Publikationsserver der RWTH Aachen University
Analyse der inflammatorischen Potenz mikro- bis nanostrukturierter Materialoberflächen auf primäre humane Makrophagen
Abstract
dc:descriptionIn this study the influence of surface topography on the inflammatory response of human macrophages was investigated. For this, differently structured polyvinylidene fluoride (PVDF) surfaces were generated; including (i) a smooth surface of PVDF spherolites serving as a control, (ii) a randomly nanotextured surface with embedded alumina particles, (iii) a structure with 500 nm wide grooves, and (iv) a microstructure with 1 µm high bumps generated by laser ablation. These structures were verified by white light interferometry and X-profile analysis, and the identical chemistry of all PVDF surfaces was demonstrated by X-ray photoelectron spectroscopy. Macrophages were cultured on the different topographies and as a control for the activation capacity cells were treated with LPS for 24 hours. After seven days of culture the macrophages’ morphology clearly differed on the different topographies. Whereas the cells on the control exhibited an amoboid morphology similar to LPS stimulated macrophages, the cultivation on the microstructure resulted in streched spindle-shaped macrophages. The macrophages on the nanotexture demonstrated a round shape. To investigate the phenotype evolved from the different topographies the macrophages were stained with monoclonal antibodies against function associated surface markers. Both the microstructure and the grooved structure induced the expression of the M1-associated 27E10 and the M2-associated CD163. The nanotexture did not alter the expression of these markers compared to control suggesting that the microstructure and the grooved structure induced an intermediate phenotype with both anti- and proinflammatory properties. To determine the activation state of the macrophages on the different topographies the concentrations of inflammatory mediators in the culture supernatant were measured. Some proinflammatory mediators like IL-1β, CCL2 and CCL3 were upregulated by the microstructure and LPS, whereas others like IL-12 and CCL5 were only induced by LPS. The LPS induced CXCL10 was downregulated by the microstructure. The anti-inflammatory cytokine IL-1RN was repressed by LPS and the microstructure. These protein data could mostly be confirmed on mRNA level using a DNA microarray. The grooved and the nanotextured topography did mostly not alter the secretion of the investigated mediators compared to the control. Additionally, the DNA microarray identified further genes regulated by topography including both M1- associated genes like IL1R1 and IL7R, and those associated with M2 responses like CD209, SEPP1 and STAB1. Some of the genes were regulated similarly by LPS like IL7R and CASP1, whereas the regulation of others was opposed like SEPP1 and HMOX1. The present results demonstrate that the microstructured topography significantly affects the activation of primary human macrophages by inducing a specific cytokine and gene expression profile. This activation resulted in a subtype of macrophages with pro- but also anti-inflammatory properties. The cytokine secretion pattern and the gene expression profile clearly differed from that induced by LPS suggesting a specific activation state of the cells. The phenotype was clearly due to this particular surface since the grooved structure and the nanotexture had nearly no effect on the activation of the macrophages compared to the control. In conclusion, our data suggest that the modification of topography could influence the inflammatory potency of a biomaterial and hence could affect the biocompatibility of implants. The establishment of a transgenic cell line was a second aim of this study. The human monocytic cell line THP-1 was transfected with a construct of the S100A8 gene (coding for one protein of the 27E10 epitope) and the coding sequence of the fluorochrome DsRed. During cultivation the gained positive clones seperated into two populations of which the smaller one (approx. 5 % of entire population) expressed DsRed constitutively. After stimulation with PMA and PMA/LPS, respectivly, inducing the expression of the 27E10 epitope the majority of the transgenic cells demonstrated DsRed fluorescence. These transgenic cells offer the opportunity to monitor inflammatory processes online and to investigate the expression kinetics of the 27E10 antigen by fluorenscence.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2008
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Paul, Nora Emilie
- Contributors dc:contributor
-
- Zwadlo-Klarwasser, Gabriele-Claudia
Subjects
dc:subject × 10Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger