Proliferation Of Saos-2 Cells Research Articles

Designing laser-modified surface structures on titanium alloy custom medical implants using a hybrid manufacturing technology.

The hybrid technology combines an efficient material-removal process and implant surface treatment by the laser reducing time of manufacture process compared to currently used machining technologies. It also permits precise structuring of the implant material surface. Six structures of the Ti6Al4V ELI surface were designed and studied how the structure topography prepared with the hybrid technology affected the Escherichia coli adhesion to the surface and viability, as well as the growth, adhesion, and viability of human osteogenic Saos-2 cells cultured on the investigated surfaces. Results have confirmed that the microtopography of medical titanium alloy plays a beneficial role in bacterial adhesion and viability (number of bacteria found on reference surface: [5.9 ± 0.44] × 106 CFU/ml, sample no. 3: [8.8 ± 0.93] × 104 CFU/ml, and sample no. 5: [1.2 ± 0.23] × 107 CFU/ml; CFU - Colony Forming Unit). All tested structured surfaces enabled good cell attachment and proliferation of Saos-2 cells (viability of Saos-2 cells [% of control] for reference surface: 81.93%; sample no. 3: 75% and sample no. 5: 100%). Transcriptome analysis of genes commonly expressed in the process of osseointegration demonstrated that the use of hybrid technology allows designing structures that enhance osseointegration but it should be coupled with other methods of preventing bacterial growth, or with a different strategy to limit microbial colonization with the satisfactory osseointegration potential.

Effects of laser processing conditions on wettability and proliferation of Saos-2 cells on CoCrMo alloy surfaces

Abstract Any processing disturbances in laser surface texturing (LST) could compromise the resulting surface topography and their desired functional response. Disturbances such as focal plane offsets and beam incident angle variations are always present in LST processing of 3D parts and can affect the surface morphology. In this research the effects of these laser processing disturbances in producing laser induced surface structures (LIPSS) on CoCrMo alloy substrates were investigated. In particular, these two disturbances were considered as laser processing variables to determine their effects on functional responses of LIPSS treated surfaces, i.e. surface wettability and the proliferation of Saos-2 osteoblast-like cells were evaluated. It was found that the changes of laser processing conditions led to a decrease in surface wettability and Saos-2 cells proliferation. In addition, a correlation between surface wettability and cell proliferation on LIPSS treated surface was identified and conclusions made about the effects of investigated process disturbances on the functional response of LIPSS treated CoCrMo substrates.

Combined effect of surface polarization and ZnO addition on antibacterial and cellular response of Hydroxyapatite-ZnO composites.

Bacterial infection is among the serious concerns in orthopaedic during/after surgery. Here, we demonstrate a novel technique to induce the antibacterial response in biomaterial substrates via surface polarization. In the present work, hydroxyapatite, HA-xZnO (x = 3.0, 4.5 and 7.5 wt. %) composites were processed by solid state sintering route at 1250 °C for 2 h. After phase evolution analyses, the detailed dielectric and electrical measurements were performed over a wide range of temperature (30-500 °C) and frequency (1 Hz-1 MHz). The impedance spectroscopic analyses suggest the activation energies for grains and grain boundaries for HA and HA-3 wt.% ZnO are (1.36, 1.44 eV), and (1.18, 1.98 eV), respectively. The sintered samples were polarized under polarizing temperature and voltage of 500 °C and 20 kV, respectively. The viability of Escherichia Coli (E. Coli) and Staphylococcus Aureus (S. Aureus) bacteria is observed to reduce significantly for polarized HA-x ZnO (x = 4.5 and 7.5 wt. %) composites as compared to their respective counterparts. On the other hand, polarization supports the proliferation of SaOS2 cells. Overall, the combination of surface polarization and optimal ZnO addition in HA has been demonstrated to significantly improve the antibacterial as well as osteoblast-like SaOS2 cellular response.

