Human Osteoblast-like Saos-2 Cells Research Articles

혈소판 농축액을 도입한 인산칼슘 시멘트의 세포친화성

We have recently demonstrated the slow release of growth factors from calcium phosphate cements (CPCs) incorporated with platelet concentrate (PC). In the present study, the cell compatibility of CPCs was evaluated by culturing human osteoblast-like cells Saos-2 (ATCC HTB85) to demonstrate the PC effect. CPCs were characterized using scanning electron microscopy (SEM), X-Ray diffraction (XRD) and energy-dispersive X-ray spectroscopy (EDS) and their mechanical properties were tested. The physical and chemical properties of CPCs were little affected by the PC incorporation within the experimented range. The cell proliferation, morphology and migration, measurement of alkaline phosphatase (ALP) levels, mRNA expression for osteopontin (OPG), osteocalcin (OC), glyceraldehyde phosphate dehydrogenase (GAPDH) were evaluated. Higher cell proliferation was performed but the cell migration was not affected by the addition of PC. Actin filaments, focal adhesion, and localized integrin were clearly visible. The PC addition increased ALP activity and expression of GAPDH, OC, and OPG mRNA.

Isoquercitrin and polyphosphate co-enhance mineralization of human osteoblast-like SaOS-2 cells via separate activation of two RUNX2 cofactors AFT6 and Ets1

Isoquercitrin, a dietary phytoestrogen, is a potential stimulator of bone mineralization used for prophylaxis of osteoporotic disorders. Here we studied the combined effects of isoquercitrin, a cell membrane permeable 3-O-glucoside of quercetin, and polyphosphate [polyP], a naturally occurring inorganic polymer inducing bone formation, on mineralization of human osteoblast-like SaOS-2 cells. Both compounds isoquercitrin and polyP induce at non-toxic concentrations the mineralization process of SaOS-2 cells. Co-incubation experiments revealed that isoquercitrin (at 0.1 and 0.3μM), if given simultaneously with polyP (as Ca2+ salt; at 3, 10, 30 and 100μM) amplifies the mineralization-enhancing effect of the inorganic polymer. The biomineralization process induced by isoquercitrin and polyP is based on two different modes of action. After incubation of the cells with isoquercitrin or polyP the expression of the Runt-related transcription factor 2 [RUNX2] is significantly upregulated. In addition, isoquercitrin causes a strong increase of the steady-state-levels of the two co-activators of RUNX2, the activating transcription factor 6 [ATF6] and the Ets oncogene homolog 1 [Ets1]. The activating effect of isoquercitrin occurs via a signal transduction pathway involving ATF6, and by that, is independent from the induction cascade initiated by polyP. This conclusion is supported by the finding that isoquercitrin upregulates the expression of the gene encoding for osteocalcin, while polyP strongly increases the expression of the Ets1 gene and of the alkaline phosphatase. We show that the two compounds, polyP and isoquercitrin, have a co-enhancing effect on bone mineral formation and in turn might be of potential therapeutic value for prevention/treatment of osteoporosis.

