Human Osteoblast-like Saos-2 Cells Research Articles

Investigation of a new implant surface modification using phosphorylated pullulan.

Various implant surface treatment methods have been developed to achieve good osseointegration in implant treatment. However, some cases remain impossible to treat with implants because osseointegration is not obtained after implantation, and the implants fail. Thus, this study focused on phosphorylated pullulan because of its adhesiveness to titanium (Ti) and bone, high biocompatibility, and early replacement with bone. In this study, the response of bone-related cells to phosphorylated pullulan was evaluated to develop a new surface treatment method. Saos-2 (human osteosarcoma-derived osteoblast-like cells), MC3T3-E1 (mouse calvaria-derived osteoblast-like cells), and RAW264.7 (mouse macrophage-like cells) were used. In evaluating cellular responses, phosphorylated pullulan was added to the culture medium, and cell proliferation and calcification induction tests were performed. The proliferation and calcification of cells on the surface of Ti disks coated with phosphorylated pullulan were also evaluated. In addition, bone morphogenetic protein-2 (BMP-2), an osteogenic factor, was used to evaluate the role of phosphorylated pullulan as a drug carrier in inducing calcification on Ti disks. Phosphorylated pullulan tended to promote the proliferation of osteoblast-like cells and the formation of calcification on Ti disks coated with phosphorylated pullulan. Ti disks coated with phosphorylated pullulan loaded with BMP-2 enhanced calcification. Phosphorylated pullulan inhibited osteoclast-like cell formation. These results are due to the properties of phosphorylated pullulan, such as adhesiveness to titanium and drug-loading function. Therefore, phosphorylated pullulan effectively promotes bone regeneration when coated on titanium implants and is useful for developing a new surface treatment method.

Effects of Daidzein, Tempeh, and a Probiotic Digested in an Artificial Gastrointestinal Tract on Calcium Deposition in Human Osteoblast-like Saos-2 Cells.

Adequate calcium intake is crucial for the prevention and treatment of bone-related issues. Developing a nutritional source of readily bioavailable calcium is particularly significant for individuals deficient in this essential element and at risk of developing osteoporosis. This research aimed to evaluate the impact of tempeh (T), daidzein (D), and Lactobacillus acidophilus (LA) within a simulated intestinal environment consisting of Caco-2 epithelial and Saos-2 cells, focusing on their implications for bone mineralization mechanisms. In the initial phase, calcium bioaccessibility from calcium citrate (CaCt), LA, D, the daidzein combination D-CaCt-LA (D1:1:1), and the tempeh combination T-CaCt-LA (T1:1:1) was assessed through digestion simulation. The calcium content of both untreated and digested samples was determined using atomic absorption spectrometry (AAS). In the subsequent stage, the digested samples were used to induce intestinal absorption in differentiated enterocyte-like Caco-2 cells. The permeable fractions were then evaluated in a culture of osteoblast-like Saos-2 cells. Preliminary cellular experiments employed the MTT assay to assess cytotoxicity. The results indicated that the analyzed products did not influence the deposition of extracellular calcium in Saos-2 cells cultured without mineralization stimulators. The combined formulations of permeable fractions of digested CaCt, LA, D, and T demonstrated the capacity to enhance the proliferation of Saos-2 cells. In Saos-2 cells, D, D1:1:1, and LA showed no discernible impact on intracellular calcium accumulation, whereas T and T1:1:1 reduced the calcium deposits. Additionally, mRNA transcripts and alkaline phosphatase (ALP) activity levels in Saos-2 cells cultured without mineralization induction were unaffected by the analyzed products. An examination of the products revealed no discernible effect on ALP activity or mRNA expression during Saos-2 cell differentiation. Our findings suggest that tempeh, daidzein, and L. acidophilus did not positively impact cellular calcium deposition in Saos-2 cells. However, tempeh, daidzein and its combination, and L. acidophilus might enhance the process of osteogenic differentiation in Saos-2 cells. Nevertheless, this study did not identify any synergistic impact on calcium deposition and the process of osteogenic differentiation in Saos-2 cells of isoflavones and probiotics.