Activating macrophages for enhanced osteogenic and bactericidal performance by Cu ion release from micro/nano-topographical coating on a titanium substrate

In the field of orthopaedics, inflammation-modulatory biomaterials are receiving increasing attentions due to their abilities to regulate innate immune response and mediate wound healing. In the current work, a Cu-containing micro/nano-topographical bio-ceramic surface (Cu-Hier-Ti surface) was employed as material model to explore the role played by Cu2+ release or material surface in regulating macrophage polarization as well as macrophage-mediated osteogenic and bactericidal effect. A Cu-free micro-topographical surface (Micro-Ti surface) generated by micro-arc oxidation was employed as control. The results showed that Cu2+ supplemented directly into the culture medium or released from Cu-Hier-Ti surface could polarize macrophages to pro-inflammatory M1 phenotype by activating Cu-transport signaling (copper transporter 1 (CTR1) and ATP7A) in macrophages, while the material characteristics exhibited anti-inflammatory effect to some extent by regulating integrin (α5, αM, β1 and β2) and TLR (TLR-3, TLR-4, Myd88 and Ticam-1/2) signaling. Macrophages grown on Cu-Hier-Ti surface or treated by Cu2+ could create a favorable inflammatory microenvironment for osteoblast-like SaOS-2 cell proliferation and differentiation. Moreover, Cu-Hier-Ti surface promoted macrophage capacity to engulf and kill bacteria, even though it did not show direct bactericidal effect against Staphylococcus aureus. In vivo results showed that Cu-Hier-Ti surface could lead to promoted osteointegraion and enhanced expression levels of M1 surface marker CD11c, growth factor BMP-6 and osteogenic makers including osteocalcin (OCN) and Runx-2 at the biomaterial/bone tissue interface in a rat model. The results indicate that Cu could be employed as a promising inflammation-modulatory agent to activate macrophages for enhanced osteogenic and bactericidal effect. Statement of significanceThe next generation of bone biomaterials should be active to regulate the local inflammatory environment such that it favors bone regeneration. For the design and development of Cu-containing inflammation-modulatory biomaterials, it is of great importance to recognize the exact role played by Cu2+ release or material surface characteristics. So far, relatively little is known about the regulatory role of Cu2+ or micro/nano-topographical surface on macrophages. The results in the current work suggest that Cu2+ release and material surface characteristics of Cu-containing micro/nano-topographical coating could activate distinct signaling pathways in macrophages. The activated M1 macrophages exhibited stimulatory effect on osteoblast maturation and enhanced bactericidal capacity against Staphylococcus aureus. This study might provide new thoughts for the development of multi-functional Cu-containing biomaterials.

Different diameters of titanium dioxide nanotubes modulate Saos-2 osteoblast-like cell adhesion and osteogenic differentiation and nanomechanical properties of the surface.

The formation of nanostructures on titanium implant surfaces is a promising strategy to modulate cell adhesion and differentiation, which are crucial for future application in bone regeneration. The aim of this study was to investigate how the nanotube diameter and/or nanomechanical properties alter human osteoblast like cell (Saos-2) adhesion, growth and osteogenic differentiation in vitro. Nanotubes, with diameters ranging from 24 to 66 nm, were fabricated on a commercially pure titanium (cpTi) surface using anodic oxidation with selected end potentials of 10 V, 15 V and 20 V. The cell response was studied in vitro on untreated and nanostructured samples using a measurement of metabolic activity, cell proliferation, alkaline phosphatase activity and qRT-PCR, which was used for the evaluation of osteogenic marker expression (collagen type I, osteocalcin, RunX2). Early cell adhesion was investigated using SEM and ELISA. Adhesive molecules (vinculin, talin), collagen and osteocalcin were also visualized using confocal microscopy. Moreover, the reduced elastic modulus and indentation hardness of nanotubes were assessed using a TriboIndenter™. Smooth and nanostructured cpTi both supported cell adhesion, proliferation and bone-specific mRNA expression. The nanotubes enhanced collagen type I and osteocalcin synthesis, compared to untreated cpTi, and the highest synthesis was observed on samples modified with 20 V nanotubes. Significant differences were found in the cell adhesion, where the vinculin and talin showed a dot-like distribution. Both the lowest reduced elastic modulus and indentation hardness were assessed from 20 V samples. The nanotubes of mainly 20 V samples showed a high potential for their use in bone implantation.

