Influence of metallic particles and TNF on the transcriptional regulation of NLRP3 inflammasome-associated genes in human osteoblasts

IntroductionThe release of mature interleukin (IL-) 1β from osteoblasts in response to danger signals is tightly regulated by the nucleotide-binding oligomerization domain leucine-rich repeat and pyrin-containing protein 3 (NLRP3) inflammasome. These danger signals include wear products resulting from aseptic loosening of joint arthroplasty. However, inflammasome activation requires two different signals: a nuclear factor-kappa B (NF-κB)-activating priming signal and an actual inflammasome-activating signal. Since human osteoblasts react to wear particles via Toll-like receptors (TLR), particles may represent an inflammasome activator that can induce both signals.MethodsTemporal gene expression profiles of TLRs and associated intracellular signaling pathways were determined to investigate the period when human osteoblasts take up metallic wear particles after initial contact and initiate a molecular response. For this purpose, human osteoblasts were treated with metallic particles derived from cobalt-chromium alloy (CoCr), lipopolysaccharides (LPS), and tumor necrosis factor-alpha (TNF) alone or in combination for incubation times ranging from one hour to three days. Shortly after adding the particles, their uptake was observed by the change in cell morphology and spectral data.ResultsExposure of osteoblasts to particles alone increased NLRP3 inflammasome-associated genes. The response was not significantly enhanced when cells were treated with CoCr + LPS or CoCr + TNF, whereas inflammation markers were induced. Despite an increase in genes related to the NLRP3 inflammasome, the release of IL-1β was unaffected after contact with CoCr particles.DiscussionAlthough CoCr particles affect the expression of NLRP3 inflammasome-associated genes, a single stimulus was not sufficient to prime and activate the inflammasome. TNF was able to prime the NLRP3 inflammasome of human osteoblasts

Label-free monitoring of uptake and toxicity of endoprosthetic wear particles in human cell cultures

The evaluation of the biological effects of endoprosthetic wear particles on cells in vitro relies on a variety of test assays. However, most of these methods are susceptible to particle-induced interferences; therefore, label-free testing approaches emerge as more reliable alternatives. In this study, impedance-based real-time monitoring of cellular viability and metabolic activity were performed following exposure to metallic and ceramic wear particles. Moreover, label-free imaging of particle-exposed cells was done by high-resolution darkfield microscopy (HR-ODM) and field emission scanning electron microscopy (FESEM). The isolated human fibroblasts were exposed to CoCr28Mo6 and alumina matrix composite (AMC) ceramic particles. HR-ODM and FESEM revealed ingested particles. For impedance measurements, cells were seeded on gold-plated microelectrodes. Cellular behavior was monitored over a period of 48 h. CoCr28Mo6 and AMC particle exposure affected cell viability in a concentration-dependent manner, i.e., 0.01 mg/mL particle solutions led to small changes in cell viability, while 0.05 mg/mL resulted in a significant reduction of viability. The effects were more pronounced after exposure to CoCr28Mo6 particles. The results were in line with light and darkfield microcopy observations indicating that the chosen methods are valuable tools to assess cytotoxicity and cellular behavior following exposure to endoprosthetic wear particles.publishedVersio

The Impact of Metal Ion Exposure on the Cellular Behavior of Human Osteoblasts and PBMCs: In Vitro Analyses of Osteolytic Processes

Osteolysis in the periprosthetic tissue can be caused by metallic wear particles and ions that can originate from implant surface corrosion. These products influence cellular behavior and stimulate the expression of proinflammatory cytokines. The purpose of this study was to evaluate the impact of CoCr29Mo6 ions on cell survival, differentiation, and cytokine expression in human osteoblasts and peripheral blood mononuclear cells (PBMCs). Thus, we exposed cells with a mixture of 200 mu g/L ion solution and determined cell viability and apoptosis/necrosis. Gene expression analyses of osteoblastic and osteoclastic differentiation markers as well as pro-osteolytic mediators (IL-6, IL-8, TNF-alpha, MCP-1, MMP1, TIMP1) were performed. These markers were also investigated in mixed cultures of adherent and non-adherent PBMCs as well as in co-cultures of human osteoblasts and PBMCs. The ion solution induced necrosis in osteoblasts and PBMCs in single cultures. All examined mediators were highly expressed in the co-culture of osteoblasts and PBMCs whereas in the single cell cultures only IL-6, IL-8, and MMP1 were found to be stimulated. While the applied concentration of the CoCr29Mo6 ion solutions had only marginal effects on human osteoblasts and PBMCs alone, the co-culture may provide a comprehensive model to study osteolytic processes in response to Co and Cr ions

The Impact of Metal Ion Exposure on the Cellular Behavior of Human Osteoblasts and PBMCs: In Vitro Analyses of Osteolytic Processes

Osteolysis in the periprosthetic tissue can be caused by metallic wear particles and ions that can originate from implant surface corrosion. These products influence cellular behavior and stimulate the expression of proinflammatory cytokines. The purpose of this study was to evaluate the impact of CoCr29Mo6 ions on cell survival, differentiation, and cytokine expression in human osteoblasts and peripheral blood mononuclear cells (PBMCs). Thus, we exposed cells with a mixture of 200 µg/L ion solution and determined cell viability and apoptosis/necrosis. Gene expression analyses of osteoblastic and osteoclastic differentiation markers as well as pro-osteolytic mediators (IL-6, IL-8, TNF-α, MCP-1, MMP1, TIMP1) were performed. These markers were also investigated in mixed cultures of adherent and non-adherent PBMCs as well as in co-cultures of human osteoblasts and PBMCs. The ion solution induced necrosis in osteoblasts and PBMCs in single cultures. All examined mediators were highly expressed in the co-culture of osteoblasts and PBMCs whereas in the single cell cultures only IL-6, IL-8, and MMP1 were found to be stimulated. While the applied concentration of the CoCr29Mo6 ion solutions had only marginal effects on human osteoblasts and PBMCs alone, the co-culture may provide a comprehensive model to study osteolytic processes in response to Co and Cr ions