Influence of scandium on mechanical properties, degradation behavior, and cytocompatibility of Zn-3Cu-0.4Li-xSc alloys for implant applications

Zinc (Zn)-based alloys have emerged as promising new biodegradable materials owing to their moderate corrosion rates and potential biological functionalities. Nevertheless, as-cast pure Zn and its alloys possess low mechanical strength, poor ductility and low hardness, which hinders their biomedical application. In this study, biodegradable Zn-3Cu-0.4Li (ZCL) and ZCL-xSc (x = 0.20, 0.35 and 0.55 wt.%) alloys were fabricated by casting and further hot-rolled (HR), with the aim of acquiring satisfactory mechanical, corrosion and biocompatibility properties for orthopedic implant applications. Results indicated that the mechanical properties of HR ZCL and ZCL-xSc alloys were significantly improved compared to the as-cast alloys. The HR ZCL-0.20Sc alloy showed the best combination of mechanical properties: yield strength of 277 MPa; ultimate tensile strength of 337 MPa; elongation of 40%; and a microhardness of 113 HV. The nanohardness of HR ZCL–xSc alloys increased from 1.64 to 2.43 GPa with increasing Sc content from 0 to 0.55 wt.%. Degradation rates of HR ZCL-xSc alloys gradually increased with increasing Sc content, with the HR ZCL-0.55Sc alloy showing the lowest corrosion resistance and the highest degradation rate of 50.1 μm/y after immersion in Hanks’ balanced salt solution for 30 d. In vitro cytocompatibility assessment using human osteoblast-like SaOS2 cells showed high cell viabilities after 1 d of exposure to undiluted extracts of HR ZCL and ZCL-xSc alloys, and also after 5 d with 50% diluted extracts. Overall, the HR ZCL-0.20Sc alloy has great potential as a biodegradable bone-implant material, due to excellent mechanical, satisfactory corrosion and good biocompatibility properties.

Nanostructured TiNb coating improves the bioactivity of 3D printed PEEK

Polyetheretherketone (PEEK) has mechanical properties suitable for the manufacturing of osseointegration implants. However, its biological properties can be significantly improved by surface modification. In this work, the macroscopic surface topography was obtained by 3D printing and then the 3D printed surface was coated with a TiNb layer. The coating made it possible to apply anodic oxidation, by which a surface with defined nanoscale topography was obtained. Thus, a material with a gradient surface topography and suitable chemical composition was prepared. The properties of the surface were analyzed using optical and scanning electron microscopy. The chemical composition was then evaluated by photoelectron spectroscopy. The roughness, the wettability, and the wear resistance of the surface were observed. The adhesion of the coating and the oxide layer was evaluated. The biological activity of samples was analyzed by seeding them with human osteoblast-like Saos-2 cells. The cell adhesion, proliferation, metabolic activity and viability was observed and was statistically evaluated. After this, the functionality of this surface modification was tested by applying it to an implant with a real geometry. The main conclusions show that the nanostructured TiNb layer on 3D printed PEEK significantly improves biological activity.

Mechanical, corrosion, nanotribological, and biocompatibility properties of equal channel angular pressed Ti-28Nb-35.4Zr alloys for biomedical applications

