Current strategies to limit macrophage adhesion, fusion and fibrous capsule formation in the foreign body response have focused on modulating material surface properties. We hypothesize that topography close to biological scale, in the micron and nanometric range, provides a passive approach without bioactive agents to modulate macrophage behavior. In our study, topography-induced changes in macrophage behavior was examined using parallel gratings (250 nm-2 μm line width) imprinted on poly(s-caprolactone) (PCL), poly(lactic acid) (PLA) and poly(dimethyl siloxane) (PDMS). RAW 264.7 cell adhesion and elongation occurred maximally on 500 nm gratings compared to planar controls over 48 hr. TNF-α and VEGF secretion levels by RAW 264.7 cells showed greatest sensitivity to topographical effects, with reduced levels observed on larger grating sizes at 48 hr. In vivo studies at 21 days showed reduced macrophage adhesion density and degree of high cell fusion on 2 μm gratings compared to planar controls. It was concluded that topography affects macrophage behavior in the foreign body response on all polymer surfaces examined. Topography-induced changes, independent of surface chemistry, did not reveal distinctive patterns but do affect cell morphology and cytokine secretion in vitro, and cell adhesion in vivo particularly on larger size topography compared to planar controls.

Implantation of biomaterial devices results in the well-known foreign body reaction consisting of monocytes, macrophages, and foreign body giant cells (FBGCs) at the material/tissue interface. We continue to address the hypothesis that material surface chemistry modulates the phenotypic expression of these cells. Utilizing our human monocyte culture system, we have used surface-modified polymers displaying hydrophobic, hydrophilic, and/or ionic chemistries to determine the cytokines/chemokines released from biomaterial-adherent macrophages/FBGCs. This study broadens our approach by using proteomic analysis to identify important factors expressed by these cells and further quantifies these molecules with ELISAs. Proteomic profiles changed over time suggesting that the adherent macrophages underwent a phenotypic switch. Macrophage/FBGC-derived proinflammatory cytokines, IL-1beta and IL-6, decreased with time, while the anti-inflammatory cytokine, IL-10, gradually increased with time. Resolution of the inflammatory response was also demonstrated by a decrease in chemoattractant IL-8 and MIP-1beta production with time. Material-dependent macrophage/FBGC activation was analyzed using cytokine/chemokine production and cellular adhesion. Monocyte/macrophage adhesion was similar on all surfaces, except for the hydrophilic/neutral surfaces that showed a significant decrease in cellular density and minimal FBGC formation. Normalizing the ELISA data based on the adherent cell population provided cytokine/chemokine concentrations produced per cell. This analysis showed that although there were fewer cells on the hydrophilic/neutral surface, these adherent cells were further activated to produce significantly greater amounts of each cytokine/chemokine tested than the other surfaces. This study clearly presents evidence that material surface chemistry can differentially affect monocyte/macrophage/FBGC adhesion and cytokine/chemokine profiles derived from activated macrophages/FBGCs adherent to biomaterial surfaces.

Matrix metalloproteinases (MMPs) can degrade structural components within the extracellular matrix and at the cellular surface producing changes in cellular behavior (i.e., adhesion and migration) and subsequent pathological responses (i.e., the foreign body reaction and wound healing). We continue to study the foreign body reaction that occurs following biomaterial implantation by investigating secretory responses of biomaterial-adherent macrophages and foreign body giant cells (FBGCs) as directed by material surface chemistry and further this research by determining whether secreted MMPs play a role in macrophage adhesion and fusion. We have identified numerous MMPs and their tissue inhibitors (TIMPs) in in vitro cell-culture supernatants using antibody arrays and quantified select MMP/TIMPs with ELISAs. MMP-9 concentrations were significantly greater than both TIMP-1 and TIMP-2 on all materials. The ratios of MMP-9/TIMP-1 and MMP-9/TIMP-2 increased with time because of an increase in MMP-9 concentrations over time, while the TIMP concentrations remained constant. Total MMP-9 concentrations in the supernatants were comparable on all materials at each timepoint, while TIMP-1 and TIMP-2 concentrations tended to be greater on hydrophilic/anionic surfaces. Analysis of the MMP/TIMP quantities produced per cell revealed that the hydrophilic/neutral surfaces, which inhibited macrophage adhesion, activated the adherent macrophages/FBGCs to produce a greater quantity of MMP-9, TIMP-1, and TIMP-2 per cell. Pharmacological inhibition of MMP-1,-8,-13, and -18 reduced macrophage fusion without affecting adhesion, while inhibitors of MMP-2,-3,-9, and -12 did not affect adhesion or fusion. These findings demonstrate that material surface chemistry does modulate macrophage/FBGC-derived MMP/TIMP secretion and implicates MMP involvement in macrophage fusion.

