E01

Effects of metallic wear and corrosion products on inflammatory processes of peri-implant bone tissue cells

Investigators

Name:Prof. Dr. Rainer Bader
PD Dr. Anika Jonitz-Heincke
Affiliation:Department of Orthopedics, Biomechanics and Implant Technology Research Laboratory, Rostock University Medical Centre
Email:rainer.bader@med.uni-rostock.de,
anika.jonitz-heincke@med.uni-rostock.de

Project description

Exposure to wear and corrosion products is associated not only with the activation of macrophages but also with the activation of osteoblasts and other stromal cells involved in the process of material-induced inflammation. In order to identify the various cellular mechanisms that lead to osteolysis, it is necessary to investigate the initial contact of the cells with the wear products. To achieve this, it must be clarified whether the binding of the wear products to receptors on the cell surface is sufficient to induce intracellular signaling cascades and inflammasome-associated processes or whether these are only initiated by active uptake of the wear products into the cell. Thereby, specific physicochemical properties of the materials for the differential recognition and uptake mechanisms have to be considered. These processes are accompanied by the intracellular activation of various signaling cascades that lead to the release of signaling molecules, which not only activate other cells in the peri-implant tissue but also trigger a systemic reaction. In addition, the intracellular uptake of the wear and corrosion products can lead to irreversible material-dependent cell damage.

Aims of the project

  1. To investigate wear-induced osteolysis by evaluating the toxicity of different particles and metal ions, taking into account exposure route, dose level, rate, and duration. Complex cell culture models are used to identify inflammation-relevant signaling pathways detected via extensive OMIC analyses.
  2. To investigate the phagocytosis capacity of different bone cells depending on material properties by characterizing the longitudinal uptake of specific particle entities with hyperspectral dark-field microscopy. Expression profiles of cellular surface receptors and other recognition mechanisms will be identified in order to conduct specific inhibition experiments on phagocytosis.
  3. To investigate the interplay of stress-induced and replicative senescence and their contribution to particle-induced implant loosening. Investigations in human bone cells after exposure to wear and corrosion products will provide information on stress and aging mechanisms. The expression of age-dependent genes in relation to telomere length, as well as DNA damage and ROS accumulation after contact with wear and corrosion products, will be determined.
E02

Influence of metallic wear and corrosion products on inflammation and cell death: deciphering the molecular signaling pathways

Investigators

Name:Prof. Dr. Barbara Bröker
Dr. VSN Murty Darisipudi
Affiliation:Institute of Immunology and Transfusion Medicine, Department of Immunology, Greifswald University Medical Centre
Email:barbara.broeker@med.uni-greifswald.de,
venkata.darisipudi@med.uni-greifswald.de

Project description

Metal wear and corrosion products from implanted biomaterials modulate the immune response. They directly or indirectly interact with immune cell receptors and induce the release of inflammatory cytokines as well as cell death. We propose that the NLRP3 inflammasome, an intracellular molecular sensor, has a central role in these processes. Activation of the NLRP3 inflammasome leads to the release of mature IL-1β via a form of cell death known as pyroptosis that is mediated by gasdermin (Gsdm) D.
Staphylococcus aureus bacteria are a major cause of prosthetic joint infection necessitating revision arthroplasty, a huge burden on patient health. Numerous bacterial compounds can activate the NLRP3 inflammasome and induce IL-1β release. This suggests that metal wear and corrosion products and S. aureus synergize in the induction of inflammation and host cell death. Finally, metals ions (Co2+, Ni2+), as well as certain S. aureus virulence factors (proteases and enterotoxins) induce “allergic” type 2 inflammation. What are the common mechanisms?
This project aims to clarify the interrelations between metal wear and corrosion products and S. aureus with inflammasome activation, cell death and allergy.

Aims of the project

  1. To identify the molecular sensors or receptors for metal particles or ions, we will screen toll-like receptors (TLRs) using reporter cell lines. Primary immune cells will then be exposed to metal corrosion products to analyze the reaction of the NLRP3 inflammasome. In an exploratory approach we will use proteomics and RNAseq to identify additional metal sensors and molecular regulators.
  2. To visualize the spacial immune landscape in peri-implant tissue sections from metal exposed and metal naïve patients. Cell composition and cell activation will be studied locally but also in the patients’ blood and bone marrow to validate the results obtained in work package 1.
  3. To shed light on the interplay of metal debris and S. aureus in the activation of the inflammasome we will expose immune cells to heat-killed S. aureus, S. aureus-derived virulence factor and/or metal ions or particles. We will read out NLRP3 activation, cytokine release and cell death. This will also provide information about T cell polarization (allergy?) induced by metal wear and corrosion products.
E03

Influence of metallic wear and corrosion products on mesenchymal stem and endothelial cells and relevant bacterial pathogens - effects of an inflammatory environment on differentiation and biofilm formation

Investigators

Name:PD Dr. Kirsten Peters
Affiliation:Institute of Cell Biology, Rostock University Medical Centre
Email:kirsten.peters@med.uni-rostock.de
  
Name:PD Dr. Tomas Fiedler
Affiliation:Institute of Medical Microbiology, Virology and Hygiene Rostock, University Medical Centre
Email:tomas.fiedler@med.uni-rostock.de

Project description

Besides the inflammatory reaction, certain metal compounds can influence cellular processes in various ways. For example, different divalent metal ions (e.g., Co2+) activate the hypoxia-induced factor 1α, which in turn affects the energy metabolism and differentiation status of exposed cells and tissue (increased glycolysis, stem cell activation, induction of vascularization). In addition, corrosion products can cause oxidative stress. Overall, exposure to metallic wear and corrosion products can trigger biochemical reactions that may promote the chronification of the inflammatory process. These reactions are difficult to control in individual cases and significantly impact the degree of differentiation or the immunological response. Further, it is still unclear to what extent released wear and corrosion products affect cells of the implant periphery in their antimicrobial activity against typical implant-infecting bacteria. In that context, it will also be studied whether metallic degradation products influence the biofilm formation of the bacteria and can thus increase susceptibility to infection.

Aims of the project

  1. To investigate the inflammation and differentiation status of human mesenchymal stem/stromal cells after contact with metallic wear and corrosion products. The impact on specific differentiation markers and the expression of toll-like receptors, as well as the release of extracellular vesicles, are being investigated in order to determine possible effects on the immunological capacity of these cells.
  2. To investigate the vascularization potential of endothelial cells and the influence of mesenchymal stem/stromal cells (in co-culture) after contact with metallic wear and corrosion products. The vascularization potential will be investigated using various in vitro models, and endothelial cell-specific extracellular vesicles will be analyzed to determine the possible effects of metallic components on regenerative capacity.
  3. To investigate the impact of wear and corrosion products on antimicrobial activity of phagocytes (neutrophils) towards S. aureus and S. epidermidis. In addition, mesenchymal stem cells, endothelial cells, and osteoblasts are treated with the same stimulants, and the culture supernatants are used to activate phagocytic cells in order to clarify whether the antimicrobial activity is influenced. Furthermore, the formation and composition of biofilms of S. epidermidis and S. aureus upon exposure to wear and corrosion products will be analyzed.