Osteoblastic SaOS-2 Cells Research Articles

Implant-Derived S. aureus Isolates Drive Strain-Specific Invasion Dynamics and Bioenergetic Alterations in Osteoblasts

Background: Implants are integral to modern orthopedic surgery. The outcomes are good, but infections remain a serious issue. Staphylococcus aureus (S. aureus), along with Staphylococcus epidermidis, are predominant pathogens responsible for implant-associated infections, as conventional antibiotic treatments often fail due to biofilm formation or the pathogens’ ability to invade cells and to persist intracellularly. Objectives: This study therefore focused on interactions of S. aureus isolates from infected implants with MG63 and SaOS2 osteoblasts by investigating the adhesion, invasion, and the impact on the bioenergetics of osteoblasts. Methods and Results: We found that the ability of S. aureus to adhere to osteoblasts depends on the isolate and was not associated with a single gene or expression pattern of characteristic adhesion proteins, and further, was not correlated with invasion. However, analysis of invasion capabilities identified better invasion conditions for S. aureus isolates with the SaOS2 osteoblastic cells. Interestingly, metabolic activity of osteoblasts remained unaffected by S. aureus infection, indicating cell survival. In contrast, respiration assays revealed an altered mitochondrial bioenergetic turnover in infected cells. While basal as well as maximal respiration in MG63 osteoblasts were not influenced statistically by S. aureus infections, we found increased non-mitochondrial respiration and enhanced glycolytic activity in the osteoblasts, which was again, more pronounced in the SaOS2 osteoblastic cells. Conclusions: Our findings highlight the complexity of S. aureus-host interactions, where both the pathogen and the host cell contribute to intracellular persistence and survival, representing a major factor for therapeutic failures.

A Porous Fluoride-Substituted Bovine-Derived Hydroxyapatite Scaffold Constructed for Applications in Bone Tissue Regeneration.

Hydroxyapatite is widely used in bone implantation because of its similar mineral composition to natural bone, allowing it to serve as a biocompatible osteoconductive support. A bovine-derived hydroxyapatite (BHA) scaffold was developed through an array of defatting and deproteinization procedures. The BHA scaffold was substituted with fluoride ions using a modified sol-gel method to produce a bovine-derived fluorapatite (BFA) scaffold. Fourier-transform infrared spectroscopy and X-ray diffraction analysis showed that fluoride ions were successfully substituted into the BHA lattice. According to energy dispersive X-ray analysis, the main inorganic phases contained calcium and phosphorus with a fluoride ratio of ~1-2 wt%. Scanning electron microscopy presented a natural microporous architecture for the BFA scaffold with pore sizes ranging from ~200-600 μm. The BHA scaffold was chemically stable and showed sustained degradation in simulated-body fluid. Young's modulus and yield strength were superior in the BFA scaffold to BHA. In vitro cell culture studies showed that the BFA was biocompatible, supporting the proliferative growth of Saos-2 osteoblast cells and exhibiting osteoinductive features. This unique technique of producing hydroxyapatite from bovine bone with the intent of producing high performance biomedically targeted materials could be used to improve bone repair.

Chitosan-Silver Thin Film-Coated Titanium Coupons using Silane Linkers Inhibit Biofilm and Planktonic Growth

Titanium is a component of many implants and orthopedic instruments, such as screws and rods; however, this and other materials may serve as a nidus for bacterial biofilm attachment. Chitosan is a biopolymer with advantages as a surface modifier, and silver ions have broad-spectrum antimicrobial properties. For this study, chitosan is bound to silver through a novel, patented process. The purpose of this research is to characterize silane-linked chitosan-silver coatings for titanium, including comparing antimicrobial efficacy. In this study, silane-linked chitosan-silver titanium coupons reduced Staphylococcus aureus (S. aureus) viability by 98% (planktonic) and 99.5% (biofilm) while supporting viability of Saos-2 osteoblast cells at levels of 75% compared with control uncoated titanium. Due to the observation of retaining osteoblast viability while reducing bacterial viability, silane-linked chitosan-silver coatings could be useful for titanium implants to reduce post-operative infection as well as support the healing process. KEYWORDS: Titanium; Staphylococcus aureus; Silver; Chitosan; Silane; Osteoblast; Antimicrobial; Coating

