The 5'-nucleotidase S5nA is dispensable for evasion of phagocytosis and biofilm formation in Streptococcus pyogenes.

5'-nucleotidases are widespread among all domains of life. The enzymes hydrolyze phosphate residues from nucleotides and nucleotide derivatives. In some pathobiontic bacteria, 5'-nucleotidases contribute to immune evasion by dephosphorylating adenosine mono-, di-, or tri-phosphates, thereby either decreasing the concentration of pro-inflammatory ATP or increasing the concentration of anti-inflammatory adenosine, both acting on purinergic receptors of phagocytic cells. The strict human pathogen Streptococcus pyogenes expresses a surface-associated 5'-nucleotidase (S5nA) under infection conditions that has previously been discussed as a potential virulence factor. Here we show that deletion of the S5nA gene does not significantly affect growth in human blood, evasion of phagocytosis by neutrophils, formation of biofilms and virulence in an infection model with larvae of the greater wax moth Galleria mellonella in S. pyogenes serotypes M6, M18 and M49. Hence, the surface-associated 5'-nucleotidase S5nA seems dispensable for evasion of phagocytosis and biofilm formation in S. pyogenes.

Effect of electric stimulation on human chondrocytes and mesenchymal stem cells under normoxia and hypoxia.

During joint movement and mechanical loading, electric potentials occur within cartilage tissue guiding cell development and regeneration. Exposure of cartilage exogenous electric stimulation (ES) may imitate these endogenous electric fields and promote healing processes. Therefore, the present study investigated the influence of electric fields on human chondrocytes, mesenchymal stem cells and the co­culture of the two. Human chondrocytes isolated from articular cartilage obtained post­mortally and human mesenchymal stem cells derived from bone marrow (BM­MSCs) were seeded onto a collagen­based scaffold separately or as co­culture. Following incubation with the growth factors over 3 days, ES was performed using titanium electrodes applying an alternating electric field (700 mV, 1 kHz). Cells were exposed to an electric field over 7 days under either hypoxic or normoxic culture conditions. Following this, metabolic activity was investigated and synthesis rates of extracellular matrix proteins were analyzed. ES did not influence metabolic activity of chondrocytes or BM­MSCs. Gene expression analyses demonstrated that ES increased the expression of collagen type II mRNA and aggrecan mRNA in human chondrocytes under hypoxic culture conditions. Likewise, collagen type II synthesis was significantly increased following exposure to electric fields under hypoxia. BM­MSCs and the co­culture of chondrocytes and BM­MSCs revealed a similar though weaker response regarding the expression of cartilage matrix proteins. The electrode setup may be a valuable tool to investigate the influence of ES on human chondrocytes and BM­MSCs contributing to fundamental knowledge including future applications of ES in cartilage repair.

Biocompatibility and Inflammatory Potential of Titanium Alloys Cultivated with Human Osteoblasts, Fibroblasts and Macrophages.

The biomaterials used to maintain or replace functions in the human body consist mainly of metals, ceramics or polymers. In orthopedic surgery, metallic materials, especially titanium and its alloys, are the most common, due to their excellent mechanical properties, corrosion resistance, and biocompatibility. Aside from the established Ti6Al4V alloy, shape memory materials such as nickel-titanium (NiTi) have risen in importance, but are also discussed because of the adverse effects of nickel ions. These might be reduced by specific surface modifications. In the present in vitro study, the osteoblastic cell line MG-63 as well as primary human osteoblasts, fibroblasts, and macrophages were cultured on titanium alloys (forged Ti6Al4V, additive manufactured Ti6Al4V, NiTi, and Diamond-Like-Carbon (DLC)-coated NiTi) to verify their specific biocompatibility and inflammatory potential. Additive manufactured Ti6Al4V and NiTi revealed the highest levels of metabolic cell activity. DLC-coated NiTi appeared as a suitable surface for cell growth, showing the highest collagen production. None of the implant materials caused a strong inflammatory response. In general, no distinct cell-specific response could be observed for the materials and surface coating used. In summary, all tested titanium alloys seem to be biologically appropriate for application in orthopedic surgery.

Direct influence of titanium and zirconia particles on the morphology and functionality of mature human osteoclasts.

Within the last ten years of biomedical implants, the focus is increasingly on bioceramics, specifically on zirconia (ZrO2 ). Hence, we analyzed the impact of ZrO2 particles in comparison to titanium particles on mature human osteoclasts (OCs) as little is known about the direct effect of wear particles on mature OCs and their role in the osteolytic process during aseptic endoprosthesis loosening. Changes in cell morphology and functionality of OCs incubated with particles in different concentrations were investigated in vitro. OCs tend to be enlarged after three days of cultivation with both types of particles, especially with high concentrations of ZrO2 , suggesting increased cell fusion. Further, we identified significantly increased expression of OC specific and bone matrix related genes: VNR, RANK, TRAP, and CTSK pointing on a direct stimulatory particle effect on the functionality of mature OCs. In completion, we quantified the bone resorption activity of particle treated mature OCs but could not detect a significant difference in bone resorption compared to OCs cultivated without particles. However, we could identify significantly higher gene expression of MMP-1 in particle treated OCs compared to untreated control OCs after three days of incubation. We also detected an impaired production of the tissue inhibitor of metalloproteinase, especially for OCs treated with high ZrO2 concentrations. In conclusion, our in vitro data show that abrasion particles could have a direct influence on mature OCs and therefore could promote increased OC-mediated bone resorption during aseptic loosening of total joint replacements. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2608-2615, 2017.