Effects of interleukin-11 on the expression of human bone sialoprotein gene

Interleukin-11 (IL-11) is a bone marrow stromal fibroblast-derived cytokine with a wide spectrum of activities in different biological systems. IL-11 and IL-6 are two cytokines known to rely on osteoblast-osteoclast communication for their effects on osteoclast differentiation. Bone sialoprotein (BSP) is a mineralized connective tissue-specific protein expressed in differentiated osteoblasts, odontoblasts, and cementoblasts. To determine the molecular basis of the transcriptional regulation of the human BSP gene by IL-11, we conducted real-time polymerase chain reactions (PCR), transient transfection analyses with chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene, gel mobility shift assays, and a chromatin immunoprecipitation assay using human osteoblast-like Saos2 cells. IL-11 (20ng/ml) increased BSP, Runx2, and Osterix mRNA levels at 6h and the alkaline phosphatase (ALP) mRNA level at 12h in osteoblast-like Saos2 cells. In a transient transfection assay, IL-11 (20ng/ml, 12h) increased luciferase activities of constructs between -60LUC and -868LUC including the human BSP gene promoter. Transcriptional stimulations by IL-11 were partially inhibited in the constructs that included 2-bp mutations in the cAMP response element 1 (CRE1, -72 to -79) and CRE2 (-667 to -674). When mutations were made in pairs of CRE1 and CRE2 in -868LUC, the effect of IL-11 on luciferase activity was almost totally abrogated. Transcriptional activities induced by IL-11 were inhibited by protein kinase A, tyrosine kinase, ERK1/2, and PI3-kinase inhibitors. Gel mobility shift analyses showed that IL-11 increased nuclear proteins binding to CRE1 and CRE2. CREB1, phospho-CREB1, c-Fos, and c-Jun antibodies disrupted the formation of CRE1 and CRE2 protein complexes. These data demonstrate that IL-11 stimulates BSP gene transcription via CRE1 and CRE2 elements in the human BSP gene promoter.

Water Soluble Components of 'Osteocare' Promote Cell Proliferation, Differentiation, and Matrix Mineralization in Human Osteoblast-Like SaOS-2 Cells.

Osteocare, a herbal formulation, has been found to be very effective in bone mineralization and support of the microstructure of bone tissue. The water-soluble components of Osteocare (WSCO) induced osteogenic activity in human osteoblast-like SaOS-2 cells. The addition of WSCO (100 μg/ml) to SaOS-2 cells was effective in increasing the cell proliferation by 41.49% and DNA content by 1.9-fold. WSCO increased matrix mineralization in SaOS-2 cells by increased alkaline phosphatase levels and calcium-rich deposits as observed by Alizarin red staining. WSCO markedly increased mRNA expression for osteopontin (OPN), osteocalcin (OCN), type I collagen (Col I) in SaOS-2 cells, and it down-regulated IL-6 mRNA levels in SaOS-2 cells. The present study showed that WSCO plays an important role in osteoblastic bone formation through enhanced activities of ALP, Col I, bone matrix proteins such as OPN and OCN, down-regulation of cytokines like IL-6, as well as promoting mineralization in SaOS-2 cells.

Melatonin regulates human bone sialoprotein gene transcription.

Melatonin is produced by the pineal gland and regulates various physiological processes including osteoblast differentiation and bone formation. Bone sialoprotein (BSP) is a mineralized connective tissue-specific protein expressed in the early stage of cementum and bone mineralization. To elucidate the effects of melatonin on human BSP gene expression, we utilized human Saos2 osteoblast-like cells. Melatonin (100 nM) increased the level of BSP mRNA at 3 h, and the level became maximal at 12 and 24 h. We then investigated the melatonin-induced transcriptional activity of luciferase constructs (between -84LUC and -868LUC) including different lengths of the human BSP gene promoter transfected into Saos2 cells. The effects of melatonin abrogated in constructs included 2-bp mutations in the two cAMP response elements (CRE1 and CRE2). The effects of melatonin were suppressed by protein kinase A, tyrosine kinase, ERK1/2 and phosphatidylinositol 3-kinase inhibitors. Gel mobility shift assays showed that melatonin increased the binding of nuclear proteins to CRE1 and CRE2, and antibodies against CRE binding protein 1 (CREB1), phospho-CREB1, c-Fos, c-Jun, JunD and Fra2 disrupted CRE1 and CRE2 protein complex formation. These data indicate that melatonin induces BSP transcription via the CRE1 and CRE2 elements in the human BSP gene promoter. (J Oral Sci 56, 67-76, 2014).

