Growth Of Saos-2 Cells Research Articles

Innovative hydroxyapatite/submicron mesoporous SiO2/HA particles composite coatings for enhanced osteogenic activity of NiTi bone implants: A comprehensive investigation of materials and biological interactions

In this study, hydroxyapatite (HA) incorporated in mesoporous SiO2 particles (SiO2/HA) was initially synthesized using a soft template method. The resulting structure owned a high surface area (796.62 m2 g-1) and a substantial pore volume (0.757 cm3 g-1). The TEM and FESEM images indicated that the synthesized mesoporous particles possessed a spherical morphology, with a diameter of approximately 200 nm, and pores of slit-shape. The mission was to improve the surface characteristics of NiTi implants by composite coatings. This investigation emphasized specifically the impact of different concentrations of mesoporous SiO2/HA particles within the electrolyte on the properties of HA/mesoporous composite coatings. The surface characteristics of the coatings were examined using ATR-FTIR, FESEM, EDS, AFM, and hydrophilicity measurements. Furthermore, the osteogenic activity of the coatings was evaluated by culturing the osteosarcoma cell line SAOS-2 on the coated samples. The introduction of mesoporous particles resulted in a decrease in the coatings’ surface roughness along with an improvement in their wettability. Moreover, there was an enhancement in the cell viability (99% after 7 days), proliferation, adhesion, and growth of SAOS-2 cells at the optimized concentration of mesoporous SiO2/HA particles (1000 mg/L). DAPI images illustrated that the mesoporous particles did not cause any adverse impact on the cellular nuclei. Finally, in the apoptosis test, the composite coating displayed a reduced number of necrotic cells compared to the pure HA coating.

Electrochemical and surface characterization of anodized and fs-laser treated Ti6Al4V for osseo-repellent bone screws and dental implants

The surfaces of titanium grade 5 (Ti6Al4V) samples were altered on a micro and nano scale by femtosecond laser irradiation and subsequent electrochemical anodization. The produced micro-cones and nano-ripples covered by the additional oxide layer were characterised and their influence on osteoblast-like Saos-2 cell growth was studied. Surface topography and morphology were studied by Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM). From the wettability experiments, it was found that the contact angle vs. roughness follows the Wenzel equation and that the wettability increases after the anodization. The composition of the formed oxide layer was determined by X-ray photoelectron spectroscopy (XPS) and oxide incorporation of phosphate ions could be confirmed. Moreover, estimated electrochemical surface area (ECSA) of the femtosecond laser treated sample was 14 times larger compared to a polished sample. The anodic oxide film formation factors for different electrolytes were determined and were in the range of 1.3 nm V−1–2.0 nm V−1. Finally, the samples anodized with 0.1 M H2SO4 and 3 M H3PO4 showed decreased Saos-2 cell growth.

Cytotoxic Impact of N-Oleoylethanolamine on Bone Cancer Cells.

Cancer is a complex disease that derives from the uncontrolled proliferation of cells. Bone cancer is a type of prevalent cancer that occurs both in young and adults. Bone cancer is most common in the long bones of the pelvis, arms and legs. Statistically, more than 200 cases of osteosarcoma have been reported annually in our country. Classical treatment with chemotherapeutics remains ineffective in the cure of this cancer type. Recent studies have shown that ceramide induces apoptosis at its increased levels in the cells. Thus, many studies have been conducted to cause the accumulation of ceramide molecules in the cell by different ways to induce apoptosis. NOE (Noleoylethanolamine) is a specific inhibitor of ceramidase enzymes that hydrolyze intracellular ceramides and prevent apoptosis. This study investigates the cytotoxic and apoptosis-inducing activities of NOE on human osteosarcoma Saos-2 cells. Cytotoxic effects were investigated by MTT colorimetric assay. For the detection of morphological and ultrastructural indicators of apoptosis, confocal and TEM techniques were used. Our finding indicated that NOE is effective in the inhibition of the growth of Saos-2 cells. Confocal and TEM findings showed morphological and ultrastructural changes as chromatin condensation, fragmentation of nuclei and mitochondria as well as damaged cytoskeleton and cell shrinkage. The results revealed that NOE exerts its cytotoxicity on Saos-2 cells through changing the ultrastructure and morphology of cells with clear apoptotic sparks.