This study systematically investigated the effect of equal channel angular pressing (ECAP) on the microstructure, mechanical, corrosion, nano-tribological properties and biocompatibility of a newly developed β Ti-28Nb-35.4Zr (hereafter denoted TNZ) alloy. Results indicated that ECAP of the β TNZ alloy refined its microstructure by forming ultrafine grains without causing stress-induced phase transformation, leading to formation of a single β phase. The ECAP-processed TNZ alloy exhibited a compressive yield strength of 960 MPa, and high plastic deformation capacity without fracturing under compression loads. Potentiodynamic polarization tests revealed the higher tendency of ECAP-processed TNZ alloys to form passive oxide films on its surface, which exhibited a lower corrosion rate (0.44±0.07 µm/y) in Hanks’ balanced salt solution compared to its as-cast counterpart (0.71±0.10 µm/y). Nanotribological testing also revealed higher resistance of the ECAP-processed TNZ alloy to abrasion, wear and scratching, when compared to its as-cast counterpart. Cytocompatibility and cell adhesion assessments of the ECAP-processed TNZ alloys showed a high viability (111%) of human osteoblast-like SaOS2 cells after 7 d of culturing. Moreover, the ECAP-processed TNZ alloy promoted adhesion and spreading of SaOS2 cells, which exhibited growth and proliferation on alloy surfaces. In summary, significantly enhanced mechanical, corrosion, and biological properties of ECAP-processed TNZ alloy advocate its suitability for load-bearing implant applications. Statement of significanceEqual channel angular pressing (ECAP) provides a unique combination of enhanced mechanical and functional properties of materials by optimizing their microstructures and phase transformations. This study investigated the mechanical, nano-tribological, corrosion, and biocompatibility properties of a newly developed β Ti-28Nb-35.4Zr (TNZ) alloy processed via ECAP. Our findings indicated that ECAP of the β TNZ alloy refined its microstructure by forming ultrafine grains without causing stress-induced phase transformation. Compared to its as-cast counterpart, ECAP-processed TNZ exhibited significantly enhanced compressive yield strength, plastic deformation capacity, hardness, wear, and corrosion properties. Moreover, in vitro cytocompatibility and cell adhesion studies revealed high cellular viabilities, growth and proliferation of osteoblast-like SaOS2 cells on the ECAP-processed TNZ alloy.

A biomechanical evaluation on Cubic, Octet, and TPMS gyroid Ti6Al4V lattice structures fabricated by selective laser melting and the effects of their debris on human osteoblast-like cells.

Lattice structures are widely used in orthopedic implants due to their unique features, such as high strength-to-weight ratios and adjustable biomechanical properties. Based on the type of unit cell geometry, lattice structures may be classified into two types: strut-based structures and sheet-based structures. In this study, strut-based structures (Cubic & Octet) and sheet-based structure (triply periodic minimal surface (TPMS) gyroid) were investigated. The biomechanical properties of the three different Ti6Al4V lattice structures fabricated by selective laser melting (SLM) were investigated using room temperature compression testing. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) were used to check the 3D printing quality with regards to defects and quantitative compositional information of 3D printed parts. Experimental results indicated that TPMS gyroid has superior biomechanical properties when compared to Cubic and Octet. Also, TPMS gyroid was found to be less affected by the variations in relative density. The biocompatibility of Ti6Al4V lattice structures was validated through the cytotoxicity test with human osteoblast-like SAOS2 cells. The debris generated during the degradation process in the form of particles and ions is among the primary causes of implant failure over time. In this study, Ti6Al4V particles with spherical and irregular shapes having average particle sizes of 36.5μm and 28.8μm, respectively, were used to mimic the actual Ti6Al4V particles to understand their harmful effects better. Also, the effects and amount of Ti6Al4V ions released after immersion within the cell culture media were investigated using the indirect cytotoxicity test and ion release test.

Rifampicin restores extracellular organic matrix formation and mineralization of osteoblasts after intracellular Staphylococcus aureus infection.

AimsBone regeneration during treatment of staphylococcal bone infection is challenging due to the ability of Staphylococcus aureus to invade and persist within osteoblasts. Here, we sought to determine whether the metabolic and extracellular organic matrix formation and mineralization ability of S. aureus-infected human osteoblasts can be restored after rifampicin (RMP) therapy.MethodsThe human osteoblast-like Saos-2 cells infected with S. aureus EDCC 5055 strain and treated with 8 µg/ml RMP underwent osteogenic stimulation for up to 21 days. Test groups were Saos-2 cells + S. aureus and Saos-2 cells + S. aureus + 8 µg/ml RMP, and control groups were uninfected untreated Saos-2 cells and uninfected Saos-2 cells + 8 µg/ml RMP.ResultsThe S. aureus-infected osteoblasts showed a significant number of intracellular bacteria colonies and an unusual higher metabolic activity (p < 0.005) compared to uninfected osteoblasts. Treatment with 8 µg/ml RMP significantly eradicated intracellular bacteria and the metabolic activity was comparable to uninfected groups. The RMP-treated infected osteoblasts revealed a significantly reduced amount of mineralized extracellular matrix (ECM) at seven days osteogenesis relative to uninfected untreated osteoblasts (p = 0.007). Prolonged osteogenesis and RMP treatment at 21 days significantly improved the ECM mineralization level. Ultrastructural images of the mineralized RMP-treated infected osteoblasts revealed viable osteoblasts and densely distributed calcium crystal deposits within the extracellular organic matrix. The expression levels of prominent bone formation genes were comparable to the RMP-treated uninfected osteoblasts.ConclusionIntracellular S. aureus infection impaired osteoblast metabolism and function. However, treatment with low dosage of RMP eradicated the intracellular S. aureus, enabling extracellular organic matrix formation and mineralization of osteoblasts at later stage.Cite this article: Bone Joint Res 2022;11(5):327–341.