Differentiation of human osteoblast-like SaOS-2 cells

Objective To observe the course of induction of human osteoblast-like SaOS-2 cells.Methods Differentation of human osteoblast-like SaOS-2 cells was induced.The change of osteoblast phenotype was observed by alkaline phosphatase (ALP) and alizarin red staining,and the difference in the osteocalcin (OC) expression was analyzed by using realtime-PCR technique during the induction of SaOS-2 cells.Results The IA values of ALP activity on the day 0,3,6,9 of osteoblast differentiation were 163 547.0 ±847.7,236 090.0 ±967.6,28 194.9 ± 185.0,and 11665.2 ± 151.0 respectively.The IA values of ALP activity on the day 3 were significantly higher than those on the day 0,6,9 of osteoblast differentiation(P ＜ 0.05).Many mineralization nodules were seen on the day 9 of osteoblast differentiation.Realtime quantitative polymerase chain reaction revealed that the expression level of OC gene in SaOS-2 cells was significantly higher on the day 6 and 9 of osteoblast differentiation than on the day 0 and 3 (P ＜0.01).Conclusion SaOS-2 cells can be induced into mature osteoblasts. Key words: Osteoblast-like SaOS-2 cells; Osteoblast differentiation; Osteocalcin

Effects of Er: YAG laser irradiation on wettability, surface roughness, and biocompatibility of SLA titanium surfaces: an in vitro study

The erbium: yttrium-aluminum-garnet (Er: YAG) laser has been introduced as an effective method in the decontamination of implant surfaces. Data concerning the effects of the Er: YAG laser on the biological and surface properties of titanium are conflicting. Cellular behavior is greatly affected by surface properties, including composition, roughness, wettability, and morphology of the titanium surface. The aim of this study was to investigate the influence of the Er: YAG laser on the biocompatibility, surface roughness, and wettability of sandblasted and acid-etched (SLA) titanium surfaces. Twenty-one SLA titanium disks were irradiated by the Er: YAG laser at a pulse energy of 100mJ, with a pulse frequency of 10Hz under water irrigation for 1min. Cell viability, surface roughness, and wettability alterations were evaluated. Thirteen nonirradiated SLA disks were used as the control groups. Human osteoblast-like SaOs-2 cells were seeded onto the disks in culture media. Cell viability was evaluated using the methylthiazol tetrazolium assay. The surface roughness and wettability of the test and control groups were measured using profilometer and tensiometer devices, respectively. A significantly higher cell viability rate was observed in the test group (p = 0.032). The surface roughness was significantly reduced in the test group compared with the control group (p = 0.008). The surface wettability was significantly higher in the test group (p = 0.004). Within the limits of this study, the application of the Er: YAG laser with the previously described properties did not appear to have adverse effects on the biocompatibility of the SLA titanium surfaces. Application of this laser decreased the surface roughness and increased the wettability of the SLA titanium surfaces.

The Effect of Carbon Dioxide (CO2) Laser on Sandblasting with Large Grit and Acid Etching (SLA) Surface.

The purpose of this study was to investigate the effect of 6W power Carbon Dioxide Laser (CO2) on the biologic compatibility of the Sandblasting with large grit and acid etching (SLA) titanium discs through studying of the Sarcoma Osteogenic (SaOS-2) human osteoblast-like cells viability. Sterilized titanium discs were used together with SaOS-2 human osteoblast-like cells. 6 sterilized SLA titanium discs of the experimental group were exposed to irradiation by CO2 laser with a power of 6W and 10.600nm wavelength, at fixed frequency of 80Hz during 45 seconds in both pulse and non-contact settings. SaOS-2 human osteoblast-like cells were incubated under 37°C in humid atmosphere (95% weather, 5% CO2) for 72 hours. MTT test was performed to measure the ratio level of cellular proliferation. The results indicated that at 570nm wavelength, the 6W CO2 laser power have not affected the cellular viability. CO2 laser in 6w power has had no effect on the biologic compatibility of the SLA titanium surface.