Comparative evaluation of surface-modified zirconia for the growth of bone cells and early osseointegration

Statement of problemRapid osseointegration between implant and bone tissue for early loading of a prosthesis with sufficient primary stability depends on the surface characteristics of the implant. The development and characterization of suitable surface coatings on dental implants is a major challenge. PurposeThe purpose of this in vitro study was to evaluate and compare the osteogenic potential and cytotoxicity of unmodified zirconia, acid-etched zirconia, bioactive glass–coated zirconia, and tamarind kernel polysaccharide with hydrophilic acrylic acid (TKP-AA) hydrogel-coated zirconia. Material and methodsThirty-six disks each of unmodified zirconia, acid-etched, 45S5 bioactive glass–coated, and TKP-AA hydrogel-coated zirconia were evaluated for osteogenic potential and cytotoxic effect by using human osteoblast Saos-2 cells. The surface topography of the disks and the morphology of the cells grown on these surfaces were examined by scanning electron microscopy (n=3). The cell attachment was evaluated by confocal imaging (n=3). The cytotoxic effect was evaluated by cell viability assay (n=9). Osteoblast maturation was assessed by alkaline phosphatase assay (n=9) and cell mineralization by alizarin red staining (n=9). ANOVA and Bonferroni multiple comparison post hoc tests were used to evaluate the statistical significance of the intergroup differences in these characteristics (α=.05). ResultsThe surface modifications resulted in distinct changes in the surface morphology of zirconia disks and the growth of Saos-2 cells. Zirconia disks coated with TKP-AA promoted higher proliferation of osteoblasts compared with unmodified disks (P<.001). Similarly, the surface modifications significantly increased the differentiation of mesenchymal stem cells to osteoblasts as compared with uncoated zirconia (P<.001). However, the rate of differentiation to osteoblasts was similar among the surface modifications. Acid-etched and TKP-AA-coated disks promoted mineralization of osteoblasts to the same extent, except bioactive glass coating, which significantly increased the rate of mineralization (P<.001). ConclusionsSurface modification of zirconia by acid etching and coating with Bioglass or TKP-AA hydrogel resulted in the improved growth and differentiation of osteoblasts. TKP-AA hydrogel coating promoted the proliferation of osteoblasts, whereas Bioglass coating showed better mineralization. TKP-AA hydrogel coating is a promising candidate for improving the osseointegration of dental implants that warrants further investigation.

Preparation, characterisation and in-vitro biocompatibility study of a bone graft developed from waste bovine teeth for bone regeneration

A bovine dentine derived hydroxyapatite (BDHA) bone graft was developed from waste bovine teeth using a successful defatting and deproteinizing procedure. The resulting BDHA material was characterised using several analytical techniques to investigate its chemical, physical, thermal and structural properties. Fourier-transform-infrared-spectroscopy spectra confirmed the presence of carbonate, hydroxyl and phosphate functional groups which is associated with hydroxyapatite. X-ray diffraction analysis suggested that the processed BDHA corresponds characteristically to hydroxyapatite. Scanning electron microscopy showed the presence of dentinal tubules with an interconnected porous architecture in conjunction with a pore diameter of ∼3-4 μm. Thermogravimetric analysis showed that the BDHA was stable up to 1000 °C and lost only 1.5% of its weight. Both Inductively-coupled-plasma-mass spectrometry and Energy-dispersive-X-ray analysis revealed that the inorganic phases of BDHA were mainly composed of calcium and phosphorous with trace amounts of sodium and magnesium. The BDHA was chemically stable and did not degrade significantly after 28 days of incubation in simulated body fluid (SBF). The BDHA material showed excellent mechanical properties in terms of yield strength and young’s modulus. In-vitro studies showed that the BDHA was non-toxic, supported the proliferative growth of Saos-2 cells and expressed the bone marker “osteonectin” after 14 days.

