Human Osteoblast-like Cell Line Saos-2 Research Articles

Chemical, Antibacterial, and Cytotoxic Properties of Four Different Endodontic Sealer Leachates Over Time

IntroductionThe management of apical periodontitis involves the elimination of bacteria to achieve healing of the periapical tissues. To this end, understanding of the antimicrobial properties and cytotoxicity of root canal sealers over time is important. This study aimed to assess the cytocompatibility and antibacterial activity of leachates obtained from selected endodontic sealers. MethodsFour sealers were used in this study; AH Plus, an epoxy resin-based sealer, and three hydraulic calcium silicate-based sealers: AH Plus Bioceramic, BioRoot RCS, and BioRoot Flow. Sealer chemistry was assessed by scanning electron microscopy and energy dispersive spectrum analysis. Leachates of the tested sealers were prepared by immersing sealer discs in Hank’s balanced salt solution for 28 and 90 days. Element release was assessed using inductively coupled plasma mass spectroscopy. Enterococcus faecalis and Fusobacterium nucleatum were exposed to the leachates followed by colony-forming unit determination, and the human osteoblast-like cell line Saos-2 was assessed with regard to cell death, caspase expression and activity of alkaline phosphatase (ALP) after stimulation with the leachates. ResultsCalcium leaching was detected in the tricalcium silicate-based sealers, which increased the leachate pH significantly (P < .0001). AH Plus Bioceramic leachate displayed antimicrobial activity at 28 days against E. faecalis (P < .01). ALP levels were reduced in response to AH Plus Bioceramic and BioRoot RCS 1-day leachates (P < .05). High cell viability was observed after exposure to all sealers. ConclusionsThe leachates of the tested sealers were cytocompatible; however, their antimicrobial activity as well as their potential to induce the bone formation marker ALP was minimal.

Cadmium Induces Apoptosis in the Human Osteoblast-like Cell Line Saos-2

Human exposure to the heavy metal cadmium has been associated with the development of bone diseases, including osteoporosis and osteomalacia. The mechanisms by which cadmium exerts a direct effect on bone remain unclear. Bone cells go through apoptosis for proper bone remodeling; therefore, it was hypothesized that cadmium disrupts this normal balance by inducing apoptosis. Human osteoblast-like cells (Saos-2) were treated with 10–200 μM cadmium chloride (CdCl2) and evaluated by trypan blue staining and phase-contrast microscopy. Exposure to CdCl2 resulted in decreased cell viability and changes in cell morphology characteristic of apoptosis. The role of apoptosis in cadmium-induced toxicity was further evaluated using the fluorescent marker annexin V, which detects externalization of cell membrane phosphatidylserine. Nuclear changes associated with apoptosis were assessed by Hoechst staining and a DNA fragmentation assay. A significant increase in annexin V-positive cells was observed following CdCl2 treatment. Nuclear changes associated with apoptosis, including marginalization and condensing of chromatin and DNA fragmentation, were also observed following CdCl2 treatment. Cadmium-induced apoptosis in Saos-2 cells was also accompanied by an increase in caspase-3 activity. The addition of the caspase-3 inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) or the known cadmium chelating agent potassium bis(2-hydroxyethy)dithiocarbamate, (K[bhedtc]), blocked caspase-3 activation induced by cadmium. Collectively, this study has identified a role for apoptosis in cadmium-induced toxicity in bone cells, and provides insight for future studies on mechanisms underlying the disruption of apoptotic signaling cascades in bone and the relationship to bone disease.

In vitro expression of proalpha1(I) collagen mRNA by human pre-osteoclastic cells.

In vitro studies have demonstrated that the extracellular matrix modulates the cell phenotype. In the present study we have investigated in vitro proalpha1(I) collagen mRNA expression in a human pre-osteoclastic cell line (FLG 29.1 cells) in basal condition and after various stimuli. In addition, in order to evaluate the effect of cell-cell interactions on collagen type I mRNA expression, we have cultured the human pre-osteoclastic cells FLG 29.1 with either the human osteoblast-like cell line Saos-2 or the bovine bone endothelial cell line BBE. We showed that the FLG 29.1 cells express proal (I) collagen mRNA, whose expression is modulated by phorbol esters (TPA). Co-culturing FLG 29.1 cells with either Saos-2 or BBE cells induced decrease of proalpha1(I) collagen mRNA expression.