Transcriptional regulation of the human bone sialoprotein gene by fibroblast growth factor 2

Fibroblast growth factor 2 (FGF2), a member of the FGF family, positively regulates bone formation and osteoblast differentiation. Bone sialoprotein (BSP) is highly expressed during early bone formation and may play a role in primary mineralization of bone. In the present study, FGF2 (10 ng/mL) was found to increase the levels of Runx2 and BSP mRNA at 3 and 12 h in human osteoblast-like Saos2 cells. Transient transfection assays were performed using chimeric constructs of the human BSP gene promoter ligated with a luciferase reporter gene. FGF2 (10 ng/mL, 12 h) induced the luciferase activities of the -84LUC and -927LUC constructs in Saos2 cells. The results of gel shift assays showed that FGF2 (10 ng/mL) increased the binding of nuclear protein to the FGF2 response element (FRE) and the activator protein 1 (AP1) binding site. Antibodies against Dlx5, Msx2, Runx2 and Smad1 blocked FRE-protein complex formation, and antibodies against CREB1, c-Jun and Fra2 interrupted AP1-protein complex formation. These results indicate that FGF2 increases BSP transcription by targeting the FRE and AP1 elements in the proximal promoter of the human BSP gene. Moreover, the transcription factors Dlx5, Msx2, Runx2, Smad1, CREB1, c-Jun and Fra2 could be key regulators of the effects of FGF2 on human BSP transcription.

Osteoblast behavior on various ultra short pulsed laser deposited surface coatings

Ultra short pulsed laser deposition technique was utilized to create amorphous diamond, alumina and carbon nitride, and two different titania coatings on silicon wafers, thus producing five different surface deposited films with variable physico-chemical properties. The surface characterizations, including the roughness, the contact angle and the zeta potential measurements were performed before we tested the growth properties of human osteoblast-like Saos-2 cells on these surfaces (three separate experiments). The average roughness and hydrophobicity were the highest on titania-deposited surfaces, while carbon nitride was the most hydrophilic one. Osteoblasts on all surfaces showed a flattened, spread-out morphology, although on amorphous diamond the cell shape appeared more elongated than on the other surfaces. On rough titania, the area covered by the osteoblasts was smaller than on the other ones. Cell proliferation assay did not show any statistically significant differences.

Role of GSH/GSSG redox couple in osteogenic activity and osteoclastogenic markers of human osteoblast‐like SaOS‐2 cells

This study comprised a comprehensive analysis of the glutathione (GSH) redox system during osteogenic differentiation in human osteoblast-like SaOS-2 cells. For the first time, a clear relationship between expression of specific factors involved in bone remodeling and the changes in the GSH/oxidized GSH (GSSG) redox couple induced during the early phases of the differentiation and mineralization process is shown. The findings show that the time course of differentiation is characterized by a decrease in the GSH/GSSG ratio, and this behavior is also related to the expression of osteoclastogenic markers. Maintenance of a high GSH/GSSG ratio due to GSH exposure in the early phase of this process increases mRNA levels of osteogenic differentiation markers and mineralization. Conversely, these events are decreased by a low GSH/GSSG ratio in a reversible manner. Redox regulation of runt-related transcription factor-2 (RUNX-2) activation through phosphorylation is shown. An inverse relationship between RUNX-2 activation and extracellular signal-regulated kinases related to GSH redox potential is observed. The GSH/GSSG redox couple also affects osteoclastogenesis, mainly through osteoprotegerin down-regulation with an increase in the ratio of receptor activator of NF-κB ligand to osteoprotegerin and vice versa. No redox regulation of receptor activator of NF-κB ligand expression was found. These results indicate that the GSH/GSSG redox couple may have a pivotal role in bone remodeling and bone redox-dysregulated diseases. They suggest therapeutic use of compounds that are able to modulate not just the GSH level but the intracellular redox system through the GSH/GSSG redox couple.