Biological Performance of Titanium Surfaces with Different Hydrophilic and Nanotopographical Features

The micro- and nanostructures, chemical composition, and wettability of titanium surfaces are essential for dental implants’ osseointegration. Combining hydrophilicity and nanostructure has been shown to improve the cell response and to shorten the healing time. This study aimed to investigate the biological response to different wettability levels and nanotopographical modifications in aged and non-aged titanium surfaces. By plasma etching titanium surfaces with the fluorine gas 2,3,3,3-tetrafluoropropene (R1234yF), additional nanostructures were created on the sample surfaces. Furthermore, this treatment resulted in sustained superhydrophilicity and fluoride accumulation. We examined the effect of various nanostructuring processes and aging using scanning electron microscopy, roughness analyses, and wettability measurement. In addition, all the surface modifications were tested for their effects on fibroblast adhesion, proliferation, and viability as well as osteoblast differentiation. Our study indicates that the plasma etching, with 2,3,3,3-tetrafluoropropene, of the machined and SLA surface neither favored nor had an adverse effect on the biological response of the SAOS-2 osteoblast cell line. Although the fluorine-plasma-etched surfaces demonstrated improved fibroblast cell viability, they did not lead to improved early osseointegration. It is still unclear which surface properties mainly influence fibroblast and osteoblast adhesion. Further physiochemical aspects, such as electrostatic interaction and surface tension, are crucial to be analyzed along with wettability and roughness.

Chemical characterization of the crude extract of Sauromatum venosum (voodoo lily) and docking study with 12-O-acetylingol 8-tiglate for cytotoxicity testing in SaOS2 (osteoblastic osteosarcoma cells).

The current study was carried out to investigate the anticancer potential of Sauromatum venosum (SV) tuber by gas chromatography with high-resolution mass spectrometry (GC-HRMS) analysis of ethanolic (eSV), hydroalcoholic (hSV), and aqueous extracts (wSV), and in silico study were performed to investigate the main targets of 12-O-acetylingol 8-tiglate by computational docking. The GC-HRMS analysis of three plant samples was carried out on a system equipped with a high-resolution mass spectrometer. The major compounds were identified in all crude extracts. Computation docking analysis was performed for the prediction of the main target of the cancer proliferation of active compound of the Sauromatum venosum tuber extract in cancer therapy. A total of 45 phytocompounds were detected including diterpenoids, esters of fatty acid, hydrocarbons, and alkanes in the tuber of SV. Among all the crude samples tested, eSV showed the lowest IC50 value treated with SaOS2 cells. 12-O-acetylingol 8-tiglate is one of the phytocompounds identified in eSV extract and has been found to exhibit cytotoxic effects against various cancer cells, as reported in the research. It shows the optimum binding affinity with -8.59kcal/mol binding energy with a molecular target protein TNF-α (PDB ID: 7PKA). The observed interactions strongly support the anticancer activity of 12-O-acetylingol 8-tiglate and its role in the medicinal efficacy of the plant. These findings highlight the potential of the compound as a valuable source for the development of a therapeutic agent aimed at combating cancer. However, it is important to note that additional in vitro and in vivo studies are required to validate these findings and establish the therapeutic potential.

Relevant Aspects of Titanium Topography for Osteoblastic Adhesion and Inhibition of Bacterial Colonization.

The influence of the surface topography of dental implants has been studied to optimize titanium surfaces in order to improve osseointegration. Different techniques can be used to obtain rough titanium, however, their effect on wettability, surface energy, as well as bacterial and cell adhesion and differentiation has not been studied deeply. Two-hundred disks made of grade 4 titanium were subjected to different treatments: machined titanium (MACH), acid-attacked titanium (AE), titanium sprayed with abrasive alumina particles under pressure (GBLAST), and titanium that has been treated with GBLAST and then subjected to AE (GBLAST + AE). The roughness of the different treatments was determined by confocal microscopy, and the wettability was determined by the sessile drop technique; then, the surface energy of each treatment was calculated. Osteoblast-like cells (SaOs-2) were cultured, and alkaline phosphatase was determined using a colorimetric test. Likewise, bacterial strains S. gordonii, S. oralis, A. viscosus, and E. faecalis were cultured, and proliferation on the different surfaces was determined. It could be observed that the roughness of the GBLAST and GBLAS + AE was higher, at 1.99 and 2.13 μm of Ra, with respect to the AE and MACH samples, which were 0.35 and 0.20 μm, respectively. The abrasive treated surfaces showed lower hydrophilicity but lower surface energy. Significant differences could be seen at 21 days between SaOS-2 osteoblastic cell adhesion for the blasted ones and higher osteocalcin levels. However, no significant differences in terms of bacterial proliferation were observed between the four surfaces studied, demonstrating the insensitivity of bacteria to topography. These results may help in the search for the best topographies for osteoblast behavior and for the inhibition of bacterial colonization.