Treatment of Saos-2 osteosarcoma cells with diallyl trisulfide is associated with an increase in calreticulin expression.

Diallyl trisulfide (DATS) is a natural organic sulfur compound that may be isolated from garlic and has strong anticancer activity. DATS has been demonstrated to upregulate the expression of calreticulin (CRT) in various types of human cancers, which is associated with the prognosis of cancer and its response to therapy. However, whether DATS has the same effect on human osteosarcoma cells is not known. Therefore, in the present study, Saos-2 human osteosarcoma cells were cultured with different concentrations of DATS (0, 25, 50 and 100 µmol/l) for 24 h, or with 50 µmol/l DATS for different time periods (0, 12, 24 and 36 h). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting and immunofluorescent staining were used to detect CRT mRNA and protein in the Saos-2 cells. Exposure to DATS changed the morphology and inhibited the growth of the Saos-2 cells, and its effects appeared to be concentration- and exposure time-dependent. The optimum concentration and exposure time of DATS were 50 µmol/l and 24 h, respectively. The levels of CRT mRNA and protein in the Saos-2 cells were significantly upregulated following exposure to DATS. The upregulation of CRT expression by DATS may be a mechanism underlying the ability of DATS to inhibit the growth of human osteosarcoma Saos-2 cells.

Long noncoding RNA MALAT1 promotes cell proliferation through suppressing miR-205 and promoting SMAD4 expression in osteosarcoma.

Increasing evidences have indicated that long non-coding RNAs (lncRNAs) play an important role in multiply biological processes including cell development, differentiation, proliferation and invasion. The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), is a highly conserved nuclear ncRNA and a key regulator of metastasis development in several cancers. However, its role in osteosarcoma progression is not well known. In this study, we sought to determine the clinical and bilogical role of MALAT1 in osteosarcoma progression. RT-qPCR analysis showed that MALAT1 expression was significantly increased in primary osteosarcoma tissues and cell lines. Kaplan-Meier analysis indicated that patients with high expression of MALAT1 was associated with poor overall survival compared with the low expressing patients. Furthermore, the gain and loss function assay showed that miR-205 was suppressed by MALAT1 in osteosarcoma and this interaction between miR-205 and MALAT1 has reciprocal effects. Cell viability assay showed that MALAT1 promoted MG-63 and SAOS-2 cell growth through suppressing miR-205. Subsequently, the downstream gene SMAD4 was identified as a direct functional target of miR-205, and miR-205 suppressed osteosarcoma cell growth through suppressing SMAD4. Finally, we demonstrated that MALAT1 promoted osteosarcoma progression via a miR-205-SMAD4 axis. In conclusion, we revealed that enhanced MALAT1 expression predicted unfavourable outcome in osteosarcoma and promoted cell proliferation through suppressing miR-205 and activating SMAD4 function. Thus, lncRNA MALAT1 may serve as a promising prognostic and therapeutic target for osteosarcoma patients.