A collagen peptide motif activates tyrosine kinase-dependent calcium signalling pathways in human osteoblast-like cells.

A collagen peptide motif (DGEA) which is a putative alpha 2 beta 1 integrin binding site was examined for its ability to activate Ca2+ signalling pathways in the human osteoblast-like cell line SaOS-2. We show that these cells express both alpha 2 beta 1 integrin subunits (by immunocytochemistry) and that an anti-beta 1 monoclonal antibody (DF5) mobilizes Ca2+ in these cells. DGEA elevated intracellular Ca2+ in fura-2-loaded cells, in a concentration- and sequence-dependent fashion, with an EC50 of 250 microM. The tyrosine kinase inhibitor herbimycin A reduced the number of cells responding to DGEA and to transforming growth factor alpha. Thrombin also stimulated a rise in intracellular Ca2+, but the number of cells responding was not reduced by herbimycin A. The DGEA response was dependent on extracellular Ca2+, but was not due to Ca2+ influx, since it was blocked by thapsigargin and not by lanthanum. Using three different anti-alpha 2 monoclonal antibodies, we were unable to show that the DGEA-induced Ca2+ signal was mediated by the alpha 2 beta 1 integrin. In summary, the DGEA collagen motif does appear to activate receptor-mediated Ca2+ signalling events in SaOS-2 cells, in a divalent cation-dependent manner, but we were unable to demonstrate a role for alpha 2 beta 1 integrin in this response.

Response of human osteoblasts to implant materials: integrin-mediated adhesion.

The initial interaction of the human osteoblast-like cell line Saos-2 with orthopaedic implant materials was analyzed to determine the mechanism by which these cells adhere to implant surfaces. Saos-2 cells were allowed to attach to disks composed of the orthopaedic implant materials Tivanium (Ti6A14V) and Zimaloy (CoCrMo) and to control disks of glass and plastic. Serum had no effect on the number of cells that attached to Tivanium and Zimaloy at 4 or 24 hours but did increase the number of cells that attached to glass at 24 hours. Collagen synthesis was determined by [3H]proline incorporation into collagenase-digestible protein and noncollagen protein. A significant increase of 19% was found for collagen synthesized in cells cultured on Zimaloy for 24 hours compared with glass, with no differences on Tivanium and plastic. However, collagenase-digestible protein and noncollagen protein were increased the most (204 and 198%, respectively) on Tivanium compared with glass. To determine if integrins were involved in cell attachment to implant materials, the peptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), which blocks integrin receptors through the Arg-Gly-Asp sequence, was added to the cells in serum-free medium. This peptide inhibited cell adhesion by 28% on Tivanium and 40% on Zimaloy but had no effect on glass and plastic. The control peptide GRADSP (Gly-Arg-Ala-Asp-Ser-Pro) had no effect on adhesion. Inhibition of protein synthesis and enzymatic removal of surface proteins did not affect the ability of Arg-Gly-Asp peptides to inhibit cell attachment to the implant materials. These results suggest that integrins are able to bind directly to Tivanium and Zimaloy. Western blot analysis of integrin protein demonstrated changes in many integrin subunits, depending on the substrate to which cells attached. In particular, the beta 1 integrin subunit was increased 3.8 to 9.5-fold at 24 hours. To determine specifically which integrins may be involved in adhesion, antibodies to integrins were added. An antibody to the fibronectin receptor, alpha 5 beta 1, significantly inhibited binding of cells to Tivanium by 63% and to Zimaloy by 49% and had no effect on glass. The vitronectin receptor antibody, alpha v beta 3/beta 5, did not alter cell adhesion. In conclusion, osteoblast-like cells appear to be capable of attaching directly to implant materials through integrins. The type of substrate determines which integrins and extracellular matrix proteins are expressed by osteoblasts. These data provide information on how implant materials may affect osteoblast differentiation and bone growth.

Regulation of parathyroid hormone/parathyroid hormone-related protein receptor expression by osteoblast-deposited extracellular matrix in a human osteoblast-like cell line.