Improved osteogenic activity of NiTi orthopedic implant by HAp-Nb2O5 composite coatings: Materials and biological points of view

The surface properties of NiTi, as an interface between the synthetic implant and living tissue, play a vital role in guaranteeing implantation success, especially during the initial stage. This contribution endeavors to enhance the surface features of NiTi orthopedic implants through the application of HAp-based coatings, placing emphasis on assessing the influence of Nb2O5 particles concentration in the electrolyte on resultant properties of HAp-Nb2O5 composite electrodeposits. The coatings were electrodeposited via pulse current mode under galvanostatic current control from an electrolyte containing 0–1 g/L of Nb2O5 particles. Surface morphology, topography, and phase composition were evaluated using FESEM, AFM, and XRD, respectively. EDS was employed to study surface chemistry. In vitro biomineralization and osteogenic activity of the samples were studied by immersing the samples in SBF and incubating them with osteoblastic SAOS-2 cells, respectively. The added Nb2O5 particles, at the optimum concentration, stimulated biomineralization, suppressed the Ni ion leaching, and improved SAOS-2 cell adhesion and proliferation. NiTi implant coated by HAp-0.50 g/L Nb2O5 layer showed tremendous osteogenic properties. Overall, the HAp-Nb2O5 composite layers bring forth fascinating coating in vitro biological performance, reducing Ni leaching, and promoting osteogenic activity, which are fundamental for the successful use of NiTi in vivo.

Effects of Hypochlorous Acid and Hydrogen Peroxide Treatment on Bacterial Disinfection Treatments in Implantoplasty Procedures.

One of the main problems in oral implantology today is peri-implantitis, which affects almost 20% of dental implants placed in patients. One of the most commonly used techniques to eliminate bacterial biofilm is the implantoplasty, that consists of the mechanical modification of the implant surface topography followed by treatments with chemical reagents for decontamination. In this study, the main aim is to evaluate the use of two different chemical treatments based on hypochlorous acid (HClO) and hydrogen peroxide (H2O2). For this purpose, 75 titanium grade 3 discs were treated with implantoplasty according to established protocols. Twenty-five discs were used as controls, 25 were treated with concentrated HClO and 25 were treated with concentrated HClO followed by treatment with 6% H2O2. The roughness of the discs was determined using the interferometric process. Cytotoxicity with SaOs-2 osteoblastic cells was quantified at 24 and 72 h, whereas bacteria proliferation using S. gordonii and S. oralis bacteria was quantified at 5 s and 1 min of treatment. The results showed an increase in the roughness values, the control discs had an Ra of 0.33 μm and those treated with HClO and H2O2 reached 0.68 μm. Cytotoxicity was present at 72 h, together with a significant proliferation of bacteria. These biological and microbiological results can be attributed to the roughness produced by the chemical agents that triggered bacterial adsorption while inhibiting osteoblast adhesion. The results indicate that even if this treatment can decontaminate the titanium surface after implantation, the produced topography will generate an environment that will not favor long-term performance.

Osteoblastic Cell Behavior and Gene Expression Related to Bone Metabolism on Different Titanium Surfaces.