An in vitro system of interleukin (IL)-4-induced foreign body giant cell (FBGC) formation was utilized to define the adhesion protein substrate(s) that promotes this aspect of the foreign body reaction on biomedical polymers. Human monocytes were cultured on cell culture polystyrene surfaces that had been pre-adsorbed with a synthetic arginine-glycine-aspartate (RGD) peptide previously found to support optimal FBGC formation, or with various concentrations of potential physiological protein substrates, i.e. complement C3bi, collagen types I or IV, fibrinogen, plasma fibronectin, fibroblast fibronectin, laminin, thrombospondin, vitronectin, or von Willebrand factor. Cultures were evaluated on days 0 (1.5 hr), 3, and 7 by May-Grünwald/Giemsa staining. Initial monocyte adhesion occurred on all adsorbed proteins. However, by day 7 of culture, only vitronectin was striking in its ability to support significant macrophage adhesion, development, and fusion leading to FBGC formation. Vitronectin supported high degrees of FBGC formation at an absorption concentration between 5 and 25 μg per ml. These findings suggest that adsorbed vitronectin is critical in the collective events that support and promote FBGC formation on biomedical polymers, and that the propensity for vitronectin adsorption may underlie the material surface chemistry dependency of FBGC formation.

Surface chemistry modulates many critical functions of monocyte/macrophages such as adhesion, fusion, spreading, phagocytosis, and secretion. In this study, we investigated the effect of silicone modification on adhesive structure development and cytoskeletal reorganization of adherent macrophages on polyurethanes. Confocal scanning laser microscopy (CSLM) was used for qualitative and quantitative evaluation of cytoskeletal reorganization of adherent macrophages. Data presented here showed less spreading for adherent cells on silicone-modified materials due to the higher hydrophobicity and protein adsorption profile. This decrease in spreading was accompanied by less F-actin content in adherent cells on silicone-modified polyurethanes and PDMS control, indicating that silicone modification reduces the strength of adhesion. With the addition of interleukin-4 (IL-4) at days 3 and 7 to our culture, adherent cell morphology dramatically changed. The change in morphology led to higher macrophage fusion and foreign body giant cell (FBGC) formation on silicone modified materials after 10 days. In addition, mannose receptor (MR) expression was up-regulated on the silicone-modified polyurethanes and PDMS control in the presence of IL-4. Up-regulation of MR expression suggests an alternatively activated phenotype for adherent macrophages, which is accompanied with an attenuated proinflammatory cytokine production and reactive oxygen secretion. It appears that silicone modification accelerates acquisition of an alternative macrophage and FBGC phenotype, which may then result in increased polyurethane biostability.

To better understand the relationship between macrophage/foreign body giant cell adhesion and activation on surface-modified biomaterials, quantitative assessment of adherent cell density (cells per mm²) and cytokine production (pgs per mL) were determined by ELISA. Further analysis to identify cellular activation was carried out by normalizing the cytokine concentration data to provide a measure of cellular activation. This method of analysis demonstrated that hydrophobic surfaces provided statistically significantly greater adherent cell densities than hydrophilic/neutral surfaces. However, when cell activation parameters were determined by normalization to the adherent cell density, the hydrophilic/neutral surfaces demonstrated statistically significantly greater levels of activation and production of IL-10, IL-1β, IL-6, IL-8, and MIP-1β. With increasing time, production of the anti-inflammatory cytokine IL-10 increased, whereas IL-1β, IL-6, and IL-8 decreased and MIP-1β was relatively constant over the culture time period. This observed dichotomy or disparity between adhesion and activation may be related to surface-induced adherent cell apoptosis. Further evaluation of macrophage activation on biomaterial surfaces indicated that an apparent phenotypic switch in macrophage phenotype occurred over the course of the in vitro culture. Analysis of cytokine/ chemokine profiles with surface-modified biomaterials revealed similarities between the classically activated macrophages and the biomaterial-adherent macrophages early (day 3) in culture, while at later timepoints the biomaterial-adherent macrophages produced profiles similar to alternatively activated macrophages. Classically activated macrophages are those commonly activated by LPS (lipopolysaccharide) or IFN-γ (interferon-γ) and alternatively activated macrophages are those activated by IL-4/IL-13 or IL-10. Surface modification of biomaterials offers an opportunity to control cellular activation and cytokine profiles in the phenotypic switch, and may provide a means by which macrophages can be induced to regulate particular secretory proteins that direct inflammation, the foreign body reaction, wound healing, and ultimately biocompatibility.