Parathyroid hormone (PTH) receptors and the biological response to PTH in osteoblasts have been shown to be influenced by glucocorticoids, growth factors, cytokines or PTH itself. Furthermore, components of extracellular matrix (ECM) appear to regulate the response to PTH as well. We investigated the effects of osteoblast-deposited ECM on PTH-related protein (PTHrP)-stimulated cAMP production, PTHrP binding and PTH/PTHrP receptor mRNA in the human osteoblast-like cell line SaOS-2. ECM was laid down by the human osteoblastic cell line MG-63. At confluence, maximal cAMP stimulation induced by 100 nmol/l PTHrP (1-34) was decreased in SaOS-2 cells grown on ECM as compared with cultures on plastic dishes, without any change in PTHrP concentration producing half-maximal stimulation. In contrast, cAMP production stimulated by PGE2 was increased in cells on ECM. Saturable 125I-PTHrP binding (as evaluated by Scatchard plot analysis) was markedly diminished in cells grown on ECM (5,600 +/- 2,010 vs. 20,700 +/- 1,710 binding sites/cell, x +/- S.E.M., P < 0.01, n = 4 experiments), without any significant change in affinity (1.3 +/- 0.4 vs. 2.5 +/- 0.5 nmol/l (NS), in cells on ECM and plastic, respectively). This apparent decrease in membrane receptor density was associated with markedly lower steady state PTH/PTHrP receptor mRNA levels as assessed by Northern blot analysis (ECM/control: 0.4 +/- 0.1). A difference in PTH/PTHrP receptor mRNA levels between cells on ECM or on plastic dishes was detectable by 8 hours but not by 4 hours, after seeding the cells at high density. By 24 hours after plating, PTH/PTHrP receptor mRNA levels were maximally decreased in cells on ECM. These results in the human osteoblast-like cell line SaOS-2 indicate that PTH/PTHrP receptors are down-regulated by growth on ECM. Thus, attachment of bone cells to bone surface could influence differentiation and function of osteoblasts.

Differences in intracellular calcium signaling after activation of the thrombin receptor by thrombin and agonist peptide in osteoblast-like cells.

Thrombin and the thrombin receptor agonist peptide (TRAP) caused a rise in intracellular calcium concentration ([Ca2+]i) in the human osteoblast-like cell line Saos-2. Striking differences in the [Ca2+]i signals elicited by these agonists were revealed. In cell populations, thrombin induced a transient increase in [Ca2+]i while TRAP caused a biphasic [Ca2+]i response consisting of an initial peak and a sustained plateau phase. In individual cells, thrombin mainly caused a single [Ca2+]i transient while TRAP induced repetitive [Ca2+]i spikes. Neither tyrosine phosphorylation, cAMP-dependent phosphorylation, nor pertussis toxin-sensitive G proteins appeared to be involved in thrombin receptor [Ca2+]i signaling in this cell line. However, the sustained [Ca2+]i response caused by TRAP was converted into a transient, thrombin-like response by pretreatment with serine/threonine phosphatase inhibitors. Pretreatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA) abrogated thrombin receptor [Ca2+]i signaling, and TRAP-induced Ca2+ entry was inhibited by the acute treatment with PMA. In contrast, Ca2+ entry stimulated by thapsigargin was not sensitive to agents affecting serine/threonine phosphorylation. The observation that thrombin and TRAP, despite being agonists for a common receptor, induce dissimilar [Ca2+]i responses indicates that binding of TRAP alone is insufficient to fully regulate the thrombin receptor in Saos-2 cells.

P13. Characterization of the receptor responsible for thrombin-induced intracellular calcium responses in osteoblast-like cells