An in vitro evaluation of the responses of human osteoblast-like SaOs-2 cells to SLA titanium surfaces irradiated by erbium:yttrium–aluminum–garnet (Er:YAG) lasers

Erbium:yttrium-aluminum-garnet (Er:YAG) laser treatment is an effective option for the removal of bacterial plaques. Many studies have shown that Er:YAG lasers cannot re-establish the biocompatibility of titanium surfaces. The aim of this study was to evaluate the responses of the human osteoblast-like cell line, SaOs-2, to sand-blasted and acid-etched (SLA) titanium surface irradiation using different energy settings of an Er:YAG laser by examining cell viability and morphology. Forty SLA titanium disks were irradiated with an Er:YAG laser at a pulse energy of either 60 or 100 mJ with a pulse frequency of 10 Hz under water irrigation and placed in a 24-well plate. Human osteoblast-like SaOs-2 cells were seeded onto the disks in culture media. Cells were then kept in an incubator with 5% carbon dioxide at 37 °C. Each experimental group was divided into two smaller groups to evaluate cell morphology by scanning electron microscope and cell viability using 3-4,5-dimethylthiazol 2,5-diphenyltetrazolium bromide test. In both the 60 and the 100 mJ experimental groups, spreading morphologies, with numerous cytoplasmic extensions, were observed prominently. Similarly, a majority of cells in the control group exhibited spreading morphologies with abundant cytoplasmic extensions. There were no significant differences among the laser and control groups. The highest cell viability rate was observed in the 100 mJ laser group. No significant differences were observed between the cell viability rates of the two experimental groups (p = 1.00). In contrast, the control group was characterized by a significantly lower cell viability rate (p < 0.001). Treatments with an Er:YAG laser at a pulse energy of either 60 or 100 mJ do not reduce the biocompatibility of SLA titanium surfaces. In fact, modifying SLA surfaces with Er:YAG lasers improved the biocompatibility of these surfaces.

Nanoscale SiO&lt;sub&gt;2&lt;/sub&gt;/ZrO&lt;sub&gt;2&lt;/sub&gt; Particulate-Reinforced Titanium Composites for Bone Implant Materials

The mechanical property of porous pure titanium (Ti) scaffold with high porosity might become poorer than that of natural bone. In this study, new Ti-based biocompatible composites were developed to simultaneously meet the requirements of low elastic modulus and appropriate strength for implant materials when they are scaffolded into a porous structure. The nanoscale particulate-reinforced Ti-based composites with different concentrations of oxide particles such as SiO2 and ZrO2 were prepared using a powder metallurgical method. The strengths of the new nanoscale particulate-reinforced titanium composites were found to be significantly higher than that of pure Ti. Cell culture results revealed that the nanoscale particulate-reinforced titanium composites showed excellent biocompatibility and cell adhesion. Human SaOS2 osteoblast-like cells grew and spread well on the surfaces of the new titanium composites. The nanoscale SiO2 and ZrO2 particulate-reinforced titanium composites are promising materials that have great potential for use as an orthopedic implant material.

Osteoblast cell response to nanoscale SiO2/ZrO2 particulate-reinforced titanium composites and scaffolds by powder metallurgy

The strength of porous pure titanium (Ti) scaffold decreases dramatically with the introduction of porosity and might become lower than that of natural bone when with high porosity. To simultaneously meet the requirements of low-elastic modulus and appropriate strength for implant materials, it is necessary to develop new biocompatible Ti-based composites that are stronger than those currently available while providing low-elastic modulus and adequate strength when they are scaffolded into a porous structure. In this study, new particulate-reinforced Ti-based composites with nanoscale oxide particles of SiO2 and ZrO2 were prepared using a powder metallurgical method. The strengths of the new particulate-reinforced titanium composites were found to be significantly higher than that of a pure Ti. Cell culture results revealed that the articulate-reinforced titanium composites showed excellent biocompatibility and cell adhesion. Human osteoblast-like SaOS2 cells grew and spread well on the surfaces of the new titanium composites. The present study illustrated the feasibility of using the particulate-reinforced titanium composites as an orthopaedic implant material.