The surface topography of titanium dental implants has a great influence on osseointegration. In this work, we try to determine the osteoblastic behavior and gene expression of cells with different titanium surfaces and relate them to the physicochemical properties of the surface. For this purpose, we have used commercial titanium discs of grade 3: as-received corresponds to machined titanium without any surface treatment (MA), chemically acid etched (AE), treated via sand blasting with Al2O3 particles (SB) and a sand-blasting treatment with acid etching (SB+AE). The surfaces have been observed using scanning electron microscopy (SEM) and the roughness, wettability and surface energy with dispersive and polar components have been characterized. Osteoblastic cultures were performed with SaOS-2 osteoblastic cells determining cell viability as well as alkaline phosphatase levels for 3 and 21 days, and osteoblastic gene expression was determined. The roughness values of the MA discs was 0.02 μm, which increases to 0.3 μm with acid attack and becomes the maximum for the sand-blasted samples, reaching values of 1.2 μm for SB and SB+AE. The hydrophilic behavior of the MA and AE samples with contact angles of 63° and 65° is superior to that of the rougher samples, being 75° for SB and 82° for SB+AE. In all cases, they show good hydrophilicity. GB and GB+AE surfaces present a higher polar component in the surface energy values, 11.96 and 13.18 mJ/m2, respectively, than AE and MA, 6.64 and 9.79 mJ/m2, respectively. The osteoblastic cell viability values at three days do not show statistically significant differences between the four surfaces. However, the viability of the SB and SB+AE surfaces at 21 days is much higher than that of the AE and MA samples. From the alkaline phosphatase studies, higher values were observed for those treated with sand blasting with and without acid etching compared to the other two surfaces, indicating a greater activity in osteoblastic differentiation. In all cases except in the Osterix (Ostx) -osteoblast-specific transcription factor-a decrease in gene expression is observed in relation to the MA samples (control). The most important increase was observed for the SB+AE condition. A decrease in the gene expression of Osteoprotegerine (OPG), Runt-related transcription factor 2 (Runx2), Receptor Activator of NF-κB Ligand (RANKL) and Alkaline Phosphatase (Alp) genes was observed in the AE surface.

Physicochemical properties, cytotoxicity and bioactivity of a ready-to-use bioceramic repair material

The aim of this study was to evaluate the physicochemical properties, cytotoxicity and bioactivity of a ready-to-use bioceramic material, Bio-C Repair (Angelus), in comparison with White MTA (Angelus) and Biodentine (Septodont). The physicochemical properties of setting time, radiopacity, pH, solubility, dimensional and volumetric changes were evaluated. Biocompatibility and bioactivity were assessed in Saos-2 osteoblast cell cultures by the MTT assay 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Neutral Red (NR), Alizarin Red (ARS), and cell migration tests. Statistical analysis was performed by ANOVA, Tukey or Bonferroni tests (α = 0.05). Bio-C Repair had the longest setting time (p < 0.05), but radiopacity and solubility were accordance with the ISO 6876/2012 standards, besides linear expansion. Bio-C Repair and MTA had similar volumetric change (p > 0.05); lower than Biodentine (p < 0.05). All the materials evaluated had an alkaline pH. Bio-C Repair was cytocompatible and promoted mineralized nodule deposition in 21 days and cell migration in 3 days. In conclusion, Bio-C Repair had adequate radiopacity above 3mm Al, solubility less than 3%, dimensional expansion, and low volumetric change. In addition, Bio-C Repair promoted an alkaline pH and presented bioactivity and biocompatibility similar to MTA and Biodentine, showing potential for use as a repair material.

Changes in the intra- and extra-mechanical environment of the nucleus in Saos-2 osteoblastic cells during bone differentiation process: Nuclear shrinkage and stiffening in cell differentiation

Osteogenic differentiation has been reportedly regulated by various mechanical stresses, including fluid shear stress and tensile and compressive loading. The promotion of osteoblastic differentiation by these mechanical stresses is accompanied by reorganization of the F-actin cytoskeleton, which is deeply involved in intracellular forces and the mechanical environment. However, there is limited information about the effect on the mechanical environment of the intracellular nucleus, such as the mechanical properties of the nucleus and intracellular forces exerted on the nucleus, which have recently been found to be directly involved in various cellular functions. Here, we investigated the changes in the intracellular force applied to the nucleus and the effect on nuclear morphology and mechanical properties during osteogenic differentiation in human osteoblast-like cells (Saos-2). We carried out cell morphological analyses with confocal fluorescence microscopy, nuclear indentation test with atomic force microscopy (AFM), and fluorescence recovery after photobleaching (FRAP) for intranuclear DNA. The results revealed that a significant reorganization of the F-actin cytoskeleton from the nuclear surfaces to the cell periphery occurred in the osteogenic differentiation processes, simultaneously with the reduction of compressive forces to the nucleus. Such changes also facilitated nuclear shrinkage and stiffening, and further intranuclear chromatin compaction. The results indicate that the reduction of the intracellular compressive force due to reorganization of the F-actin cytoskeleton affects the intra- and extra-mechanical environment of the nucleus, and this change may affect gene expression and DNA replication in the osteogenic differentiation process.

