH terminated surfaces after 1 day immersion. After 7 days, all SAM surfaces were covered with uniformly dispersed and denser Ca-P precipitates. The underlying Ca-P layer showed cracks on the -NH2-terminated surface. Rutherford backscattering spectrometry (RBS) data analysis confirmed that Ca/P ratio is in excellent agreement with the theoretical value of 1.67 for hydroxyapatite. X-ray diffraction (XRD) analysis also showed evidence of apatite formation on all the surfaces, with stronger evidence on the -OH-terminated surface. Highly porous Ca-P precipitates were observed on the SAM surfaces portrayed by the AFM scans with nanoscale RMS roughness. Thus, using highly controlled surface chemistry, under physiological conditions, in vitro, this study demonstrates that a hydroxylated surface enhances Ca-P nucleation and growth relative to other surfaces, thereby supporting the concept of its beneficial effect on bone tissue formation and growth.

Amines↗
PubMed2005

Stem cells in craniofacial and dental tissue engineering.

Mesenchymal stem cells (MSC) have been identified in a variety of adult tissues as a population of pluripotential self-renewing cells. Based on their adherence and colony forming properties, a small number of MSC can be isolated from most mesenchymal tissues as well as bone marrow. In the presence of one or more growth factors, these cells commit to lineages that lead to the formation of bone, cartilage, muscle, tendon and adipose tissue; recent studies indicate that stem cells for cementum, dentine and the periodontal ligament also exist. All of these cells can be expanded in vitro, and, embedded in a scaffold, inserted into defects to promote healing and tissue replacement. Increased understanding of the molecular mechanism directing lineage specification and morphogenesis is providing a rational approach for the regeneration of craniofacial tissues and oral structures.

Adult↗
PubMed2004

Model surfaces engineered with nanoscale roughness and RGD tripeptides promote osteoblast activity.

Cell adhesion to biomaterials is a prerequisite for tissue integration with the implant surface. Herein, we show that we can generate a model silica surface that contains a minimal-length arginine-glycine-aspartic acid (RGD) peptide that maintains its biological activity. In the first part of this study, attachment of MC3T3-E1 osteoblast-like cells was investigated on silicon oxide, amine terminated substrates [i.e., 3-aminopropyl triethoxysilane (APTS)], grafted RGD, and physisorbed RGD control. The APTS layer exhibited nanoscale roughness and presented amine functional groups for grafting a minimal RGD tripeptide devoid of any flanking groups or spacers. Contact angle measurements indicated that the hydrophobicity of the APTS surface was significantly lower than that of the surface with grafted RGD (RGD-APTS). Atomic force microscopy showed that surfaces covered with RGD-APTS were smoother (Ra = 0.71 nm) than those covered with APTS alone (Ra = 1.59 nm). Focusing mainly on cell morphology, experiments showed that the RGD-APTS hybrid provided an optimum surface for cell adhesion, spreading, and cytoskeletal organization. Discrete focal adhesion plaques were also observed consistent with successful cell signaling events. In a second set of experiments, smooth, monolayers of APTS (Ra = 0.1 nm) were used to prepare arginine-glycine-aspartic acid-serine (RGDS)-APTS and arginine-glycine-glutamic acid-serine (RGES)-APTS (control) substrates. Focusing mainly on cell function, integrin and gene expression were all enhanced for rate osteosarcoma cells on surfaces containing grafted RGDS. Both sets of studies demonstrated that grafted molecules of RGD(S) enhance both osteoblast-like cell adhesion and function.

Animals↗
PubMed2004

Ionizing radiation sensitizes bone cells to apoptosis.