Dose-dependent enhancement of bioactivity by surface ZnO nanostructures on acid-etched pure titanium

ABSTRACTZinc (Zn) is found to be essential in biologic osseous functions, and deficiency of Zn may cause delayed skeletal growth and osteoporosis. Additionally, Zn-based coatings are reported to be effective to promote the bioactivity of implants. In this study, we employed the hydrothermal treatment to incorporate Zn into the surface of acid-etched pure Ti. The process was conducted in ammonia solution with an increased Zn precursor concentration (0.0002 M, 0.002 M and 0.02 M, respectively). XPS analysis demonstrated that the nanostructures were composed of ZnO. Proliferation and alkaline phosphatase (ALP) activity of osteoblast-like SaOS-2 cells were enhanced dose-dependently, compared to those on the acid-etched pure Ti without ZnO nanostructures. This study addresses a favourable surface modification method to improve the bioactivity of implants.

Let-7b acts as a tumor suppressor in osteosarcoma via targeting IGF1R.

MicroRNAs serve crucial functions in cancer progression by inhibiting the translation of target genes and causing mRNA degradation. However, the underlying regulatory mechanism of Let-7b in osteosarcoma (OS) has not, to the best of our knowledge, been comprehensively elucidated. The aim of the present study was to investigate the function of Let-7b in OS and clarify the regulation of insulin-like growth factor 1 receptor (IGF1R) by Let-7b. It was observed that Let-7b was significantly downregulated in OS tissues and cell lines compared with the matched adjacent non-tumorous tissues and human normal osteoblastic cell line hFOB 1.19. Overexpression of Let-7b significantly inhibited the proliferation and invasion of U2OS and SAOS-2 cells. A luciferase reporter assay validated that IGF1R was a downstream and functional target of Let-7b. Let-7b was also able to decrease the expression levels of IGF1R protein. Functional studies revealed that the antitumor effect of Let-7b was probably due to targeting and suppressing IGF1R expression. Furthermore, in OS tissues, IGF1R was identified to be significantly upregulated and negatively correlated with Let-7b levels. In conclusion, the results of the present study indicated that Let-7b suppresses OS cellular proliferation and invasion via targeting IGF1R. A novel candidate prognostic factor was identified and it is suggested that the Let-7b/IGF1R axis may represent a novel anti-metastasis therapeutic target in OS.

Proliferation inhibition and apoptosis promotion by dual silencing of VEGF and Survivin in human osteosarcoma.

Simultaneous silencing of multiple upregulated genes is an attractive and viable treatment strategy for many incurable diseases including cancer. Herein we used a dual gene-targeted siRNA conjugate composed of VEGF and Survivin siRNA sequences in the same backbone to inhibit proliferation and angiogenesis in two human osteosarcoma cell lines. We synthesized siRNA sequences targeting the VEGF and Survivin genes individually (VEGF siRNA and Survivin siRNA) or simultaneously (one-chain-double-target siRNA: dual siRNA). VEGF and Survivin mRNA and protein expression levels in human osteosarcoma MG-63 and Saos-2 cells were detected by qRT-PCR and western blot analysis. VEGF and Survivin protein location and expression were evaluated by immunohistochemistry and immunofluorescence staining. MG-63 and Saos-2 cell migration, proliferation, apoptosis, and angiogenesis were detected by scratch test, MTT assay, flow cytometry, and capillary tube assay respectively. The dual siRNA induced similar downregulation of VEGF and Survivin mRNA and protein levels, compared with VEGF siRNA or Survivin siRNA alone. The dual siRNA caused greater suppression of MG-63 and Saos-2 cell migration, proliferation and angiogenesis, and promoted more cell apoptosis than VEGF siRNA or Survivin siRNA alone, suggesting that the effects of the dual siRNA on inhibiting cell proliferation, migration, and angiogenesis and promoting apoptosis were superior to those of the single-target siRNAs. Simultaneous silencing of VEGF and Survivin using the dual siRNA may be an advantageous alternative for the development of therapeutic strategies against human osteosarcoma.

SIRT6 inhibits proliferation and invasion in osteosarcoma cells by targeting N-cadherin.