Boron-incorporated biocomposite coatings on 316L and NiTi alloys: Enhanced structural, antibacterial activity, and cell viability performances.

Boron doped (5 %, 10%, and 15 wt.%) Hydroxyapatite (B-HA) biocomposites were syntesized and coated on 316L SS and NiTi (Ni-45Ti) metallic substrates by using the electrophoretic deposition process (EPD). The morphological and structural characterization of the coatings was executed using scanning electron microscopy (SEM) and X-ray diffraction devices (XRD). Antibacterial tests were conducted using Escherichia coli (E. coli, JM103) and Staphylococcus aureus (S. aureus, ATCC29293) microorganisms. The mitochondrial activity assay (MTT)-[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was used to examine cell viability and cytotoxicity in Saos-2 osteoblast cells. HA and boron peaks, as well as B-TCP and metallic components, were detected in XRD examinations. Porous morphologies were generated on the surface with boron doped B-HA coatings, as revealed by SEM views. Antibacterial activity studies revealed that both metallic coating groups, notably with boron doping, demonstrated antibacterial activity against gram-negative E. coli and gram-positive S. aureus. The antibacterial activity of the 316L group was shown to be better than that of the NiTi group in comparisonal testing. The syntesized boron-doped biocomposite coatings did not have any detrimental effects on living cells, according to cell viability studies. The cell viability rate was found to be greater in NiTi coatings than in 316 SS coatings, and the impact was amplified by the addition of boron.

Effects of silver/selenium/chitosan doped hydroxyapatite coatings on REX-734 alloy: Morphology, antibacterial activity, and cell viability

The goal of this study was to improve the antibacterial activity and cell viability of the Rex-734 alloy in biomedical applications. Various biocomposite coatings, such as single-layer hydroxyapatite (HAp), HAp/silver(Ag), and HAp/selenium(Se)/chitosan(Chit), were applied to Rex-734 alloy at various film thicknesses. The surface coatings were characterized by scanning electron microscopy, SEM, EDX, XRD and coating thickness and hardness of surface coatings were measured. The in vitro antibacterial activities of the coated and uncoated substrates were tested against Esherichia coli JM 103 and Staphylococcus aureus ATCC29293. In vitro cell–material interactions using Saos-2 osteoblast cells were characterized by the 3-(4,5-dimethylthiazol2-yl)− 2,5-diphenyltetrazolium bromide (MTT) assay for cell viability. The post-coating sintering parameters were shown to have a significant effect on the surface morphology. When compared to single HAp and HAp/Ag coatings, Se and Chit additions to HAp powders revealed good crack-free coating surfaces. Crack-free coating morphologies were achieved with a sintering speed of 3 °C/min. While single-HA coatings had an antimicrobial efficiency of 83% against E. coli, this value increased to 94% with Ag additive and to 85% with Se additive. Similarly, Ag and Se additives improved HAp's antimicrobial efficiency against Staphylococcus aureus. Cell viability decreased in the HAp/Ag coating at 86.5% and 90.93%, but increased significantly (p < 0.05) in the HA/Se-Chit coating at 137,43% and 158.23% at the end of the first and seventh days of incubation, respectively. The addition of Ag/Se/Chit to HAp increased the viability of the Rex-734 cells. Furthermore, it was revealed that the HAp-doped Se/Chit duplex biocomposite coating groups increased bone cell viability at much higher rates than the single-layer HAp coatings.