The role of lymphocytes in the biological response to synthetic polymers is poorly understood despite the transient appearance of lymphocytes at the biomaterial implant site. To investigate cytokines, chemokines, and extracellular matrix (ECM) proteins produced by lymphocytes and macrophages in response to biomaterial surfaces, human peripheral blood monocytes and lymphocytes were co-cultured on polyethylene terephthalate (PET)-based material surfaces displaying distinct hydrophobic, hydrophilic/neutral, hydrophilic/anionic, and hydrophilic/cationic chemistries. Antibody array screening showed the majority of detected proteins are inflammatory mediators that guide the early inflammatory phases of wound healing. Proteomic ELISA quantification and adherent cell analysis were performed after 3, 7, and 10 days of culture. IL-2 and IFN-γ were not detected in any co-cultures suggesting lack of lymphocyte activation. The hydrophilic/neutral surfaces increased IL-8 relative to the hydrophobic PET surface (p<0.05). The hydrophilic/anionic surfaces promoted increased TNF-α over hydrophobic and cationic surfaces and increased MIP-1β compared to hydrophobic surfaces (p<0.05). Since enhanced macrophage fusion was observed on hydrophilic/anionic surfaces, the production of these cytokines likely plays an important role in the fusion process. The hydrophilic/cationic surface promoted IL-10 production and increased matrix metalloproteinase (MMP)-9/tissue inhibitor of MMP (TIMP) relative to hydrophilic/neutral and anionic surfaces (p<0.05). These results suggest hydrophilic/neutral and anionic surfaces promote pro-inflammatory responses and reduced degradation of the ECM, whereas the hydrophilic/cationic surfaces induce an anti-inflammatory response and greater MMP-9/TIMP with an enhanced potential for ECM breakdown. The study also underscores the usefulness of protein arrays in assessing the role of soluble mediators in the inflammatory response to biomaterials.

Adherent macrophages and foreign body giant cells (FBGCs) are known to release degradative molecules that can be detrimental to the long-term biostability of polyurethanes. The modification of polyurethanes using surface modifying endgroups (SMEs) and/or the incorporation of silicone into the polyurethane soft segments may alter macrophage adhesion, fusion and apoptosis resulting in improved long-term biostability. An in vitro study of macrophage adhesion, fusion and apoptosis was performed on polyurethanes modified with fluorocarbon SMEs, polyethylene oxide (PEO) SMEs, or poly(dimethylsiloxane) (PDMS) co-soft segment and SMEs. The fluorocarbon SME and PEO SME modifications were shown to have no effect on macrophage adhesion and activity, while silicone modification had varied effects. Macrophages were capable of adapting to the surface and adhering in a similar manner to the silicone-modified and unmodified polyurethanes. In the absence of IL-4, macrophage fusion was comparable on the modified and unmodified polyurethanes, while macrophage apoptosis was promoted on the silicone modified surfaces. In contrast, when exposed to IL-4, a cytokine known to induce FBGC formation, silicone modification resulted in more macrophage fusion to form foreign body giant cells. In conclusion, fluorocarbon SME and PEO SME modification does not affect macrophage adhesion, fusion and apoptosis, while silicone modification is capable of mediating macrophage fusion and apoptosis. Silicone modification may be utilized to direct the fate of adherent macrophages towards FBGC formation or cell death through apoptosis.