SIRT6, is a member of the NAD-dependent sirtuin family of enzymes, and has been reported as a novel tumor suppressor gene or oncogene, dependent on the type of cancer. However, the role of SIRT6 in osteosarcoma has not been investigated. The present study demonstrated that the expression of SIRT6 was downregulated in osteosarcoma tissues and osteosarcoma cell lines when compared with adjacent tissues or osteoblastic cell lines. Kaplan-Meier analysis was performed to evaluate the prognostic significance of SIRT6. The overall survival of patients with higher expression of SIRT6 was significantly longer than patients with lower expression. Subsequently, MTT and invasion assays were performed to detect the biological functions of SIRT6 in osteosarcoma cells in vitro. The results revealed that overexpression of SIRT6 inhibited SAOS-2 and MG-63 cell proliferation and invasion. Knockdown of SIRT6 enhanced cell ability for the proliferation and invasion. A qChIP assay, luciferase reporter assay, reverse transcription-quantitative polymerase chain reaction and western blotting confirmed that CDH2 (N-cadherin) was a target of SIRT6. SIRT6 overexpression suppressed N-cadherin on the mRNA and protein levels. In addition, it was confirmed that the promotional effect of Si-SIRT6 on OS cell growth and invasion was suppressed by downregulating N-cadherin. The present study suggested that SIRT6 may serve as a tumor suppressor during the development of osteosarcoma. In addition, N-cadherin may be a promising therapeutic target for osteosarcoma.

In vivo study on scaffolds based on chitosan, collagen, and hyaluronic acid with hydroxyapatite

Scaffolds based on chitosan, collagen, and hyaluronic acid supplemented with nano-hydroxyapatite were obtained with the use of the freeze-drying method. Composites swelling behavior was assessed by the liquid uptake test. The adhesion and proliferation of human osteosarcoma SaOS-2 cells on the scaffolds were examined in 4-day culture. The biocompatibility of the chosen scaffolds was further studied by in vivo implantation into subcutaneous tissue of rabbits.The results showed low stability of the scaffolds based on chitosan, collagen, and hyaluronic acid supplemented with hydroxyapatite. The addition of hydroxyapatite delayed the degradation process of the obtained scaffolds. The X-ray images of the tissues surrounding the scaffolds showed that both, the control scaffold without hydroxyapatite (HAp) and those with addition of 50% wt. HAp underwent degradation after 6 months. However, the scaffolds supplemented with 80% wt. HAp premained in the implanted place. The results showed satisfactory tissue response on the implanted scaffolds.

MiR‑590‑5p suppresses osteosarcoma cell proliferation and invasion via targeting KLF5.

Recently, microRNA (miR)‑590‑5p has been shown to inhibit tumorigenesis in colorectal and breast cancer; however, its function in osteosarcoma (OS) requires further investigation. In the present study miR‑590‑5p expression was poorly expressed in OS samples and cell lines when compared with that observed in normal cells. In addition, overexpression of miR‑590‑5p significantly reduced the proliferation, migration and invasion of SAOS2 and U2OS cells invitro, as well as inhibiting tumor sizes invivo. The results revealed that miR‑590‑5p directly targeted Kruppel‑like factor 5 (KLF5) in SAOS2 and U2OS cells. Their expression was inversely correlated with OS tissues. Finally, it was demonstrated that overexpression of KLF5 rescued the inhibitory effects of miR‑590‑5p on cell proliferation, migration and invasion. Overall, the results of the present study suggested that the miR‑590‑5p/KLF5 axis may regulate OS progression and thus, may be a novel therapeutic target for the treatment of patients with OS.

Preparation of micro/nanopatterned gelatins crosslinked with genipin for biocompatible dental implants.

Background: Collagen is a basic component of the periodontium and plays an important role in the function of the periodontal unit. Therefore, coating with collagen/gelatin has been applied to enable dental implants to positively interact with peri-implant tissues. Although the micro/nanoscale topography is an important property of the surface of dental implants, smaller collagen/gelatin surface patterns have not been sufficiently developed. Furthermore, only few reports on the behavior of cells on gelatin surfaces with different patterns and sizes exist. In this study, we developed micro/nanometer-scaled gelatin surfaces using genipin crosslinking, with the aim of understanding the use of patterning in surface modification of dental implants.Results: Grooves, holes, and pillars, with widths or diameters of 2 µm, 1 µm, or 500 nm were fabricated using a combination of molding and genipin crosslinking of gelatin. The stability of the different gelatin patterns could be controlled by the degree of genipin crosslinking. The gelatin patterns at 20 mM concentration of genipin and 41% crosslinking maintained a stable, patterned shape for at least 14 days in a cell culture medium. A cell morphology study showed that the cells on groves were aligned along the direction of the grooves. In contrast, the cells on pillars and holes exhibited randomly elongated filopodia. The vinculin spots of the cells were observed on the top of ridges and pillars or the upper surface of holes. The results of a cell attachment assay showed that the number of surface-attached cells increased with increasing patterning of the gelatin surface. Unlike the cell attachment assay, the results of a cell proliferation assay showed that Saos-2 cells prefer grooves with diameters of approximately 2 µm and 1 µm and pillars with diameters of 1 µm and heights of 500 nm. The number of cells on pillars with heights of 2 µm was larger than those of the other gelatin surface patterns tested.Conclusion: These data support that a detailed design of the gelatin surface pattern can control both cell attachment and proliferation of Saos-2 cells. Thus, gelatin surfaces patterned using genipin crosslinking are now an available option for biocompatible material patterning.

Boron-containing micro/nano-structured TiO2/bioceramics coatings with modulatory effects on SaOS-2 cell response

In order to take the advantages of micro/nano-topography and benefits of boron (B), B-containing micro/nano-structured TiO2/bioceramics coatings were developed on titanium (Ti) substrates for enhanced osteogenesis. The micro-arc oxidation was employed to fabricate a micro-topographical TiO2 layer (referred as Micro-Ti). Then, the second layer of nano-rods was prepared on Micro-Ti surface via hydrothermal treatment (referred as Hier-Ti). These nano-rods transformed to nano-particles after a post heat treatment (referred as Hier-HT-Ti). The adhesion, proliferation and differentiation of SaOS-2 cells were inhibited on Hier-Ti, but promoted on Hier-HT-Ti. The results indicate that the development of B-containing micro/nano-topographical coatings on Ti substrates could lead to enhanced osteogenesis.

Restoration of microRNA-130b expression suppresses osteosarcoma cell malignant behavior in vitro.

The alteration of microRNA (miR)-130b expression has been associated with promoting or suppressing numerous types of human cancer. A previous study evaluated the expression level of miR-130b in osteosarcoma tissues, and subsequently investigated the effects of miR-130b on the regulation of osteosarcoma cells malignant behavior in vitro. The study revealed that miR-130b expression levels were significantly reduced in osteosarcoma tissues and cell lines, compared with in adjacent tissues or normal cell lines. The expression of miR-130b inhibited the proliferation of osteosarcoma U-2OS and Saos-2 cells and impaired their ability to migrate, invade and form colonies. Furthermore, analysis using TargetScan and a dual-luciferase reporter assay demonstrated that miR-130b directly interacted with the 3′-untranslated region of transforming growth factor α (TGFA) and suppressed TGFA expression. TGFA and miR-130b were also inversely expressed in osteosarcoma tissues. In addition, expression of TGFA was able to alter miR-130-regulated osteosarcoma cell proliferation, migration and invasion. Thus, the present study demonstrated that miR-130b was downregulated in osteosarcoma tissues and cell lines, whereas the expression of miR-130b suppressed osteosarcoma cell malignant behavior. At the gene level, miR-130 directly targets and inhibits TGFA expression, in addition to phosphorylated protein kinase B and epidermal growth factor receptor expression levels. Further study is required to evaluate miR-130b antitumor activity in osteosarcoma.

The osteogenic, inflammatory and osteo-immunomodulatory performances of biomedical Ti-Ta metal-metal composite with Ca- and Si-containing bioceramic coatings.

It is known that good mechanical properties, low modulus to reduce stress-shielding effect, favorable osteogenic activity and limited inflammatory response are critical factors for orthopedic implants to induce excellent osteointegration. In this study, Ti-20% Ta metal-metal composite (referred as Ti-Ta) which consisted of Ti- and Ta-rich phases was fabricated via the strategy of powder metallurgy. Micro-arc oxidation (MAO) was employed to modify the surface of Ti-Ta composite. The surfaces of Ti-Ta composite after MAO treatment at an applied voltage of 250 (referred as MAO-250 V) or 300 V (referred as MAO-300 V) exhibited three distinct zones with significantly different morphological features and surface chemistry. Osteoblast-like SaOS-2 cells were found to be preferential to attach on the Ta-rich phase and its surrounding areas, exhibiting an area-dependent adhesion tendency. However, the attachment of Raw 264.7 macrophages was found to be insensitive to the surface characteristics. The proliferation and differentiation of SaOS-2 cells cultured on various surfaces basically followed the trend: MAO-modified surfaces > Ti-Ta surface > Ti surface. The Ti-Ta and MAO-modified surfaces were found to inhibit the inflammatory response and polarize macrophages to anti-inflammatory M2 phenotype compared to Ti surface. Moreover, the microenvironments created by Ti-Ta, MAO-250 V and MAO-300 V/macrophage interactions promoted the proliferation and differentiation of SaOS-2 cells compared to that created by Ti/macrophage interactions. MAO-300 V surface exhibited further enhanced positive osteo-immunomodulatory effects compared to Ti-Ta surface. Together, the Ti-20% Ta metal-metal composite modified by MAO at an applied voltage of 300 V is considered as a promising implant material for orthopedic applications.