Growth of a TiNb adhesion interlayer for bioactive coatings

Surface bioactivity has been under intensive study with reference to its use in medical implants. Our study is focused on coatings prepared from an electroactive material which can support bone cell adhesion. Until now, hydroxyapatite films have usually been utilized as a chemically-active surface agent. However, electrically-active films could set a new direction in hard tissue replacement. As a base for these films, it is necessary to prepare an intermediate film, which can serve as a suitable barrier against the possible diffusion of some allergens and toxic elements from the substrate. The intermediate film also improves the adaptation of the mechanical properties of the basic material to an electroactive film. The aim of our work was to select an implantable and biocompatible material for this intermediate film that is suitable for coating several widely-used materials, to check the possibility of preparing an electroactive film for use on a material of this type, and to characterize the structure and several mechanical properties of this intermediate film. TiNb was selected as the material for the intermediate film, because of its excellent chemical and mechanical properties. TiNb coatings were deposited by magnetron sputtering on various substrates, namely Ti, Ti6Al4V, stainless steel, and bulk TiNb (as standard), and important properties of the layers, e.g. surface morphology and surface roughness, crystalline structure, etc., were characterized by several methods (SEM, EBSD, X-ray diffraction, nanoindentation and roughness measurement). It was found that the structure and the mechanical properties of the TiNb layer depended significantly on the type of substrate. TiNb was then used as a substrate for depositing a ferroelectrically active material, e.g., BaTiO3, and the adhesion, viability and proliferation of human osteoblast-like Saos-2 cells on this system were studied. We found that the electroactive BaTiO3 film was not only non-cytotoxic (i.e. it did not affect the cell viability). It also enhanced the growth of Saos-2 cells in comparison with pure TiNb and with standard tissue culture polystyrene wells, and also in comparison with BaTiO3 films deposited on Ti, i.e. a material clinically used for implantation into the bone.

JPH203, a selective L-type amino acid transporter 1 inhibitor, induces mitochondria-dependent apoptosis in Saos2 human osteosarcoma cells.

Most normal cells express L-type amino acid transporter 2 (LAT2). However, L-type amino acid transporter 1 (LAT1) is highly expressed in many tumor cells and presumed to support their increased growth and proliferation. This study examined the effects of JPH203, a selective LAT1 inhibitor, on cell growth and its mechanism for cell death in Saos2 human osteosarcoma cells. FOB human osteoblastic cells and Saos2 cells expressed LAT1 and LAT2 together with their associating protein 4F2 heavy chain, but the expression of LAT2 in the Saos2 cells was especially weak. JPH203 and BCH, a non-selective L-type amino acid transporter inhibitor, potently inhibited L-leucine uptake in Saos2 cells. As expected, the intrinsic ability of JPH203 to inhibit L-leucine uptake was far more efficient than that of BCH in Saos2 cells. Likewise, JPH203 and BCH inhibited Saos2 cell growth with JPH203 being superior to BCH in this regard. Furthermore, JPH203 increased apoptosis rates and formed DNA ladder in Saos2 cells. Moreover, JPH203 activated the mitochondria-dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bad, Bax, and Bak, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. These results suggest that the inhibition of LAT1 activity via JPH203, which may act as a potential novel anti-cancer agent, leads to apoptosis mediated by the mitochondria-dependent intrinsic apoptotic signaling pathway by inducing the intracellular depletion of neutral amino acids essential for cell growth in Saos2 human osteosarcoma cells.

Hydroxyapatite-based materials of marine origin: a bioactivity and sintering study.

Single phase hydroxyapatite (HAp) and biphasic material hydroxyapatite/β-tricalcium phosphate (HAp/β-TCP) were obtained from a marine source (Atlantic cod fish bones). Here we report a study on the biological properties of these materials, including cytotoxicity, bioactivity and haemocompatibility. Results showed that the materials are not cytotoxic, neither in their powder nor in pellet form; indeed growth of Saos-2 cells was comparable to that of commercial. The haemolysis rate was lower than 2%; hence the materials can be classified as non-haemolytic. Moreover, when immersed in Simulated Body Fluid (SBF), crystal formation was observed on the surface of both materials. The sintering behaviour of the samples was also studied; both powders showed very high sinterability (density higher than 95% of the theoretical value). Overall, these results confirm the suitability of these materials for biomedical applications.