Fibroblast growth factor 2 and forskolin induce mineralization-associated genes in two kinds of osteoblast-like cells

Fibroblast growth factor 2 (FGF2) and cyclic AMP (cAMP) play critical roles in controlling the differentiation of osteoblasts and mineralization of bone. We have previously reported that each of FGF2 and forskolin (FSK) alone increase transcription of the bone sialoprotein (BSP) gene, and that together (FGF/FSK) they upregulate BSP gene expression synergistically in rat osteoblast-like ROS 17/2.8 cells. However, other genes that are upregulated after stimulation by FGF2, FSK or FGF/FSK remain unclear. In the present study, we investigated candidate genes associated with mineralization after stimulation by FGF2, FSK and FGF/FSK in two kinds of osteoblast-like cells using microarray and real-time PCR. In ROS17/2.8 cells, FGF2 and FSK each increased the gene expression of c-FOS (7.2-fold and 10.7-fold, respectively). However, FGF/FSK did not induce c-FOS gene expression. FGF2 increased the expression of the dentin matrix protein 1 (DMP1, 129.8-fold) gene. In contrast, FGF/FSK increased the expression of the amphiregulin (AREG, 73-fold) gene maximally. In human osteoblast-like Saos2 cells, FGF2 increased the expression of the osteopontin (SPP1, 16.7-fold), interleukin-8 (IL8, 6.4-fold) and IL11 (4.8-fold) genes. FSK induced the expression of the IL6 (2.6-fold), IL11 (4.0-fold), chemokine ligand 13 (CXCL13, 2.8-fold) and bone morphogenetic protein 2 (BMP2, 2.5-fold) genes. These results suggest that FGF2 and FSK might be crucial regulators of mineralization and bone formation.

Intrinsically superparamagnetic Fe-hydroxyapatite nanoparticles positively influence osteoblast-like cell behaviour

BackgroundSuperparamagnetic nanoparticles (MNPs) have been progressively explored for their potential in biomedical applications and in particular as a contrast agent for diagnostic imaging, for magnetic drug delivery and more recently for tissue engineering applications. Considering the importance of having safe MNPs for such applications, and the essential role of iron in bone remodelling, this study developed and analysed novel biocompatible and bioreabsorbable superparamagnetic nanoparticles, that avoid the use of poorly tolerated magnetite based nanoparticles, for bone tissue engineering applications.ResultsMNPs were obtained by doping hydroxyapatite (HA) with Fe ions, by directly substituting Fe2+ and Fe3+ into the HA structure yielding superparamagnetic bioactive phase. In the current study, we have investigated the effects of increasing concentrations (2000 μg/ml; 1000 μg/ml; 500 μg/ml; 200 μg/ml) of FeHA MNPs in vitro using Saos-2 human osteoblast-like cells cultured for 1, 3 and 7 days with and without the exposure to a static magnetic field of 320 mT. Results demonstrated not only a comparable osteoblast viability and morphology, but increased in cell proliferation, when compared to a commercially available Ha nanoparticles, even with the highest dose used. Furthermore, FeHA MNPs exposure to the static magnetic field resulted in a significant increase in cell proliferation throughout the experimental period, and higher osteoblast activity.In vivo preliminary results demonstrated good biocompatibility of FeHA superparamagnetic material four weeks after implantation into a critical size lesion of the rabbit condyle.ConclusionsThe results of the current study suggest that these novel FeHA MNPs may be particularly relevant for strategies of bone tissue regeneration and open new perspectives for the application of a static magnetic field in a clinical setting of bone replacement, either for diagnostic imaging or magnetic drug delivery.

Collagen with simvastatin promotes cell metabolism in osteoblast-like SaOS-2 cells

Background:Simvastatin (SMV) is one of the cholesterol-lowering pharmacological drugs. Recent studies demonstrate that it has a bone stimulatory effect. The present study was designed to investigate the effect of SMV along with collagen membrane on osteoblast-like SaOS-2 cells and also to standardize the dosage of SMV to be incorporated into the collagen membrane to achieve regeneration.Materials and Methods:SMV at doses of 0.5, 1, 1.5, and 2 mg was incorporated into the collagen membrane and cell metabolism was assessed by (3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide) (MTT) assay for 24 h.Results:SMV enhanced cell metabolism dose dependently at 24-h time and the maximum effect was obtained at a concentration of 1.5 mg of SMV.Conclusion:These results indicate that collagen with 1.5 mg SMV exhibits positive effect on cell metabolism of human osteoblast-like SaOS-2 cells.