Biological behavior of titanium processed by severe plastic deformation

Hydrostatic Extrusion (HE) was successfully applied to achieve grain refinement in titanium Grade 2. Cellular response (WST-1 assay) and protein adsorption tests (colloidal gold method) were performed to investigate the effects of severe plastic deformation on the biocompatibility of titanium. STEM observations were performed in order to characterize the microstructure of the deformed metal followed by correlation with it properties. This also made it possible to better understand the phenomena occurring at the surface. The surface characteristics, including wettability, surface energy and X-ray photoelectron spectroscopy (XPS), were used to determine those factors responsible for the increase in the biocompatibility of the extruded samples. The nanostructure had beneficial effect on proliferation and attachment of SaOS-2 cells. Grain refinement affected protein adsorption behavior. Non-specific adsorption represented by albumin was favored as a result of the high density of non-equilibrium defects. After the HE process, the biocompatibility of the titanium increased due to microstructural changes affecting the properties of the native passive oxide layer. Grain refinement promoted the formation of charged sites on the surface which improved protein adsorption and increased the amount of hydroxyl (OH−) groups. Surface free energy measurements revealed that this was attributed to a high acid-base component, γAB. Indirect XPS studies demonstrated that, after the HE process, the oxide layer on the titanium surface was thinner, which contributed to greater homogeneity and better corrosion resistance. The most possible explanations for the improved biocompatibility of the nanostructured materials are also discussed in this paper.

Enhanced SaOS-2 cell adhesion, proliferation and differentiation on Mg-incorporated micro/nano-topographical TiO2 coatings

Insufficient osteointegration still remains as one of the major problems associated with implant failures. Recently, modifying implant surface with hierarchical micro/nano-topography has attracted a great deal of attentions due to its stimulatory effect on osteogenesis. In the current work, micro-arc oxidation (MAO) was applied to create micro-scale topography on titanium substrate (referred as MAO surface). The MAO surface was mainly composed of porous TiO2 matrix distributed with CaSiO3 and CaO amorphous compounds. Subsequently, the hydrothermal treatment was employed to enable the formation of MgO nano-rods on MAO-fabricated surface (referred as MAO-HT surface). Thereafter, a post heat treatment was carried out for MAO-HT surface (referred as MAO-HT-800 surface). The rod-like nano-structures transformed to nano-particles after post heat treatment. The MAO-HT-800 surface exhibited significantly reduced release of Ca, Si and Mg ions compared to MAO-HT surface. In addition, the introduction of nano-structures onto MAO surface significantly increased surface hydrophilicity. Moreover, SaOS-2 cell behaviors such as adhesion, proliferation and differentiation in response to various surfaces basically followed the order: MAO surface < MAO-HT surface < MAO-HT-800 surface. The results indicate that modifying titanium surface with hierarchical micro/nano-topographies and tuning the morphological features of nano-structures could lead to enhanced osteogenesis.

MicroRNA-338-3p inhibits proliferation, migration, invasion, and EMT in osteosarcoma cells by targeting activator of 90\xa0kDa heat shock protein ATPase homolog 1