N2/H2o Plasma Assisted Functionalization of Poly(ε‐caprolactone) Porous Scaffolds: Acidic/Basic Character versus Cell Behavior

A primary research focus in tissue engineering addresses the factors that drive cell growth in scaffolds. Due to the inherent complexity of phenomena at the cell/biomaterial interface in cell‐culture media, it is not easy to unambiguously disentangle the effects of different surface properties (e.g., chemical composition, roughness, porosity, etc.) in promoting specific cell behaviors, especially on porous scaffold materials. We have investigated fast low‐pressure plasma treatments of porous PCL scaffolds for tissue engineering with neutral, acidic, and basic groups to obtain stable water absorbent materials. An increase of Saos2 cell growth was observed on plasma‐treated scaffolds with respect to native samples; amphoteric surfaces were found very suitable for cell proliferation.

GDC-0152 attenuates the malignant progression of osteosarcoma promoted by ANGPTL2 via PI3K/AKT but not p38MAPK signaling pathway.

Angiopoietin-like protein 2 (ANGPTL2) plays an important role in inflammatory carcinogenesis and tumor metastasis. The compound GDC-0152 is a peptidomimetic small molecule antagonist of inhibitor of apoptosis (IAP) proteins with antitumor activity. However, the interaction between ANGPTL2 and GDC-0152 has not been studied. It has been proven that ANGPTL2 promotes metastasis of osteosarcoma. Therefore, in the present study, the effect of GDC-0152 on the malignant progression of osteosarcoma promoted by ANGPTL2 was investigated. Human osteosarcoma cell line SaOS2 cells were pre-treated or non-treated with GDC-0152 and then exposed to recombinant human ANGPTL2. The viability of SaOS2 cells was determined by MTT assay, the migration of SaOS2 cells was analyzed by chamber migration assay kit, and the SaOS2 cell apoptosis was determined by fluorescence-activated cell sorting (FACS) and nuclear staining. Treatment with ANGPTL2 increased SaOS2 cell growth and migration and decreased cell apoptosis. The increased cell growth and decreased cell apoptosis were significantly attenuated in SaOS2 cells receiving GDC-0152. However, the ANGPTL2-increased SaOS2 cell migration was not inhibited by GDC-0152 treatment. Furthermore, western blot analysis showed that the activation of phosphatidyl inositol 3-kinase (PI3K) (p85), PI3K (p110), protein kinase B (Akt) (Ser473), Akt (Thr308) and p38 mitogen-activated protein kinase (p38MAPK) were upregulated by ANGPTL2. Quantitative real-time polymerase chain reaction (qTR-PCR) and gelatin zymography showed that the mRNA expression and activity of matrix metalloproteinase-9 (MMP-9) and matrix metalloproteinase-2 (MMP-2) were also increased by ANGPTL2. The upregulated activation of PI3K and Akt were significantly suppressed by the treatment of GDC-0152. In contrast, GDC-0152 did not suppress ANGPTL2-induced p38MAPK phosphorylation, MMP-9/MMP-2 mRNA expression or MMP-9/MMP-2 activity. Taken together, these data indicate that GDC-0152 attenuates the malignant progression of osteosarcoma promoted by ANGPTL2 via PI3K/AKT but not p38MAPK signaling pathway. The present study indicated a novel therapeutic strategy to inhibit tumor growth by indirectly preventing ANGPTL2 signaling.

Induction of apoptosis by diphenyldifluoroketone in osteogenic sarcoma cells is associated with activation of caspases