Silver/selenium/chitosan co‐substituted bioceramic coatings of Ni–Ti alloy: Antibacterial efficiency and cell viability

The effects of silver (Ag), selenium (Se), and chitosan (Cht) additives on the antibacterial activity and cell viability of NiTi were investigated. Three different hydroxyapatite (HA)-based bioceramic coatings to improve the antibacterial activity and cell viability of NiTi; HA, HA/Ag, and HA/Se–Cht were applied to NiTi substrates by a sol–gel method. The coated samples were characterized by scanning electron microscopy–electron-dispersive spectroscopy and X-ray diffraction and surface hardnesses were measured. The antimicrobial efficiency of the coatings and uncoated surfaces were tested against Escherichia coli-JM 103 and Staphylococcus aureus-ATCC29293. In vitro cell–material interactions using Saos-2 osteoblast cells were characterized by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mitochondrial activity test) assay for cell viability. A homogeneous, crack-free, and porous surface morphology is achieved in coatings. It has been shown that the Se–Cht additives did not have a significant effect on the surface hardness of the HA coating, but the Ag additive increased the hardness. Through in vitro antibacterial activity and cell viability tests, it was shown that Ag additive to bioceramic coatings significantly increased (p < .05) antibacterial properties but caused a decrease in cell viability. However, although Se–Cht additives did not have a significant effect on antibacterial properties (p < .05), it was observed that they increased cell viability.

The Effects of Tetrapeptides Designed to Fit the Androgen Binding Site of ZIP9 on Myogenic and Osteogenic Cells.

Simple SummaryPro-androgenic substances such as testosterone are often used to treat muscle- or bone-related disorders. Their interactions with the classical androgen receptor, however, can trigger a number of undesirable effects. It would therefore be of great benefit if the positive androgenic effects could be obtained by circumventing the classical androgen receptor. ZIP9 is a recently identified membrane-bound androgen receptor of physiological significance. Using in silico methods, we identified and verified the extracellular localization of its androgen binding site and designed small peptides that fit in it that do not interact with the AR. All peptides were found to be pro-androgenic; they stimulate mineralization in osteoblastic cells and myogenesis in myoblasts. Thus, these peptides might serve as testosterone surrogates in the treatment of osteogenic or myogenic disorders.ZIP9 is a recently identified membrane-bound androgen receptor of physiological significance that may mediate certain physiological responses to androgens. Using in silico methods, six tetrapeptides with the best docking properties at the testosterone binding site of ZIP9 were synthesized and further investigated. All tetrapeptides displaced T-BSA-FITC, a membrane-impermeable testosterone analog, from the surface of mouse myogenic L6 cells that express ZIP9 but not the classical androgen receptor (AR). Silencing the expression of ZIP9 with siRNA prevented this labeling. All tetrapeptides were found to be pro-androgenic; in L6 cells they stimulated the expression of myogenin, triggered activation of focal adhesion kinase, and prompted the fusion of L6 myocytes to syncytial myotubes. In human osteoblastic SAOS-2 cells that express AR and ZIP9, they reduced the expression of alkaline phosphatase and stimulated mineralization. These latter effects were prevented by silencing ZIP9 expression, indicating that the osteoblast/osteocyte conversion is exclusively mediated through ZIP9. Our results demonstrate that the synthetic tetrapeptides, by acting as ZIP9-specific androgens, have the potential to replace testosterone or testosterone analogs in the treatment of bone- or muscle-related disorders by circumventing the undesirable effects mediated through the classical AR.

3D Environment Is Required In Vitro to Demonstrate Altered Bone Metabolism Characteristic for Type 2 Diabetics.

A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings.

Long Noncoding RNA HOXA11-AS and Transcription Factor HOXB13 Modulate the Expression of Bone Metastasis-Related Genes in Prostate Cancer.

Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression, which play fundamental roles in cancer development. In this study, we found that homeobox A11 antisense RNA (HOXA11-AS), a highly expressed lncRNA in cell lines derived from prostate cancer bone metastases, promoted the cell invasion and proliferation of PC3 prostate cancer cells. Transcription factor homeobox B13 (HOXB13) was identified as an upstream regulator of HOXA11-AS. HOXA11-AS regulated bone metastasis-associated C-C motif chemokine ligand 2 (CCL2)/C-C chemokine receptor type 2 (CCR2) signaling in both PC3 prostate cancer cells and SaOS2 osteoblastic cells. The HOXB13/HOXA11-AS axis also regulated integrin subunits (ITGAV and ITGB1) specific to prostate cancer bone metastasis. HOXB13, in combination with HOXA11-AS, directly regulated the integrin-binding sialoprotein (IBSP) promoter. Furthermore, conditioned medium containing HOXA11-AS secreted from PC3 cells could induce the expression of CCL2 and IBSP in SaOS2 osteoblastic cells. These results suggest that prostate cancer HOXA11-AS and HOXB13 promote metastasis by regulation of CCL2/CCR2 cytokine and integrin signaling in autocrine and paracrine manners.