Osteoradionecrosis is a common sequelae of radiation therapy for head and neck cancer. To test the hypothesis that radiation induces osteoradionecrosis by induction of bone cell apoptosis, we exposed MC3T3-E1 osteoblast-like cells to gamma-radiation and evaluated cell viability. Twenty-four hours postirradiation, measurement of osteoblast dehydrogenase activity suggested that there was a small decrease in cell viability. However, TUNEL and flow cytometric analysis indicated that the viability loss was caused by inhibition of cell proliferation and not by induction of apoptosis. The effect of irradiation on osteoblast function was examined by Western blot and flow cytometric analysis. It was found that irradiated osteoblasts underwent G2 cell cycle arrest. In addition, we observed changes in expression of molecules that regulate the cell cycle. Thus, there was an increase in p53 transcription, a raised level of MDM2 dephosphorylation, and elevation in p21 and GADD153 protein levels. Since these proteins are concerned with the regulation of the cell cycle, the observed changes in expression would be expected to disturb cyclin activity and cause G2M arrest. The arrested cells displayed a dramatic increase in sensitivity to specific apoptogens. Thus, when irradiated, and then treated with Ca2+Pi or staurosporine, agents that cause mitochondrial dysfunction, more osteoblasts underwent apoptosis than with the apoptogen alone. In contrast, irradiated cells treated with anti-Fas antibody showed no change in apoptotic sensitivity; apoptosis was inhibited when osteoblasts were treated with etoposide. Similar alterations in sensitivity were observed when cells were arrested in G2/M by pretreatment with colchicine and then challenged with apoptogens. It was concluded that activation of radiation-induced G2 arrest sensitizes osteoblasts to agents that mediate apoptosis through a mitochondrial-dependent death pathway.

Animals↗
PubMed2004

Bone cell survival in microgravity: evidence that modeled microgravity increases osteoblast sensitivity to apoptogens.

Studies were performed to evaluate the effects of modeled microgravity on the induction of osteoblast apoptosis. MC3T3-E1 osteoblast-like cells were cultured in alginate carriers in the NASA-approved high aspect ratio vessel (HARV). This system subjects the cells to a time-averaged gravitational field (vector-averaged gravity) to simulate low gravity conditions. Cells were cultured in the HARV for five days, and then examined for apoptosis. In simulated microgravity, the cells remained vital, although analysis of expressed genes indicated that there was loss of the mature osteoblast phenotype. Additionally, we noted that there was a loss of the mitochondrial membrane potential, a low level of the antiapoptotic protein Bcl-2, as well as Akt protein, and the redox status of the cells was disturbed. All of these parameters indicated that vector-averaged gravity disrupts mitochondrial function, thereby sensitizing osteoblasts to apoptosis. We then used a challenge assay to evaluate the apoptotic sensitivity of the cells subjected to vector-averaged gravity. When challenged with staurosporine, cells subjected to vector-averaged gravity evidenced elevated levels of cell death relative to control cell populations. Another objective of the study was to improve upon conventional carriers by using alginate encapsulation to support cells in the HARV. We have demonstrated that the alginate carrier system affords a more robust system than surface-seeded carriers. This new system has the advantage of shielding cells from mechanical damage and fluid shear stresses on cells in the HARV, permitting carefully controlled studies of the effects of vector-averaged gravity.

3T3 Cells↗
PubMed2003

Induction of apoptosis in skeletal tissues: phosphate-mediated chick chondrocyte apoptosis is calcium dependent.

In an earlier study, we have shown that Pi induced apoptosis of terminally differentiated hypertrophic chondrocytes. To ascertain whether Ca2+ modulates Pi-induced cell death, we asked the following two questions: First, can we prevent Pi-induced apoptosis by removing Ca2+ from the culture medium; alternatively, can we potentiate cell death by increasing the Ca2+ concentration? Second, can we inhibit chondrocyte apoptosis by blocking Pi transport? We also explored the mechanism of apoptosis by evaluating mitochondrial activity and reactive oxygen species (ROS) generation in cells treated with the ion pair. We noted that EDTA and EGTA blocked Pi-induced apoptosis in a dose-dependent manner. While high levels of Ca2+ alone had little effect on chondrocyte viability, the cation enhanced Pi-dependent cell death and greatly increased Pi uptake. When Pi transport was blocked, there was complete inhibition of cell killing. The process of cell death was characterized by mitochondrial hyperpolarization; two hours following apoptogen treatment, there was a significant decrease in the mitochondrial membrane potential. Coincident with the changes in mitochondrial function, there was an increase in intracellular Ca2+ that was maintained throughout the experimental period. A raised Ca2+ signal was observed in blebs at the cell membrane. Finally, we noted that, 75 minutes after treatment with the ion pair, there was a six-fold elevation in ROS levels. This increase declined to baseline values after three hours. Based on these observations, we suggest that, at the cartilage mineralization front, an elevation in local environmental Ca2+ and Pi concentrations modulates oxidative metabolism, and triggers apoptosis of terminally differentiated chondrocytes.

Animals↗
PubMed2003

Mechanisms by which extracellular matrix components induce osteoblast apoptosis.

Bone cell apoptosis is seen at sites of active turnover. We hypothesize that at these sites, factors released from resorbing bone induce apoptosis of vicinal cells. Related to this observation, earlier studies indicate that an elevation in the level of inorganic phosphate ions combined with a modest increase in the calcium (Ca2+) concentration, or a rise in the local concentration of RGD-containing peptides promote osteoblast apoptosis. The aim of the current investigation is to elucidate the mechanism by which these extracellular matrix components induce bone cell apoptosis. The data presented in this study clearly demonstrate that osteoblasts are sensitive to peptide fragments and solubilized mineral ions. It is reasonable to expect that these apoptogens would be generated by osteoclasts during resorption of the extracellular bone matrix. We suggest that these components conspire to regulate bone cell function. In terms of the mechanism by which these agents activate apoptosis, it is clear that while they share common pathways, there are some differences in the mechanism of apoptosis. These differences appear to be upstream of caspase activation. The observation that two such pathways exist lends strength to the notion that apoptosis is carefully regulated in bone and that signals from both matrix components act together to trigger the remodeling process.

Animals↗
PubMed2002

RGD peptides immobilized on a mechanically deformable surface promote osteoblast differentiation.

The major objective of this work was to attach bone cells to a deformable surface for the effective transmission of force. We functionalized a silastic membrane and treated it with 3-aminopropyltriethoxysilane (APTS). A minimal RGD peptide was then covalently linked to the aminated surface. MC3T3-E1 osteoblast-like cells were cultured on the arginine-glycine-aspartic acid (RGD)-treated membrane for 3-15 days and cell attachment and proliferation was evaluated. We observed that cells were immediately bound to the membrane and proliferated. After 8 days on the material surface, osteoblasts exhibited high levels of ALP staining, indicating that the cells were undergoing maturation. Alizarin red staining and Fourier transform infrared (FTIR) analysis showed that the mineral formed by the cells was a biological apatite. The second objective was to apply a mechanical force to cells cultured on the modified silicone membrane. Dynamic equibiaxial strain, 2% magnitude, and a 0.25-Hz frequency were applied to bone cells for 2 h. Osteoblasts elicited increased phalloidin fluorescence, suggesting that there was reorganization of the cytoskeleton. Furthermore, the applied strain elicited increased expression of the alpha(v)beta3 integrin receptor. We concluded that the covalent binding of RGD peptides to a silicone membrane provides a compatible surface for the attachment and subsequent differentiation of osteoblasts. Moreover, the engineered surface transduces applied mechanical forces directly to the adherent cells via integrin receptors.

3T3 Cells↗
PubMed2001

Matrix regulation of skeletal cell apoptosis. Role of calcium and phosphate ions.

Previously, we noted that inorganic phosphate (P(i)), a major component of bone extracellular matrix, induced osteoblast apoptosis (Meleti, Z., Shapiro, I. M., and Adams, C. S. (2000) Bone (NY) 27, 359-366). Since Ca(2+) along with P(i) is released from bone during the resorption process, we advanced the hypothesis that Ca(2+) modulates P(i)-mediated osteoblast apoptosis. To test this hypothesis, osteoblasts were incubated with both ions, and cell death was determined. We noted that a modest increase in the medium Ca(2+) concentrations ([Ca(2+)](e)) of 0.1-1 mm caused a profound and rapid enhancement in P(i)-dependent death of cultured osteoblasts. An elevation in [Ca(2+)](e) alone had no effect on osteoblast viability, whereas Ca(2+) channel blockers failed to inhibit killing of ion pair-treated cells. These results indicated that P(i)-mediated cell death is not dependent on a sustained increase in the cytosolic Ca(2+) concentration. Terminal dUTP nick-end labeling analysis and measurement of caspase-3 activity of the ion pair-treated cells suggested that death was apoptotic. Apoptosis was confirmed using caspase-3 and endonuclease inhibitors. The mitochondrial membrane potential and cytosolic Ca(2+) status of the treated cells were evaluated. After incubation with [Ca(2+) ](e) and P(i), a decrease in mitochondrial fluorescence was noted, suggesting that the ions decreased the mitochondrial transmembrane potential. Subsequent to the fall in mitochondrial membrane potential, there was a transient elevation in the cytosolic Ca(2+) concentration. Results of the study suggest that the ion pair conspire at the level of the plasma membrane to induce intracellular changes that result in loss of mitochondrial function. The subsequent increase in the cytosolic Ca(2+) concentration may trigger downstream events that transduce osteoblast apoptosis.

Apoptosis↗
PubMed2001

Phosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism.

In a previous investigation we showed that phosphate ions (Pi) induced apoptosis of terminally differentiated hypertrophic chondrocytes. To explore the mechanism by which Pi induces cell death, we asked the following two questions. First, can we prevent Pi-induced apoptosis by inhibiting plasma membrane Na-Pi cotransport? Second, which specific Na-Pi transporters are expressed in chondrocytes and are they developmentally regulated? Terminally differentiated hypertrophic chondrocytes were isolated from chick tibial cartilage and cell death was measured in the presence of 3-7 mmol/L Pi. To ascertain whether apoptosis was linked to a rise in cellular Pi loading, we examined the effect of phosphonoformic acid (PFA), a competitive inhibitor of Na-Pi cotransport on Pi-induced apoptosis in chondrocytes. We found that 1 mmol/L PFA blocked anion-induced cell death and prevented an increase in the cell Pi content. In a parallel study, we determined that the bisphosphonate, alendronate, also protected chondrocytes from death, albeit at a lower concentration than PFA. Using a DNA end-labeling procedure, we showed that the Pi-treated cells were apoptotic and, as might be predicted, the presence of PFA blocked induction of the death sequence. Next, we examined the expression of two Pi transporters in relation to chondrocyte maturation and anion treatment. We noted that there was expression of the constitutive transporter, Glvr-1, and a type II cotransporter in chick growth plate cells. Although these transport systems are active in terminally differentiated cells, it is probable that the initiation of apoptosis may require the induction of other Pi-transport systems. It is concluded that, at the mineralization front, cell death is linked directly to the elevation in environmental anion concentration and the concomitant rise in intracellular Pi levels.

Alendronate↗
PubMed2001

Phosphate-induced chondrocyte apoptosis is linked to nitric oxide generation.

An elevation in inorganic phosphate (P(i)) concentration activates epiphyseal chondrocyte apoptosis. To determine the mechanism of apoptosis, tibial chondrocytes were treated with P(i), and nitrate/nitrite (NO/NO) levels were determined. P(i) induced a threefold increase in the NO/NO concentration; inhibitors of nitric oxide (NO) synthase activity and P(i) transport significantly reduced NO/NO levels and prevented cell death. Furthermore, a dose-dependent increase in cell death was observed after exposure of chondrocytes to S-nitrosoglutathione. P(i) increased caspase 3 activity 2.7-fold. Both caspase 1 and caspase 3 inhibitors protected chondrocytes from P(i)-induced apoptosis. P(i) caused a significant decrease in the mitochondrial membrane potential, while NO synthase inhibitors maintained mitochondrial function. While P(i) caused thiol depletion, inhibition of P(i) uptake or NO generation served to maintain glutathione levels. The results suggest that NO serves to mediate key metabolic events linked to P(i)-dependent chondrocyte apoptosis.

Animals↗
PubMed2001

[Search for frequently encountered mutations in genes predisposing to breast cancer].