The aim of the present study was to investigate and compare the effects of diferuloylmethane (curcumin) and diphenyldifluoroketone (EF-24) on cell growth and apoptosis induction in human osteogenic sarcoma cells. This was examined by MTT assay, nuclear DAPI staining, caspase-activation assay, flow cytometry analysis and immunoblotting in Saos2 human osteogenic sarcoma cells. Curcumin and EF-24 inhibited the growth of Saos2 cells in a dose-dependent manner. The apparent potency of EF-24 was more than 3-fold higher that of curcumin. Treatment with curcumin or EF-24 resulted in nuclear condensation and fragmentation in the cells. The caspase-3/-7 activities were detected in living cells treated with curcumin or EF-24. Flow cytometry showed that the rate of apoptosis was increased by curcumin and EF-24 compared to the control. Curcumin and EF-24 promoted the proteolytic cleavages of procaspase-3/-7/-8/-9 with increases in the amount of cleaved caspase-3/-7/-8/-9. The curcumin- or EF-24-induced apoptosis in the Saos2 cells was mediated by the expression of Fas and activation of caspase-8, caspase-3 and poly(ADP-ribose) polymerase. Immunoblotting revealed the Bid and Bcl-2 proteins to be downregulated, and truncated-Bid, Bax and p53 proteins to be upregulated by curcumin and EF-24. Curcumin and EF-24 increased the Bax/Bcl-2 ratio significantly. These results suggest that the curcumin and EF-24 inhibit cell proliferation and induce apoptotic cell death in Saos2 human osteogenic sarcoma cells via both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway, and may have potential properties for anti-osteosarcoma drug discovery.

Epigenetic regulation of the pro-apoptosis gene TSSC3 in human osteosarcoma cells

Promoter hypermethylation can lead to a loss of genetic imprinting in carcinogenesis. The mechanism for the loss of expression of the imprinted gene TSSC3 has not been investigated in cases of osteosarcoma. In this study, we treated osteosarcoma cell lines with 5-Aza-CdR, which is a widely-used DNA methyltransferase inhibitor, and found dose-dependent reduction in cell growth, conversion of cell morphology to a non-motile phenotype, and obvious increase in apoptosis. In addition, we also found that 5-Aza-CdR reactivated TSSC3 expression through demethylation of the promoter regions. These findings indicate that the TSSC3 gene is silenced through hypermethylation of the promoter regions, a mechanism commonly associated with gene silencing in cancer. Finally, we examined the role of TSSC3 in human osteosarcoma SaOS2 cells. We showed that TSSC3 overexpression suppressed SaOS2 cell growth and increased apoptosis through caspase-3 upregulation, thereby, suggesting that TSSC3 may play a pro-apoptosis role to maintain the normal balance of growth. Taken together, these observations suggest that the epigenetic regulation of TSSC3, a pro-apoptosis gene, provides valuable insights into possible osteosarcoma therapies.

Histone deacetylase inhibits the growth and migration of human osteosarcoma cells

To explore the effects of histone deacetylase inhibitors (HDACIs) on human osteosarcoma in vitro and in vivo. HDACI suberoylanilide hydroxamic acid (SAHA) and sodium butyrate (SB) were employed to examine the effects on human osteosarcoma in vitro and in vivo. The in vitro effects of HDACI SAHA and SB were evaluated in SaOS2 and U2OS human osteosarcoma cell lines. Cell growth, cell cycle progression and histone acetylation were examined by methyl tolyl sulfide (MTS), flow cytometry and Western blot respectively.In addition, SAHA or SB was administered for 4 weeks in a murine xenograph model for assessing the in vitro effects. MTS assay revealed that SAHA and SB significantly suppressed the growth of SaOS2 and U2OS cells in a concentration-dependent manner.Western blot analysis indicated that the levels of acetylated H3 increased after HDACI treatment.Flow cytometry showed that SAHA arrested the cell cycle in G1 and G2/M phase, while SB arrested the cell cycle in G2/M phase. The tumor growth of murine xenograph model with SaOS2 was inhibited by SAHA and SB compared with vehicle control. HDACI SAHA and SB significantly inhibit the growth of human osteosarcoma cells and induce cell cycle arrest. Meanwhile, the tumor inhibitory effects were also validated in a murine xenograft model.