Effect of nanocoating with rhamnogalacturonan‐I on surface properties and osteoblasts response

Long-term stability of titanium implants are dependent on a variety of factors. Nanocoating with organic molecules is one of the methods used to improve osseointegration. Therefore, the aim of this study is to evaluate the in vitro effect of nanocoating with pectic rhamnogalacturonan-I (RG-I) on surface properties and osteoblasts response. Three different RG-Is from apple and lupin pectins were modified and coated on amino-functionalized tissue culture polystyrene plates (aminated TCPS). Surface properties were evaluated by scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy. The effects of nanocoating on proliferation, matrix formation and mineralization, and expression of genes (real-time PCR) related to osteoblast differentiation and activity were tested using human osteoblast-like SaOS-2 cells. It was shown that RG-I coatings affected the surface properties. All three RG-I induced bone matrix formation and mineralization, which was also supported by the finding that gene expression levels of alkaline phosphatase, osteocalcin, and collagen type-1 were increased in cells cultured on the RG-I coated surface, indicating a more differentiated osteoblastic phenotype. This makes RG-I coating a promising and novel candidate for nanocoatings of implants.

Cellular proliferation, cellular viability, and biocompatibility of HA‐ZnO composites

One of the important issues in the development of hydroxyapatite (HA)-based biomaterials is the prosthetic infection, which limits wider use of monolithic HA despite superior cellular response. Recently, we reported that ZnO addition to HA can induce bactericidal property. It is therefore important to assess how ZnO addition influences the cytotoxicity property and cell adhesion/proliferation on HA-ZnO composite surfaces in vitro. In the above perspective, the objective of this study is to investigate the cell type and material composition dependent cellular proliferation and viability of pressureless sintered HA-ZnO composites. The combination of cell viability data as well as morphological observations of cultured human osteoblast-like SaOS2 cells and mouse fibroblast L929 cells suggests that HA-ZnO composites containing 10 Wt % or lower ZnO exhibit the ability to support cell adhesion and proliferation. Both SaOS2 and L929 cells exhibit extensive multidirectional network of actin cytoskeleton and cell flattening on the lower ZnO containing (≤10 Wt %) HA-ZnO composites. The in vitro results illustrate how variation in ZnO content can influence significantly the cell vitality, as evaluated using MTT biochemical assay. Also, the critical statistical analysis reveals that ZnO addition needs to be carefully tailored to ensure good in vitro cytocompatibility. The underlying reasons for difference in biological properties are analyzed. It is suggested that surface wettability as well as dissolution of ZnO, both contribute to the observed differences in cellular viability and proliferation.

AMP-activated protein kinase attenuates Wnt/β-catenin signaling in human osteoblastic Saos-2 cells

AMP-activated protein kinase (AMPK) is a key sensor of cellular energetic conditions. Recent studies suggest that AMPK affects osteoblast differentiation, although its role and mechanism are not fully understood. One of the most important signals in osteoblast differentiation is the Wnt/β-catenin pathway which induces T-cell transcription factor 1 (TCF)-dependent transcription. Using human osteoblast-like Saos-2 cells, we determined whether AMPK modulates Wnt/β-catenin signaling in osteoblasts. Chemical activators of AMPK (AICAR [5-aminoimidazole-4-carboxamide riboside], metformin) suppressed Wnt3a-induced TCF-dependent transcriptional activity. Transactivation by Wnt was potentiated by inhibiting β-catenin degradation with lithium chloride (LiCl). LiCl-induced Wnt transactivation was suppressed by addition of metformin. Metformin increased the phosphorylation of β-catenin and decreased β-catenin protein levels leading to suppression of Wnt/β-catenin signaling. Our present study showed that AMPK attenuates Wnt/β-catenin signaling by reducing β-catenin protein levels in osteoblast-like cells.