BackgroundOsteosarcoma (OS) is a rare, malignant bone tumor that primarily affects adolescents and has a high degree of malignancy and high incidence of recurrence and metastasis. Our study aimed to explore the role of miR-338-3p in OS cells.MethodsqRT-qPCR was performed to quantify miR-338-3p expression levels in OS tissue samples and in three common OS cell lines. MG-63 and Saos2 cells were separately transfected with miR-338-3p or NC mimics and miR-338-3p expression levels was determined by qRT-PCR. Cell proliferation was monitored using the Cell Counting Kit-8. Flow cytometer analysis was carried out to determine the distribution of cell cycle stages and apoptosis. Transwell assay was performed to measure the migratory and invasive capacities of MG-63 and Saos2 cells. The expression of Vimentin and E-cadherin was detected by western blot. Luciferase reporter assay, qRT-PCR and western blotting were performed to confirm the target of miR-338-3p.ResultsAnalysis by qRT-PCR revealed that miR-338-3p was downregulated in the tissue samples of 20 OS patients when compared with that in their matched adjacent non-tumor tissues. Furthermore, miR-338-3p was significantly downregulated in three common OS cell lines, namely, MG-63, Saos2, and HOS, when compared with that in the human osteoblast cell line hFOB1.19. Analysis by luciferase reporter assay, qRT-PCR, and western blotting revealed that activator of 90 kDa heat shock protein ATPase homolog 1 (AHSA1) is a direct target of miR-338-3p. miR-338-3p overexpression led to significant reduction in AHSA1 protein levels in MG63 and Saos2 cells. miR-338-3p overexpression reduced cell viability and migration and invasion behavior of MG63 and Saos2 cells. In addition, miR-338-3p overexpression suppressed epithelial–mesenchymal transition (EMT), induced a significant G1-phase arrest and did not affect the apoptosis in both MG-63 and Saos2 cells. Moreover, overexpression of AHSA1 reversed the inhibitory effect of miR-338-3p overexpression on proliferation, cell cycle, apoptosis, EMT, migration, and invasion of MG63 and Saos2 cells, thereby suggesting that miR-338-3p acts as a tumor suppressor in OS cells by targeting AHSA1.ConclusionsmiR-338-3p/AHSA1 can serve as a potential therapeutic target for OS therapy.

MicroRNA-134 inhibits osteosarcoma angiogenesis and proliferation by targeting the VEGFA/VEGFR1 pathway.

Vascular endothelial growth factor (VEGF) A and vascular endothelial growth factor receptor 1 (VEGFR1) signaling is crucial for angiogenesis and progression of osteosarcoma (OS). However, the regulation of the VEGF/VEGFR1 expression is still unclear in OS. Here, we show lower levels of miRNA-134 (miR-134) in OS tissues and cells. Induction of miR-134 overexpression significantly reduced the proliferation of Saos-2 cells and their secretion of pro-angiogenic factors, but increased the frequency of apoptotic Saos-2 cells. Treatment with conditioned medium from the cells transfected with miR-134 reduced the tube formation in human umbilical vein endothelial cells, which was abrogated by a combination of VEGF and conditioned medium. Furthermore, miR-134 significantly inhibited the growth of implanted OS tumors invivo and attenuated the VEGFA and VEGFR1 expression and angiogenesis in the tumors. In addition, higher levels of VEGFA and VEGFR1 were detected and miR-134 inhibited the expression of VEGFA and VEGFR1 in Saos-2 cells and OS tumors. Bioinformatic analysis indicated that the 3'-UTR of VEGFA and VEGFR1 contained the motif for miR-134 binding. Co-transfection with the luciferase reporter containing the wild-type, but not the mutant, of the 3'-UTR of VEGFA or VEGFR1 together with miR-134 decreased the luciferase activity in Saos-2 cells. Finally, miR-134 dramatically inhibited AKT activation and proliferating cell nuclear antigen expression in Saos-2 cells. Collectively, these findings indicate that miR-134 is a potential tumor suppressor by targeting VEGFA/VEGFR1 signaling to attenuate the progression and angiogenesis in OS. Therefore, miR-134 may be a novel biomarker for the prognosis of OS and a target for the design of new therapies for OS.

Liposomal formulation of vitamin A for the potential treatment of osteoporosis.

Vitamin A targeting to bones is a suitable treatment for osteoporosis. In this study, we have developed vitamin A-encapsulated liposomes that can be useful to deliver vitamin A to the bones in a selective manner. This liposomal formulation of vitamin A has been found to be stable for >6 months as no significant change in size and charge occurred. Vitamin A liposomes-induced cell proliferation in SaOS-2 (human osteosarcoma cell line) and release kinetics study concluded that the liposomal formulation of vitamin A gives a controlled release of vitamin A in comparison to the free vitamin. The MTT assay showed the proliferation of SaOS-2 cells after their treatment with vitamin A liposomes.