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.

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A common component of the foreign-body response to implanted materials is the presence of adherent macrophages that fuse to form foreign-body giant cells (FBGCs). These multinucleated cells have been shown to concentrate the phagocytic and degradative properties of macrophages at the implant surface and are responsible for the damage and failure of the implant. Therefore, the modulation of the presence or actions of macrophages and FBGCs at the material-tissue interface is an extensive area of recent investigations. A possible mechanism to achieve this is through the induction of the apoptosis of adherent macrophages, which results in no inflammatory consequence. We hypothesize that the induction of the apoptosis of biomaterial adherent cells can be influenced by the chemistry of the surface of adhesion. Herein, we demonstrate that surfaces displaying hydrophilic and anionic chemistries induce apoptosis of adherent macrophages at a higher magnitude than hydrophobic or cationic surfaces. Additionally, the level of apoptosis for a given surface is inversely related to that surface's ability to promote the fusion of macrophages into FBGCs. This suggests that macrophages fuse into FBGCs to escape apoptosis.

The host foreign body response ensues immediately following implantation of medical devices and prostheses. We have previously identified the role of macrophages in adhering to biomaterial surfaces and guiding the foreign body response while fusing into foreign body giant cells (FBGCs) and concentrating degradative and phagocytic activities. Despite their early and transient presence around implanted biomaterials, few studies have focused on the role of lymphocytes in the foreign body response and biocompatibility. To address this, an in vitro human lymphocyte/macrophage coculture system has been developed. Using this system, it has been shown that when lymphocytes are present during the initial adhesion of monocytes, the rate of monocyte adhesion and fusion is significantly increased (1,500 cells/mm2 and 60%, respectively) when compared to either no lymphocytes present (500 cells/mm2 adhesion and 0% fusion). Although lymphocytes adhered to the tissue culture polystyrene surface, 90% of the lymphocytes were associated with adherent macrophages. However, these cell-cell direct interactions were not necessary to influence macrophage adhesion or fusion as separating the two cell types by a Transwell insert still resulted in significantly increased levels of macrophage adhesion (p < 0.05 when compared to macrophage only cultures). Conversely, the presence of macrophages in Transwell experiments increased lymphocyte proliferation rates at all time points tested. These studies begin to detail the interactions between lymphocytes and macrophages in the absence of known antigen that appropriately relates to the scenarios experienced upon implantation of biomedical devices and the initiation of the foreign body response.

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.

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.

A cytokine-based, in vitro model of foreign body giant cell (FBGC) formation was utilized to examine the effect of biomaterial surface chemistry on the adhesion, motility, and fusion of monocytes and macrophages. Human monocytes were cultured for 10 days on 14 different silane-modified glass surfaces, during which time the cells assumed the macrophage phenotype. The adhesion of monocytes and macrophages during the culture period decreased by an average of approximately 50%, with the majority of cell loss observed during days 1-3. Most important, the adhesion of monocytes and macrophages was surface independent except for two surfaces containing terminal methyl groups, which decreased adhesion levels. Interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were added to the medium to induce FBGC formation and enhance macrophage adhesion, respectively. Surprisingly, GM-CSF decreased long-term monocyte/macrophage adhesion. IL-4-induced FBGC density was strongly influenced by the surface carbon content, as determined by X-ray photoelectron spectroscopy (XPS). In contrast, contact angle and surface energy displayed no correlation with FBGC formation. The motility of adherent macrophages, as measured by time-lapse confocal microscopy, was not affected significantly by differences in surface chemistry or the addition of cytokines. The surface dependence of FBGC formation is hypothesized to be the result of varying levels of silane-derived surface carbon.