Inflammatory Response of Human Peripheral Blood Mononuclear Cells and Osteoblasts Incubated With Metallic and Ceramic Submicron Particles

Inflammatory reactions associated with osteolysis and aseptic loosening are the result of wear particles generated at the articulating surfaces of implant components. The aim of the present study was to analyze the biological response of human osteoblasts and peripheral blood mononuclear cells (PBMCs) after exposure to metallic and alumina ceramic particles regarding cellular differentiation, cytokine release, and monocyte migration. Cells were exposed to particles (0.01 and 0.05 mg/ml) from an alumina matrix composite (AMC) ceramic and a CoCr28Mo6 alloy with an average size of 0.5 µm over 48 and 96 h. The expression rates of osteogenic (Col1A1, ALP) and pro-osteoclastic (RANK, Trap5b) differentiation markers as well as pro-osteolytic mediators (MMP-1, TIMP-1, IL-6, IL-8, MCP-1) were determined and soluble protein concentrations of active MMP-1, IL-6, IL-8, and pro-collagen type 1 in cell culture supernatants were evaluated. Additionally, the capacity of particle-treated osteoblasts to attract potentially pro-inflammatory cells to the site of particle exposure was investigated by migration assays using osteoblast-conditioned media. The cellular morphology and metabolism of human osteoblasts and adherent PBMCs were influenced by particle type and concentration. In human osteoblasts, Col1A1 expression rates and protein production were significantly reduced after exposing cells to the lower concentration of cobalt-chromium (CoCr) and AMC particles. Exposure to AMC particles (0.01 mg/ml) resulted in increased mRNA levels of RANK and Trap5b in adherent PBMCs. For MMP-1 gene expression, elevated levels were more prominent after incubation with CoCr compared to AMC particles in osteoblasts, which was not reflected by the protein data. Interleukin (IL)-6 and IL-8 mRNA and protein were induced in both cell types after treatment with AMC particles, whereas exposure to CoCr particles resulted in significantly upregulated IL-6 and IL-8 protein contents in PBMCs only. Exposure of osteoblasts to CoCr particles reduced the chemoattractant potential of osteoblast-conditioned medium. Our results demonstrate distinct effects of AMC and CoCr particles in human osteoblasts and PBMCs. Complex cell and animal models are required to further evaluate the impact of cellular interactions between different cell types during particle exposure

The Effect of Structural Design on Mechanical Properties and Cellular Response of Additive Manufactured Titanium Scaffolds

Restoration of segmental defects in long bones remains a challenging task in orthopedic surgery. Although autologous bone is still the ‘Gold Standard’ because of its high biocompatibility, it has nevertheless been associated with several disadvantages. Consequently, artificial materials, such as calcium phosphate and titanium, have been considered for the treatment of bone defects. In the present study, the mechanical properties of three different scaffold designs were investigated. The scaffolds were made of titanium alloy (Ti6Al4V), fabricated by means of an additive manufacturing process with defined pore geometry and porosities of approximately 70%. Two scaffolds exhibited rectangular struts, orientated in the direction of loading. The struts for the third scaffold were orientated diagonal to the load direction, and featured a circular cross-section. Material properties were calculated from stress-strain relationships under axial compression testing. <em>In vitro</em> cell testing was undertaken with human osteoblasts on scaffolds fabricated using the same manufacturing process. Although the scaffolds exhibited different strut geometry, the mechanical properties of ultimate compressive strength were similar (145–164 MPa) and in the range of human cortical bone. Test results for elastic modulus revealed values between 3.7 and 6.7 GPa. <em>In vitro</em> testing demonstrated proliferation and spreading of bone cells on the scaffold surface

New Coating Technique of Ceramic Implants with Different Glass Solder Matrices for Improved Osseointegration-Mechanical Investigations

Ceramics are a very popular material in dental implant technology due to their tribological properties, their biocompatibility and their esthetic appearance. However, their natural surface structure lacks the ability of proper osseointegration, which constitutes a crucial process for the stability and, thus, the functionality of a bone implant. We investigated the application of a glass solder matrix in three configurations—consisting mainly of SiO2, Al2O3, K2O and Na2O to TZP-A ceramic specimens. The corresponding adhesive strength and surface roughness of the coatings on ceramic specimens have been analyzed. Thereby, high adhesive strength (70.3 ± 7.9 MPa) was found for the three different coatings. The obtained roughness (Rz) amounted to 18.24 ± 2.48 µm in average, with significant differences between the glass solder configurations. Furthermore, one configuration was also tested after additional etching which did not lead to significant increase of surface roughness (19.37 ± 1.04 µm) or adhesive strength (57.2 ± 5.8 MPa). In conclusion, coating with glass solder matrix seems to be a promising surface modification technique that may enable direct insertion of ceramic implants in dental and orthopaedic surgery