The receptor responsible for the increase in intracellular calcium concentration ([Ca 2+ ] i ) after the addition of thrombin to the human osteoblast-like cell line Saos-2 has been characterized. Thrombin caused a dose-dependent increase in [Ca 2+ ] i ; a half-maximal stimulation was observed with 3.2 ± 1.1 nM thrombin. The human platelet thrombin receptor is activated by thrombin cleavage to create a new NH 2 terminus that acts as a tethered ligand, and peptides based on the tethered ligand can activate the receptor independently of thrombin. Northern analysis indicated the presence of mRNA encoding the platelet receptor in Saos-2 cells, and surface expression of the receptor was demonstrated by immunocytochemistry. A tethered ligand peptide (SFLLRNPNDKYEPF, single-letter amino acid code) was found to increase [Ca 2+ ] i . The maximal response to the peptide was similar to that observed with thrombin, and a half-maximal response was observed with 22 ± 6 μM peptide. The time course of the increase in [Ca 2+ ] i with the peptide was different than that observed with thrombin; a pronounced shoulder was observed after an initial sharp rise. The phenylalanine in the second position of the agonist peptide and the arginine in the fifth position were shown to be essential for its activity. The requirement for proteolysis of the receptor for the thrombin-dependent increase in [Ca 2+ ] i was demonstrated by two methods. Antibodies that reacted with the cleavage site of the receptor abolished the effect of thrombin on [Ca 2+ ] i . In addition, a mutant of thrombin without catalytic activity as well as chemically inactivated thrombin failed to cause an increase in [Ca 2+ ] i . Similar results were obtained with the rat osteoblast-like cell line UMR-106; a tethered ligand peptide based on the rat sequence induced an increase in [Ca 2+ ] i , and antibodies to the cleavage site of the rat receptor inhibited the effect of thrombin.

Characterization of the receptor responsible for thrombin-induced intracellular calcium responses in osteoblast-like cells.

The receptor responsible for the increase in intracellular calcium concentration ([Ca2+]i) after the addition of thrombin to the human osteoblast-like cell line Saos-2 has been characterized. Thrombin caused a dose-dependent increase in [Ca2+]i; a half-maximal stimulation was observed with 3.2 +/- 1.1 nM thrombin. The human platelet thrombin receptor is activated by thrombin cleavage to create a new NH2 terminus that acts as a tethered ligand, and peptides based on the tethered ligand can activate the receptor independently of thrombin. Northern analysis indicated the presence of mRNA encoding the platelet receptor in Saos-2 cells, and surface expression of the receptor was demonstrated by immunocytochemistry. A tethered ligand peptide (SFLLRNPNDKYEPF, single-letter amino acid code) was found to increase [Ca2+]i. The maximal response to the peptide was similar to that observed with thrombin, and a half-maximal response was observed with 22 +/- 6 microM peptide. The time course of the increase in [Ca2+]i with the peptide was different than that observed with thrombin; a pronounced shoulder was observed after an initial sharp rise. The phenylalanine in the second position of the agonist peptide and the arginine in the fifth position were shown to be essential for its activity. The requirement for proteolysis of the receptor for the thrombin-dependent increase in [Ca2+]i was demonstrated by two methods. Antibodies that reacted with the cleavage site of the receptor abolished the effect of thrombin on [Ca2+]i. In addition, a mutant of thrombin without catalytic activity as well as chemically inactivated thrombin failed to cause an increase in [Ca2+]i. Similar results were obtained with the rat osteoblast-like cell line UMR-106; a tethered ligand peptide based on the rat sequence induced an increase in [Ca2+]i, and antibodies to the cleavage site of the rat receptor inhibited the effect of thrombin.

Effects of parathyroid hormone on ornithine decarboxylase activity in human osteosarcoma cells

Ornithine decarboxylase (ODC) is a rate-limiting enzyme of the biosynthesis of polyamines, which are important in cell growth and differentiation. Here, we studied whether parathyroid hormone (PTH) affects the induction of ODC and the proliferation of the human osteoblast-like cell line SaOS2, which is sensitive to PTH. In confluent cells, ODC activity was not detected, but activity was significantly induced by fresh medium, with maximum activity 6 h after the change. PTH potentiated this enzyme induction in a dose-dependent manner at 10 −9 and 10 −8 m at which range the intracellular cAMP level also rose. Dibutyryl cAMP, cholera toxin, and 3-isobutyl-1-methylxanthine each caused an increase in ODC activity similar to that with PTH. The half-life of enzyme activity was about 30 min and was not changed by the addition of PTH. mRNA coding for ODC was detected in the confluent cells and its concentration was increased two- to threefold by the fresh medium. No further increase in mRNA occurred when PTH was added. At 48 h after the change of medium, PTH inhibited the DNA synthesis induced by fresh medium. These results suggest that the increase in ODC activity caused by PTH was caused by enhancement of cAMP synthesis, and that this augmentation involves post-transcriptional regulation.