DNA of oncological patients, including Ashkenazi Jews and Slavs, living in St. Petersburg was collected, and the resultant collection was screened for three common mutations of genes BRCA1 and BRCA2 by means of heteroduplex analysis. The mutation 5382insC in exon 20 of the BRCA1 gene was found in four unrelated patients, including three Slavs and one Ashkenazi Jew, with a positive family history of breast cancer. The mutations 185delAG and 6174delT in the BRCA1 and BRCA2 genes, respectively, which are typical of Ashkenazi Jewish patients with breast cancer, were not found in the patients of either ethnicity living in St. Petersburg, although the 6174delT mutation was found in the control group of Ashkenazi Jews. A new 12-nucleotide duplication g.71741ins12nt found in intron 20 of the BRCA1 gene was described. The high frequency of the 5382insC mutation in the BRCA1 gene in patients with familial breast cancer in both St. Petersburg and Moscow indicates that Russian families with the history of breast cancer should be primarily tested for this mutation.

BRCA2 Protein↗
PubMed2000

Effect of varying physical properties of porous, surface modified bioactive glass 45S5 on osteoblast proliferation and maturation.

The objective of this study was to determine the effect of porous bioactive glass (45S5) substrate characteristics on the expression and maintenance of the osteoblastic phenotype. We cultured ROS 17/2. 8 cells on substrates with different pore size and porosity for periods up to 14 days and analyzed the characteristics of the cells and extracellular matrix. Results of the study show that the glass substrates supported the proliferation and growth of osteoblast-like cells. Although the morphologies of the cells differed on the various substrates, their shape and the extent of membrane ruffling suggested that they maintained high levels of metabolic activity. Cells on all substrates expressed high levels of alkaline phosphatase activity and produced extracellular matrices that mineralized to form nonstoichiometric, carbonated, calcium-deficient apatites. An important finding was that at a given porosity of 44%, the pore size neither directed nor modulated the in vitro expression of the osteoblastic phenotype. In contrast, porosity did affect cellular function. We noted that at an average pore size of 92 microm, as the porosity increased from 35 to 59%, osteoblast activity was reduced. As designed in this experiment, an increase in the porosity led to a corresponding increase in total surface area of the specimens. With increasing porosity and surface area, glass reactions in the media may persist for longer durations at higher intensities, thereby affecting local media composition. As such, we suggest that extensive conditioning treatments before cell seeding can reduce this effect. Our results also revealed that the expression of the osteoblastic phenotype is enhanced by the ongoing glass dissolution. The reaction pathway at the origin of this effect still needs to be elucidated. Taken together, the findings support the overall hypothesis that in vitro cell activity can be controlled by a careful selection of substrate properties.

Alkaline Phosphatase↗
PubMed2000

Bioactive glass serves as a substrate for maintenance of phenotype of nucleus pulposus cells of the intervertebral disc.

The objective of the study was to investigate the capability of bioactive glass 45S5 to serve as a substrate for nucleus pulposus cells in vitro. Nucleus pulposus cells were isolated from adult rabbit discs and seeded onto bioactive glass. At selected time intervals, the cells and glass were evaluated. We found that the cells rapidly attached to the substrate, colonizing it within 12 h. By 21 days, they had formed a lawn of cells over the glass substrate. DNA measurements showed a progressive increase in cell number with time. The phenotype was maintained; the cells expressed aggrecan, and collagen type II and I, but were negative for collagen type X. CD44, a cell-surface glycoprotein that binds hyaluronate, was also expressed by these cells. Electron dispersive X-ray analysis and Fourier transform infrared spectroscopy revealed calcium phosphate-rich layer formation on the substrate surface. The results of this study suggest that nucleus pulposus cell proliferation may be an anchorage dependent event, and that the cells use the calcium phosphate-rich layer to facilitate cell adhesion, and subsequent proliferation. These findings point to the importance of the conditioned bioactive glass as a substrate for nucleus pulposus cells.

Aggrecans↗
PubMed2000

Mercury-induced apoptosis in human lymphoid cells: evidence that the apoptotic pathway is mercurial species dependent.

There is growing evidence that heavy metals, in general, and mercurial compounds, in particular, are toxic to the human immune system. In this regard, we have previously shown that both inorganic and organic mercurials are potent human T-cell apoptogens; moreover, mitochondria appear to be a target organelle for the induction of cell death. To ascertain whether both mercury species utilize the same molecular pathway to trigger the apoptotic cascade, cells were treated with MeHgCl or HgCl2 and mitochondrial activity was examined. We show that both mercury species affect mitochondrial activity by inducing the development of a membrane permeability transition. This state is characterized by a decline in both the transmembrane potential and the intracellular pH, as well as the generation of reactive oxygen species. We also determined that mercury exposure results in a decline in the T-cell GSH content. Since mitochondrial dysfunction and the development of a permeability transition may result in the release of cytochrome c, a factor that promotes apoptosis, we assessed the abilities of both species of mercury to induce the translocation of cytochrome c from mitochondria to the cytosol. We noted that MeHgCl caused a significant increase in cytosolic cytochrome c. Surprisingly, however, HgCl2 did not alter the level of cytosolic cytochrome c. We next determined whether the mercurials could alter the level of the anti-apoptotic protein Bcl-2. Our results demonstrate that HgCl2 induces a significant elevation in the Bcl-2 content of T-cells; in contrast, T-cells treated with MeHgCl did not exhibit altered levels of this anti-apoptotic protein. Regardless of whether cytochrome c is released from the mitochondria, both mercurial species were capable of activating the caspase cascade, as evident by cleavage of poly (ADP-ribose) polymerase. Thus, our study shows that, whereas each of the mercury species shares common features in the apoptotic process, profound differences exist in a number of key steps in the pathway. The significance of these differences is discussed.

Adult↗
PubMed2000

Inorganic phosphate induces apoptosis of osteoblast-like cells in culture.

The major goal of this investigation was to test the hypothesis that one of the major products of bone resorption, inorganic phosphate (Pi), activates osteoblast apoptosis. Osteoblast-like cells were isolated from explants of human bone. In monolayer culture, these cells showed an osteogenic phenotype. Thus, the cells exhibited raised alkaline phosphatase activity, expressed osteogenic messenger RNA transcripts, and formed biological mineral. When these cells were treated with 1-7 mmol/L Pi there was a dose- and time-dependent decrease in cell viability. Accordingly, after 48 h, 5 mmol/L Pi reduced the number of viable osteoblast-like cells by 25%; 7 mmol/L Pi reduced the number of cells by 60%. By 96 h, following treatment with 5 mmol/L Pi, the percentage of viable cells was 30%, whereas 7 mmol/L Pi caused an almost complete loss of osteoblast viability. Osteoblast death was blocked by treating the cells with phosphonoformic acid, an inhibitor of the plasma-membrane Na-Pi transporter. Using morphological and end-labeling procedures, we confirmed that cell death was through apoptosis. To probe the mechanism of cell death, osteoblast-like cells were probed with rhodamine 123, a dye that is responsive to the membrane potential. We noted that Pi-treated cells displayed a profound loss of mitochondrial membrane potential, suggesting that the anion activated the death program through the induction of a mitochondrial membrane permeability transition. We conclude that high levels of osteoblast apoptosis observed at sites of bone resorption may be linked to release of Pi from bone mineral.

Apoptosis↗
PubMed2000

Evaluation of osteoblast response to porous bioactive glass (45S5) substrates by RT-PCR analysis.

Previous studies have shown that neonatal rat calvaria osteoblasts elaborate substantial amounts of extracellular material with bone-like characteristics when cultured on porous bioactive glass substrates in vitro. However, the osteoblastic response to this material has not been fully characterized. The objective of this study was to characterize osteoblast response to porous bioactive glass substrates following the expression of the classical markers for osteoblast differentiation. In this study we synthesized porous bioactive glass substrates, seeded them with osteoblast-like cells (ROS 17/2.8) and followed the temporal expression of alkaline phosphatase (AP) activity, as well as the expression of mRNA for collagen type I (Coll-1), osteonectin (OSN), osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP). The data confirm that porous bioactive glass substrates are capable of supporting the in vitro growth and maturation of osteoblast-like cells. At a porosity of 42% and an average pore size of 80 microm, the substrates promote the expression and maintenance of the osteoblastic phenotype. The results additionally suggest that there is both a solution-mediated and a surface-controlled effect on cell activity.

Alkaline Phosphatase↗