Osteogenic differentiation of encapsulated cells in dexamethasone-loaded phospholipid-induced silk fibroin hydrogels.

The tissue engineering triad comprises the combination of cells, scaffolds and biological factors. Therefore, we prepared cell- and drug-loaded hydrogels using in situ silk fibroin (SF) hydrogels induced by dimyristoyl glycerophosphoglycerol (DMPG). DMPG is reported to induce rapid hydrogel formation by SF, facilitating cell encapsulation in the hydrogel matrix while maintaining high cell viability and proliferative capacity. In addition, DMPG can be used for liposome formulations in entrapping drug molecules. Dexamethasone (Dex) was loaded into the DMPG-induced SF hydrogels together with human osteoblast-like SaOS-2 cells, then the osteogenic differentiation of the entrapped cells was evaluated in vitro and compared to cells cultured under standard conditions. Calcium production by cells cultured in DMPG/Dex-SF hydrogels with Dex-depleted osteogenic medium was equivalent to that of cells cultured in conventional osteogenic medium containing Dex. The extended-release of the entrapped Dex by the hydrogels was able to provide a sufficient drug amount for osteogenic induction. The controlled release of Dex was also advantageous for cell viability even though its dose in the hydrogels was far higher than that in osteogenic medium. The results confirmed the possibility of using DMPG-induced SF hydrogels to enable dual cell and drug encapsulation to fulfil the practical applications of tissue-engineered constructs.

The Effects of Three Chlorhexidine-Based Mouthwashes on Human Osteoblast-Like SaOS-2 Cells. An In Vitro Study.

Several decontamination methods for removing biofilm from implant surfaces during surgical peri-implantitis treatment have been reported, including the intraoperative usage of chlorhexidine (CHX)-based antiseptics. There is a lack of information on possible adverse effects on bone healing. The study aimed to examine the impact of three CHX-based mouthwashes on osteoblast-like cells (SaOS-2) in vitro. Cells were cultured for three days in 96-well binding plates. Each well was randomly treated for either 30, 60 or 120 s with 0.05% CHX combined with 0.05% cetylpyridinium chloride (CPC), 0.1% CHX, 0.2% CHX or sterile saline (NaCl) as control. Cell viability, cytotoxicity and apoptosis were assessed at day 0, 3 and 6. Cell viability resulted in being higher in the control group at all time points. At day 0, the CHX 0.2 group showed significantly higher cytotoxicity values compared to CHX 0.1 (30 s), CHX + CPC (30 s, 60 s and 120 s) and control (60 s and 120 s), while no significant differences were identified between CHX + CPC and both CHX 0.1 and NaCl groups. All test mouthwashes were found to induce apoptosis to a lower extent compared to control. Results indicate that 0.2% CHX presented the highest cytotoxic effect. Therefore, its intraoperative use should be carefully considered.

