Elsevier

Biomaterials

Volume 29, Issue 30, October 2008, Pages 4056-4064
Biomaterials

Topographical control of human macrophages by a regularly microstructured polyvinylidene fluoride surface

https://doi.org/10.1016/j.biomaterials.2008.07.010Get rights and content

Abstract

In this study we investigated the influence of surface topography on the inflammatory response of human macrophages. We generated different polyvinylidene fluoride (PVDF) surfaces including (i) a smooth surface of PVDF spherulites as a control, (ii) a randomly nanotextured surface with alumina particles, and (iii) a microstructure using laser ablation. The identical chemistry of all PVDF surfaces was demonstrated by X-ray photoelectron spectroscopy. The topography was evaluated by white light interferometry and X-profile analysis. Macrophages were cultured on the different surfaces including lipopolysaccharide (LPS) treatment as an inflammatory activator. Our results demonstrate that the microstructured surface but not the nanotexured significantly affects the activation of primary human macrophages by inducing a specific cytokine and gene expression pattern. This activation resulted in a subtype of macrophages with pro- but also anti-inflammatory properties. Interestingly, the response on the topography differed from that triggered by LPS, pointing to a different activation state of the cells. Our data clearly show that a particular topography induces an inflammatory response. This suggests that the modification of topography could influence the inflammatory potency of a biomaterial and hence could affect the biocompatibility of implants.

Introduction

It is well known and accepted that the surface topography of an implanted material exerts a significant influence on the function of cells – a term known as ’topographical control’ (reviewed in [1]). However, only 2% of all implants are made of specially optimized biomaterials and many polymers have a micrometre or submicrometre scaled topography by accident due to the production process that could trigger undesired cell responses after implantation. In particular, the interaction of inflammatory cells with biomaterial surfaces is believed to affect the biocompatibility of a given biomaterial.

Among inflammatory cells macrophages play a key role in the host response to foreign bodies therefore being crucial for determining the biocompatibility of an implant [2], [3]. Macrophages mediate inflammatory processes especially chronic inflammation, the most frequent adverse effect after implantation. Moreover, macrophages are crucially involved in guiding tissue repair and wound healing [4]. The multiple functions of macrophages mirror their heterogeneity. During inflammation macrophages exhibit two different phenotypes: M1 and M2 macrophages [5], [6], [7]. M1 macrophages are classically activated proinflammatory subtypes induced by lipopolysaccharide (LPS). They secrete proinflammatory cytokines like interleukin (IL)-1 and IL-6 as well as chemokines and can be stained with the 27E10 antibody directed against a heterodimer formed of the two calcium-binding proteins S100A8 and S100A9 [8], [9]. The term M2 macrophages refers to various subtypes of non-classically activated macrophages [6]. A common feature of M2 macrophages is the release of anti-inflammatory cytokines. The scavenger receptor CD163 is a specific surface marker expressed by M2 cells [10], [11].

Because of their important role in the foreign body response macrophages are widely used to study the biocompatibility of implanted materials. Several studies point to the significance of the surface chemistry on the inflammatory response of macrophages in particular on cytokine release [12], [13]. The influence of topography, however, is poorly investigated. Using murine macrophage cell lines it was reported that rough titanium surfaces induce the expression of proinflammatory cytokines and chemokines on mRNA as well as on protein level [14], [15]. With respect to human macrophages Meyle et al. demonstrated contact guidance on a micro-grooved substratum but no further cell parameters were investigated in this context [16].

In this study, the influence of surface topography on the cellular and molecular response of primary human macrophages is being investigated using different micro- and nanostructured (poly)vinylidene fluoride (PVDF) textures demonstrating the same chemistry. PVDF is a partially fluorinated homopolymer with outstanding biocompatibility demonstrated for several cell types and already in use in clinical applications [17], [18].

We could show that the microstructured but not the nanotextured topography influenced the activation state of primary human macrophages resulting in a specific cytokine and gene expression pattern different from that elicited by LPS.

Section snippets

PVDF surface preparation

PVDF SOLEF™ 1008 pellets were purchased from Solvay Adv. Polym. (Dusseldorf, Germany). [2], [2]-Paracyclophane was obtained from Fluka (Sigma-Aldrich, Munich, Germany). All solvents and reagents were purchased from Fluka and Riedel-deHaën (Sigma-Aldrich). Glass cover slips (Marienfeld, Germany), with 15 mm diameter, were used as substrate materials. The substrates were cleaned by sonification for 5 min in an ethanol/hexane solution (21:79; m/m) and dried under nitrogen flow. PVDF pellets were

Generation and characterisation of PVDF surfaces

Three chemically identical PVDF substrates with different topography were produced using different techniques. First sample type was spin coated and annealed PVDF crystallised in spherulitic α phase PVDF with a supermolecular morphology [20], [21]. The average diameter of spherulites was estimated from scanning white light interferometry (SWIM) graphs to be 80 μm (Fig. 1A). The spherulitic PVDF surface demonstrated tip-like heights representing the primary nucleation area of each spherulite.

Discussion

Materials for biomedical or tissue engineering purposes should provide physico-chemical properties matching the surrounding tissue to support the desired cell response, e.g. adhesion, proliferation or differentiation. Moreover, the inflammatory response, the most important adverse reaction to biomaterials, should be prevented. Besides the well-documented effects of material chemistry on the function of cells [23], it is now generally accepted that the surface topography influences the cellular

Conclusions

In conclusion, our data demonstrate that the microstructured surface significantly affected the activation of primary human macrophages by inducing a specific cytokine and gene expression pattern. This activation resulted in a subtype of macrophages with pro- and anti-inflammatory properties. The data obtained with the nanotextured surface confirmed that this activation is due to this particular topography because the response to this surface is similar to that elicited on the control.

Acknowledgments

Supported by a grant from the Interdisciplinary Centre for Clinical Research (IZKF-BIOMAT) within the faculty of Medicine at the RWTH Aachen (TVB105) and a grant from the DFG research training group “Biointerface” (GRK1035).

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