TSSC3 overexpression associates with growth inhibition, apoptosis induction and enhances chemotherapeutic effects in human osteosarcoma

Loss of expression of TSSC3, an apoptosis-related imprinted gene, has been reported in several cases of malignant tumors. However, the roles and mechanisms of TSSC3 in human osteosarcoma remain to be defined. In this study, we found TSSC3 to be downregulated during osteosarcoma transformation and progression in osteosarcoma cell lines and tissues. The SaOS2 cell line was used to further evaluate the precise role of TSSC3 in osteosarcoma development. Overexpression of TSSC3 markedly reduced cell vitality and growth, colony formation, Ki67 expression as well as cell cycle arrest in the G(0)/G(1) phase. Consistently, TSSC3 overexpression was associated with increased apoptosis assayed by annexin V/propidium iodide and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining. Subcutaneous injection of TSSC3 overexpressing SaOS2 cells into athymic nude mice showed that TSSC3 also inhibited tumorigenesis through growth inhibition and apoptosis induction in vivo. Further mechanistic studies revealed that the mitochondrial apoptosis pathway was required for TSSC3-mediated cell apoptosis. These findings support a suppressor role for TSSC3 in osteosarcoma development by regulating apoptosis. In addition, constitutive TSSC3 expression greatly enhanced the sensitivity of human osteosarcoma cells to the chemotherapeutic drugs cisplatin and epirubicin. Conversely, TSSC3 knockdown increased SaOS2 cell growth and decreased apoptosis in vitro and in vivo and reduced sensitivity of the cells to chemotherapy. This is the first study to demonstrate that TSSC3 has a potent tumor suppressor role in osteosarcoma, probably by inhibition of growth and induction of apoptosis via the mitochondrial apoptosis pathway.

In search of representative models of human bone-forming cells for cytocompatibility studies

Osteosarcoma-derived cells have been routinely used for studying osteoblastic functions, but it remains unclear to what extent they mimic the behavior of primary osteoblasts in the study of cells and materials interactions. This study reports comparatively on the responses of three human osteosarcoma cell lines, MG-63, Saos-2 and U-2 OS, and human primary osteoblasts cultured on Ti6Al4V surfaces or exposed to Ti particles. Phenotypic characterization of the cell lines revealed that Saos-2 cells and primary osteoblasts displayed similar expression patterns of Cbfa1, SP7 and osteocalcin. Unlike primary cells, the cell lines expressed markers of undifferentiated cells, had high proliferative rates and poor fibronectin matrix assembly. None of the three cell lines faithfully reproduced the adhesive behavior of primary osteoblasts when cultured on Ti6Al4V surfaces or exposed to Ti particles. Differences in cell growth between the cell lines and primary osteoblasts cultured on Ti6Al4V surfaces were also observed. Ti particles inhibited the growth of Saos-2 cells and primary osteoblasts to a similar extent, while no such effect was observed in U-2 OS and MG-63 cells. Saos-2 cells reproduced the alkaline phosphatase (ALP) activity profile of primary osteoblasts cultured on metallic surfaces or exposed to particles. Altogether, these results show that none of the osteoblast-like cells studied perfectly mimic the behavior of human osteoblast cells (hOB) on Ti6Al4V surfaces or exposed to Ti particles. Saos-2 cells reproduce some of the hOB responses such as the profile of enzymatic ALP activity when cultured on the surfaces or treated with particles as well as cell growth inhibition when exposed to Ti particles. Although in vitro cytocompatibility studies involve the evaluation of multiple parameters, Saos-2 cells may be used as representative of human osteoblasts when these standard tests are evaluated.