Beta-Titanium Alloy Covered by Ferroelectric Coating–Physicochemical Properties and Human Osteoblast-Like Cell Response

Beta-titanium alloys are promising materials for bone implants due to their advantageous mechanical properties. For enhancing the interaction of bone cells with this perspective material, we developed a ferroelectric barium titanate (BaTiO3) coating on a Ti39Nb alloy by hydrothermal synthesis. This coating was analyzed by scanning electron and Raman microscopy, X-ray diffraction, piezoresponse force microscopy, X-ray photoelectron spectroscopy, nanoindentation, and roughness measurement. Leaching experiments in a saline solution revealed that Ba is released from the coating. A progressive decrease of Ba concentration in the material was also found after 1, 3, and 7 days of cultivation of human osteoblast-like Saos-2 cells. On day 1, the Saos-2 cells adhered on the BaTiO3 film in higher initial numbers than on the bare alloy, but they were less spread, and their initial proliferation rate was slower. These cells also contained a lower amount of beta1-integrins and vinculin, i.e., molecules involved in cell adhesion, and produced a lower amount of collagen I. This cell behavior was attributed to a higher surface roughness of BaTiO3 film rather than to its potential cytotoxicity, because the cell viability on this film was very high, reaching almost 99%. The amount of alkaline phosphatase, an enzyme involved in bone matrix mineralization, was similar in cells on the BaTiO3-coated and uncoated alloy, and on day 7, the cells on BaTiO3 film attained a higher final cell population density. These results indicate that after some improvements, particularly in its roughness and stability, the hydrothermal ferroelectric BaTiO3 film could be promising coating for improved osseointegration of bone implants.

Electroless Palladium-Coated Polymer Scaffolds for Electrical Stimulation of Osteoblast-Like Saos-2 Cells.

Three-dimensional porous scaffolds offer some advantages over conventional treatments for bone tissue engineering. Amongst all non-bioresorbable scaffolds, biocompatible metallic scaffolds are preferred over ceramic and polymeric scaffolds, as they can be used as electrodes with different electric field intensities (or voltages) for electric stimulation (ES). In the present work we have used a palladium-coated polymeric scaffold, generated by electroless deposition, as a bipolar electrode to electrically stimulate human osteoblast-like Saos-2 cells. Cells grown on palladium-coated polyurethane foams under ES presented higher proliferation than cells grown on foams without ES for up to 14 days. In addition, cells grown in both conditions were well adhered, with a flat appearance and a typical actin cytoskeleton distribution. However, after 28 days in culture, cells without ES were filling the entire structure, while cells under ES appeared rounded and not well adhered, a sign of cell death onset. Regarding osteoblast differentiation, ES seems to enhance the expression of early expressed genes. The results suggest that palladium-coated polyurethane foams may be good candidates for osteoblast scaffolds and demonstrate that ES enhances osteoblast proliferation up to 14 days and upregulate expression genes related to extracellular matrix formation.

Influence of roughness on initial in vitro response of cells to Al2O3/Ce-TZP nanocomposite

ABSTRACT Al2O3/Ce-tetragonal zirconia polycrystal (TZP) nanocomposite was synthesized by a colloidal processing route and sintered in air atmosphere. Sandblasting treatment was made to alumina toughened zirconia (ATZ) nanocomposite in order to evaluate the influence of surface roughness on the osteogenic differentiation performing in vitro growing a human osteoblast-like cell line, SaOs-2, and human adipose-derived mesenchymal stem cells (hADMSC) osteogenic differentiated. Smooth roughness values around Ra = 0.5 µm were obtained when the abrasive material was below 90 µm increasing the expression of BGLAP and IBSP genes and Ra = 1.5 µm was found with particles of sizes between 90 and 250 µm upregulating SPARC gene. The non-cytotoxicity and haemocompatibility of ATZ nanocomposite were proved. Alumina-ceria-stabilized zirconia nanocomposite presented in this work exhibits a high potential for application in the fabrication of dental implants due to their biological behavior and very promising mechanical properties.

Human osteoblast-like SAOS-2 cells on submicron-scale fibers coated with nanocrystalline diamond films