In Vitro and In Vivo Evaluation of Carboxymethyl Cellulose Scaffolds for Bone Tissue Engineering Applications.

The present study involves the development of citric acid-cross-linked carboxymethyl cellulose (C3CA) scaffolds by a freeze-drying process. Scaffolds were fabricated at different freezing temperatures of −20, −40, or −80 °C to investigate the influence of scaffold pore size on bone regeneration. All three scaffolds were porous in structure, and the pore size was measured to be 74 ± 4, 55 ± 6, and 46 ± 5 μm for −20, −40, and −80 °C scaffolds. The pores were larger in scaffolds processed at −20 °C compared to −40 and −80 °C, indicating the reduction in pore size of the scaffolds with a decrease in freezing temperature. The cytocompatibility, cell proliferation, and differentiation in C3CA scaffolds were assessed with the Saos-2 osteoblast cell line. These scaffolds supported the proliferation and differentiation of Saos-2 cells with significant matrix mineralization in scaffolds processed at −40 °C. Subcutaneous implantation of C3CA scaffolds in the rat model was investigated for its ability of vascularization and new matrix tissue formation. The matrix formation was observed at the earliest of 14 days in the scaffolds when processed at −40 °C while it was observed only after 28 days of implantation with the scaffolds processed at −20 and −80 °C. These results suggest that the citric acid-cross-linked CMC scaffolds processed at −40 °C can be promising for bone tissue engineering application.

The influence of periostin on osteoblastic adhesion and proliferation on collagen matrices - An in vitro study.

Purpose:The purpose of the study was to evaluate the ability of periostin when impregnated onto varied collagen matrices to influence osteoblast cell adhesion, proliferation, and activity.Materials and Methods:Saos-2 osteoblast cells were cultured and seeded onto two different collagen matrices as follows: Group A: absorbable collagen sponge (ACS), Group B: ACS impregnated with recombinant human periostin, Group C: nanocrystalline hydroxyapatite collagen (NcHC), and Group D: NcHC impregnated with recombinanant human periostin. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to evaluate cell viability as well as adhesion and proliferation on 2nd, 5th, and 7th day. Osteoblast activity was studied using alkaline phosphatase (ALP) assay for the study groups.Results:The periostin-treated absorbable collagen matrices showed a statistically significant increase in the osteoblast adhesion compared to periostin-treated NcHC on days 2, 5, and 7 (P < 0.001). The osteoblast activity as evaluated by ALP assay showed that there is increased activity in the periostin-treated ACS compared to the periostin-treated NcHC.Conclusion:From the observations of this study, it is evident that Periostin has a significant role in the modulating cellular response of the osteoblast cells. Further, incorporation of periostin into the ACS has been shown to increase the cell viability, proliferation, and adhesion of osteoblast-like Saos-2 cells.

USF1 Suppresses Expression of Fibrillar Type I, II, and III Collagen and pNP Adamts-3 in Osteosarcoma Cells

Collagens are the main components of human tissues. Various regulatory factors and cytokines may influence expression levels for collagen-encoding genes, and, therefore, contrubite to some collagen-associated pathologies. In this study, we demonstrate regulatory effects of USF1 on expression of genes encoding fibrillar collagen types I, II, and III in osteoblastic Saos-2 and MG-63 cells. An ectopic expression of the human USF1 led to a decrease in both mRNA and protein expression levels of the collagen-encoding genes mentioned above. ADAMTS-3 is a proteinase primarily responsible for the amino-terminal cleavage of type I and type II collagen precursors. The ADAMTS-3 promoter region contains potential binding sites for USF1. Here we show that an overexpression of USF1 lead to a decrease in ADAMTS-3 mRNA and protein expression levels. In co-transfection studies, USF1 negatively regulated ADAMTS-3 promoter activity. Further, in EMSA studies, we showed that USF1 binds to the ADAMTS-3 promoter region. In conclusion, it seems that ADAMTS-3 and USF1 contribute to the regulation of collagen encoding genes in osteosarcoma.