Osteogenic Potential of Biosilica on Human Osteoblast-Like (SaOS-2) Cells

Biosilica is a natural polymer, synthesized by the poriferan enzyme silicatein from monomeric silicate substrates. Biosilica stimulates mineralizing activity and gene expression of SaOS-2 cells. To study its effect on the formation of hydroxyapatite (HA), SaOS-2 cells were grown on different silicatein/biosilica-modified substrates (bone slices, Ca-P-coated coverslips, glass coverslips). Growth on these substrates induced the formation of HA nodules, organized in longitudinal arrays or spherical spots. Nodules of sizes above 1 μm were composed of irregularly arranged HA prism-like nanorods, formed by aggregates of three to eight SaOS-2 cells. Moreover, growth on silicatein/biosilica-modified substrates elicited increased [(3)H]dT incorporation into DNA, indicative of enhanced cell proliferation. Consequently, an in vitro-based bioassay was established to determine the ratio between [(3)H]dT incorporation and HA formation. This ratio was significantly higher for cells that grew on silicatein/biosilica-modified substrates than for cells on Ca-P-coated coverslips or plain glass slips. Hence, we propose that this ratio of in vitro-determined parameters reflects the osteogenic effect of different substrates on bone-forming cells. Finally, qRT-PCR analyses demonstrated that growth of SaOS-2 cells on a silicatein/biosilica matrix upregulated BMP2 (bone morphogenetic protein 2, inducer of bone formation) expression. In contrast, TRAP (tartrate-resistant acid phosphatase, modulator of bone resorption) expression remained unaffected. We conclude that biosilica shows pronounced osteogenicity in vitro, qualifying this material for studies of bone replacement also in vivo.

The role of biosilica in the osteoprotegerin/RANKL ratio in human osteoblast-like cells

Earlier studies have demonstrated that biosilica, synthesized by the enzyme silicatein, induces hydroxyapatite formation in osteoblast-like SaOS-2 cells. Here we study the effect of biosilica on the expressions of osteoprotegerin [ OPG] and the receptor activator for NF- κB ligand [ RANKL] in the SaOS-2 cell model. We show that during growth of SaOS-2 cells on biosiliceous matrices hydroxyapatite formation is induced, while syntheses of cartilaginous proteoglycans and sulfated glycosaminoglycans are down-regulated. Furthermore, quantitative real-time RT-PCR analysis revealed a strong time-depended increase in expression of OPG in biosilica exposed SaOS-2 cells while the steady-state expression level of RANKL remained unchanged. These results have been corroborated on the protein level by ELISA assays. Therefore, we propose that biosilica stimulated OPG synthesis in osteoblast-like cells counteracts those pathways that control RANKL expression and function (e.g. maturation of pre-osteoclasts and activation of osteoclasts). Hence, the data obtained in the present study reveal the considerable biomedical potential of biosilica for treatment and prophylaxis of osteoporotic disorders.

Different effects of ribosome biogenesis inhibition on cell proliferation in retinoblastoma protein‐ and p53‐deficient and proficient human osteosarcoma cell lines

To evaluate the effects of rRNA synthesis inhibition on cell cycle progression and cell population growth according to the RB and p53 status. RB- and p53-proficient U2OS cells and the RB- and p53-deficient SAOS-2 cells were used, rRNA transcription hindered by actinomycin D, and cell cycle analysed by flow cytometry. One hour of actinomycin D treatment induced in U2OS cells a block at the cell cycle checkpoints G(1)-S and G(2)-M, which was removed only after rRNA synthesis was resumed. rRNA synthesis inhibition did not influence cell cycle progression in SAOS-2 cells. No effect on cell cycle progression after actinomycin D-induced rRNA inhibition was also found in U2OS cells silenced for RB and p53 expression. A mild perturbation of cell cycle progression was observed in U2OS cells silenced for the expression of either RB or p53 alone. We also treated U2OS and SAOS-2 cells with actinomycin D for 1 h/day for 5 days. This treatment lightly reduced growth rate of the U2OS cell population, whereas cell population growth of SAOS-2 cells was completely inhibited. A marked reduction of ribosome content occurred in SAOS-2 cells after the long-term actinomycin D treatment, whereas no modification was observed in U2OS cells. These results demonstrate that inhibition of ribosome biogenesis does not hinder cell cycle progression in RB- and p53-deficient cells. A daily-repeated transitory inhibition of ribosome biogenesis leads to a progressive reduction of ribosome content with the consequent extinction of cancer cell population lacking RB and p53.