A unique composite nanodiamond-based porous material with a hierarchically-organized submicron-nano-structure was constructed for potential bone tissue engineering. This material consisted of submicron fibers prepared by electrospinning of silicon oxide (SiOx), which were oxygen-terminated (O-SiOx) and were hermetically coated with nanocrystalline diamond (NCD) films. The NCD films were then terminated with hydrogen (H-NCD) or oxygen (O-NCD). The materials were tested as substrates for the adhesion, growth and osteogenic differentiation of human osteoblast-like Saos-2 cells. The number and the spreading area of the initially adhered cells, their growth rate during 7 days after seeding and the activity of alkaline phosphatase (ALP) were significantly higher on the NCD-coated samples than on the uncoated O-SiOx samples. In addition, the concentration of type I collagen was significantly higher in the cells on the O-NCD-coated samples than on the bare O-SiOx samples. The observed differences could be attributed to the tunable wettability of NCD and to the more appropriate surface morphology of the NCD-coated samples in contrast to the less stable, rapidly eroding bare SiOx surface. The H-NCD coatings and the O-NCD coatings both promoted similar initial adhesion of Saos-2 cells, but the subsequent cell proliferation activity was higher on the O-NCD-coated samples. The concentration of beta-actin, vinculin, type I collagen and alkaline phosphatase (ALP), the ALP activity, and also the calcium deposition tended to be higher in the cells on the O-NCD-coated samples than on the H-NCD-coated samples, although these differences did not reach statistical significance. The improved cell performance on the O-NCD-coated samples could be attributed to higher wettability of these samples (water drop contact angle less than 10°), while the H-NCD-coated samples were hydrophobic (contact angle >70°). NCD-coated porous SiOx meshes can therefore be considered as appropriate scaffolds for bone tissue engineering, particularly those with an O-terminated NCD coating.

Effect of diamond-like carbon doped with chromium on cell differentiation, immune activation and apoptosis.

Diamond-like carbon (DLC) is a biocompatible material that has many potential biomedical applications, including in orthopaedics. DLC layers doped with Cr at atomic percent (at.%) of 0, 0.9, 1.8, 7.3, and 7.7 at.% were evaluated with reference to their osteoinductivity with human bone marrow mesenchymal stromal cells (hMSCs), immune activation potential with RAW 264.7 macrophage-like cells, and their effect on apoptosis in Saos-2 human osteoblast-like cells and neonatal human dermal fibroblasts (NHDFs). At mRNA level, hMSCs on DLC doped with 0.9 and 7.7 at.% of Cr reached higher maximum values of both RUNX2 and alkaline phosphatase. An earlier onset of mRNA production of type I collagen and osteocalcin was also observed on these samples; they also supported the production of both type I collagen and osteocalcin. RAW 264.7 macrophages were screened using a RayBio™ Human Cytokine Array for cytokine production. 10 cytokines were at a concentration more than 2 × as high as the concentration of a positive control, but the values for the DLC samples were only moderately higher than the values on glass. NHDF cells, but not Saos-2 cells, had a higher expression of pro-apoptotic markers Bax and Bim and a lower expression of anti-apoptotic factor BCL-XL in proportion to the Cr content. Increased apoptosis was also proven by annexin V staining. These results show that a Cr-doped DLC layer with a lower Cr content can act as an osteoinductive material with relatively low immunogenicity, but that a higher Cr content can induce cell apoptosis.

Preparation of highly wettable coatings on Ti\u20136Al\u20134V ELI alloy for traumatological implants using micro-arc oxidation in an alkaline electrolyte

Pulsed micro-arc oxidation (MAO) in a strongly alkaline electrolyte (pH > 13), consisting of Na2SiO3⋅9H2O and NaOH, was used to form a thin porous oxide coating consisting of two layers differing in chemical and phase composition. The unique procedure, combining MAO and removal of the outer layer by blasting, enables to prepare a coating suitable for application in temporary traumatological implants. A bilayer formed in an alkaline electrolyte environment during the application of MAO enables the formation of a wear-resistant layer with silicon incorporated in the oxide phase. Following the removal of the outer rutile-containing porous layer, the required coating properties for traumatological applications were determined. The prepared surfaces were characterized by scanning electron microscopy, X-ray diffraction patterns, X-ray photoelectron spectroscopy, atomic force microscopy and contact angle measurements. Cytocompatibility was evaluated using human osteoblast-like Saos-2 cells. The newly-developed surface modifications of Ti–6Al–4V ELI alloy performed satisfactorily in all cellular tests in comparison with MAO-untreated alloy and standard tissue culture plastic. High cell viability was supported, but the modifications allowed only relatively slow cell proliferation, and showed only moderate osseointegration potential without significant support for matrix mineralization. Materials with these properties are promising for utilization in temporary traumatological implants.

The effect of TNF-α on osteoblasts in metal wear-induced periprosthetic bone loss.

AimsThis study aimed to examine the effects of tumour necrosis factor-alpha (TNF-α) on osteoblasts in metal wear-induced bone loss.MethodsTNF-α immunoexpression was examined in periprosthetic tissues of patients with failed metal-on-metal hip arthroplasties and also in myeloid MM6 cells after treatment with cobalt ions. Viability and function of human osteoblast-like SaOs-2 cells treated with recombinant TNF-α were studied by immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, western blotting, and enzyme-linked immunosorbent assay (ELISA).ResultsMacrophages, lymphocytes, and endothelial cells displayed strong TNF-α immunoexpression in periprosthetic tissues containing metal wear debris. Colocalization of TNF-α with the macrophage marker CD68 and the pan-T cell marker CD3 confirmed TNF-α expression in these cells. Cobalt-treated MM6 cells secreted more TNF-α than control cells, reflecting the role of metal wear products in activating the TNF-α pathway in the myeloid cells. While TNF-α did not alter the immunoexpression of the TNF-receptor 1 (TNF-R1) in SaOs-2 cells, it increased the release of the soluble TNF-receptor 1 (sTNF-R1). There was also evidence for TNF-α-induced apoptosis. TNF-α further elicited the expression of the endoplasmic reticulum stress markers inositol-requiring enzyme (IRE)-1α, binding-immunoglobulin protein (BiP), and endoplasmic oxidoreductin1 (Ero1)-Lα. In addition, TNF-α decreased pro-collagen I α 1 secretion without diminishing its synthesis. TNF-α also induced an inflammatory response in SaOs-2 cells, as evidenced by the release of reactive oxygen and nitrogen species and the proinflammatory cytokine vascular endothelial growth factor.ConclusionThe results suggest a novel osteoblastic mechanism, which could be mediated by TNF-α and may be involved in metal wear debris-induced periprosthetic bone loss.Cite this article: Bone Joint Res 2020;9(11):827–839.

Simultaneous Visualization of Wet Cells and Nanostructured Biomaterials in SEM using Ionic Liquids.

This work presents a successful methodology to image mammalian cells adhered to nanostructured titanium by using scanning electron microscopy (SEM) operating in low-vacuum mode following ionic liquid treatment. Human osteoblast-like Saos-2 cells were treated with a room-temperature ionic liquid, 1-ethyl-3-methylimidazolium tetrafluoroborate, and subsequently imaged on titanium by SEM. Titanium substrates were modified to create laser-induced periodic surface structures (LIPSS) for visualization at the submicron scale. By using a combination of fluorescence-based cell metabolism along with light microscopy and SEM image analysis, the shape and location of irradiated cells were confirmed to be unchanged after multiple irradiation sessions; the viability of minimally irradiated cells was also unaltered. The wet imaging conditions combined with a rapid facile protocol using ionic liquid allows this technique to fulfill a niche in examining cellular behavior on biomaterials with submicron surface features. The demonstrated method to track observed cell adhesion to submicron surface features by SEM has great implications for understanding cell migration on nanostructured surfaces as well as the exploration of simpler SEM preparation methods for cellular imaging.

In Vitro Comparison of Macrophage Polarization and Osteoblast Differentiation Potentials between Granules and Block Forms of Deproteinized Bovine Bone Mineral.

Deproteinized bovine bone mineral (DBBM) bone grafts are commonly utilized for guided bone regeneration (GBR) techniques in regenerative dentistry. It has been hypothesized that different forms (blocks versus particulates) might demonstrate the varying properties of cell behavior during the regenerative process. Therefore, the aim of the present study was to investigate DBBM granules and blocks for their effects on osteoblasts and macrophages (Mφs). DBBM granules and blocks were filled to the same size (φ6.4 mm in diameter × 2.0 mm in height) in cell culture wells and assessed for cell viability and cell differentiation of human osteoblast-like Saos-2 cells, and Mφs derived from human monocyte THP-1 cells. The two groups were first characterized by micro-CT analysis, which demonstrated that DBBM granules had a two-fold greater material volume and a four-fold larger surface area than the blocks. DBBM blocks showed superior viability for both osteoblasts and Mφs. Osteoblast experiments were then utilized to better characterize the influence of DBBM shapes/forms on osteoblast differentiation. Alkaline phosphatase (ALP) staining on the undecalcified frozen sections was observed throughout the DBBM granule surface, yet this staining was only observed on the upper portion of the DBBM blocks. Furthermore, DBBM blocks showed M1-Mφ polarization trends with higher IL-1 and IL-6 mRNA expression in Mφs, while the conditioned media from Mφs cultured on DBBM granules promoted osteoblast differentiation with higher mRNA levels of Runx 2, ALP and osteocalcin. In conclusion, the DBBM granules showed more regenerative effects, lower M1 marker expression, and higher osteoblast differentiation potential when compared with the blocks, which might be related to the larger material volume, higher surface area and greater ability for the cells to penetrate through the scaffold.

Improving the Tribological Properties and Biocompatibility of Zr-Based Bulk Metallic Glass for Potential Biomedical Applications.

Zr-based bulk metallic glasses (Zr-BMGs) are potentially the next generation of metallic biomaterials for orthopaedic fixation devices and joint implants owing to their attractive bulk material properties. However, their poor tribological properties and long-term biocompatibility present major concerns for orthopaedic applications. To this end, a novel surface modification technology, based on ceramic conversion treatment (CCT) in an oxidising medium between the glass transition temperature and the crystallisation temperature, has been developed to convert the surface of commercially available Zr44Ti11Cu10Ni11Be25 (Vitreloy 1b) BMG into ceramic layers. The engineered surfaces were fully characterised by in-situ X-ray diffraction, glow-discharge optical emission spectroscopy, scanning electron microscopy, transmission electron microscopy, and scanning transmission electron microscopy. The mechanical, chemical, and tribological properties were evaluated respectively by nano-indentation, electrochemical corrosion testing, tribological testing and the potential biocompatibility assessed by a cell proliferation assay. The results have demonstrated that after CCT at 350 °C for 40 h and at 380 °C for 4.5 h the original surfaces were converted into to a uniform 35–55-nm-thick oxide layer (with significantly reduced Ni and Cu concentration) followed by a 200–400-nm-thick oxygen-diffusion hardened case. The surface nano hardness was increased from 7.75 ± 0.36 to 18.32 ± 0.21 GPa, the coefficient of friction reduced from 0.5–0.6 to 0.1–0.2 and the wear resistance improved by more than 60 times. After 24 h of contact, SAOS-2 human osteoblast-like cells had increased surface coverage from 18% for the untreated surface to 46% and 54% for the 350 °C/40 h and 380 °C/4.5 h treated surfaces, respectively. The significantly improved tribological properties and biocompatibility have shown the potential of the ceramic conversion treated Zr-BMG for orthopaedic applications.

Construction of eukaryotic expression plasmid of pcDNA3.1(+)- CTGF and its expression in human osteoblast-like cells SaOS-2

To construct pcDNA3.1(+) eukaryotic expression plasmid of connective tissue growth factor(CTGF), and detected its expression in human osteoblast-like cells SaOS-2, which provides a technical support for further research on the mechanism of CTGF gene in bone development and bone repair process. ;Methods: The whole sequence of CTGF gene was cloned in vitro by polymerase chain reaction(PCR) method and connected to the linear pcDNA3.1(+) vector for constructing pcDNA3.1(+)-CTGF eukaryotic expression plasmid by homologous recombination technology. The plasmid was identified by sequencing. After identification, it was transfected into SaOS-2 cells and its expression was detected at 48 h. ;Results: pcDNA3.1(+)-CTGF eukaryotic expression recombinant plasmid was successfully constructed, which was confirmed by sequencing. Compared with the control group, CTGF expression level was significantly up-regulated after transfection of SaOS-2 cells for 48 h, up to five times as much as the control group. ;Conclusion: pcDNA3.1(+)-CTGF eukaryotic expression plasmid was successfully constructed and could be stably expressed in human osteoblasts-like cell SaOS-2, which laid a foundation for further study on the regulatory mechanism of CTGF gene on bone formation.