Influence of biomaterial surface chemistry on the apoptosis of adherent cells

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.     

Inhibition of macrophage development and foreign body giant cell formation by hydrophilic interpenetrating polymer network

The ability of monocytes to adhere, differentiate into macrophages, and fuse to form foreign body giant cells (FBGCs) on an implanted material surface is a critical step toward biomaterial degradation. Novel homogeneous surfaces were utilized to mediate adhesion. These surfaces consisted of N-(2 aminoethyl)-3-aminopropyltrimethoxysilane (EDS) and an interpenetrating polymer network (IPN) of polyacrylamide and poly(ethylene glycol). These surfaces were designed to control cell adhesion and morphology and mediate cell differentiation, activation, metabolic ability, and apoptosis, resulting in a reduced or controlled inflammatory response. The EDS surface promotes cell adhesion and the IPN minimizes protein adsorption and subsequent cell adhesion. Both surfaces had similar cellular adhesion rates at each respective time point. However, the adherent macrophage morphology was similar at 2 h and day 3, and at days 7 and 10 adherent macrophages on the EDS surface formed FBGCs (46% at day 7 and 40% at day 10). Adherent cells on the IPN surface did not form FBGCs but instead formed monocyte aggregates (73% of adherent cells formed aggregates at day 7 and 63% at day 10). It is indicated that the two surface chemistries differentially controlled monocyte differentiation into macrophages and subsequent macrophage fusion to form FBGCs.     

Macrophage behavior on surface-modified polyurethanes

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.     

Proteomic analysis and quantification of cytokines and chemokines from biomaterial surface-adherent macrophages and foreign body giant cells

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.     

Macrophage behavior on surface-modified polyurethanes

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.     

In vivo leukocyte cytokine mRNA responses to biomaterials are dependent on surface chemistry

An in vivo mouse cage implant system was used to determine whether leukocyte cytokine mRNA responses to implanted biomaterials were dependent on surface chemistry. Surfaces displaying various chemistries (hydrophobic, hydrophilic, anionic, and cationic) were placed into stainless steel cages and implanted subcutaneously. Semiquantitative RT-PCR analyses revealed that hydrophilic surfaces showed a decreased expression of proinflammatory cytokines, IL-6 and IL-8, and pro-wound healing cytokines, IL-10 and TGF-beta by adherent cells, and mRNA levels of the proinflammatory cytokine, IL-1beta, and the pro-wound healing cytokine IL-13 were decreased in surrounding, exudate cells. Cytokine responses by adherent and exudate cells to hydrophobic, anionic and cationic surfaces were similar and indicative of a strong inflammatory response at the earliest time point followed by a wound healing response at later time points. However, no differences in the types or levels of exudate cells for any of the surfaces or the empty cage at each of the respective time points were observed, indicating their respective biocompatibility. These studies identify hydrophilic surface chemistries as having significant effects on leukocyte cytokine responses in vivo by decreasing the expression of inflammatory and wound healing cytokines by inflammatory cells adherent to the biomaterial as well as present in the surrounding exudate.     

Matrix metalloproteinases and their inhibitors in the foreign body reaction on biomaterials

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.     

Lymphocyte/macrophage interactions: biomaterial surface-dependent cytokine, chemokine, and matrix protein production

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-gamma 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-alpha over hydrophobic and cationic surfaces and increased MIP-1beta 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.     

Fibronectin modulates macrophage adhesion and FBGC formation: the role of RGD, PHSRN, and PRRARV domains.

To probe the role of human plasma fibronectin in modulating human blood-derived macrophage adhesion and fusion to form multinucleated foreign-body giant cells (FBGC), a series of biomimetic oligopeptides based on the functional structure of fibronectin was designed and synthesized. Peptides incorporated the RGD and PHSRN integrin-binding sequences from FIII-10 and FIII-9 modules, respectively, and the PRRARV sequence from the C-terminal heparin-binding domain, either alone or in combination. Peptides were immobilized onto a polyethyleneglycol-based polymer substrate. The following conclusions were reached. Fibronectin modulated macrophage adhesion and the extent (i.e., size) of FBGC formation on control surfaces in the presence of serum proteins. Macrophages adhered to all substrates with relatively subtle differences between adhesion mediated by RGD, PHSRN, PRRARV, or combinations thereof. beta1-integrin subunit was essential in macrophage adhesion to peptide-grafted networks in a receptor-peptide specific manner, whereas beta3-integrin subunit was less important. Macrophage adhesion to PRRARV was mediated primarily by the direct interaction with integrins. RGD or PHSRN alone did not provide an adequate substrate for macrophage fusion to form FBGCs. However, the PHSRN synergistic site and the RGD site in a single oligopeptide provided a substrate for FBGC formation that was statistically comparable to that on the positive control material in the presence of serum proteins. This response was highly dependent upon the relative orientation between RGD and PHSRN. PRRARV did not support FBGC formation. These results demonstrate the importance of fibronectin and, specifically, the synergy between RGD and PHSRN domains, in supporting macrophage fusion to form FBGCs.     

Paracrine and juxtacrine lymphocyte enhancement of adherent macrophage and foreign body giant cell activation

Lymphocytes have been shown to be involved in modulating monocyte and macrophage behavior in the foreign body reaction. Lymphocyte effects on biomaterial-adherent macrophage and foreign body giant cell (FBGC) behavior were further investigated by culturing monocytes alone or together with lymphocytes, either in direct co-cultures or indirectly in transwells, on a series of polyethylene terephthalate-based photograft co-polymerized material surfaces displaying distinct hydrophobic, hydrophilic/neutral, hydrophilic/anionic, and hydrophilic/ cationic chemistries. After periods of 3, 7, and 10 days, cytokine production was quantified by enzyme-linked immunosorbent assay and normalized to adherent macrophage/FBGC density to yield a measure of adherent macrophage/FBGC activation. Interactions with lymphocytes enhanced adherent macrophage and FBGC production of pro-inflammatory IL-1beta, TNF-alpha, IL-6, IL-8, and MIP-1beta on the hydrophobic and hydrophilic/cationic surfaces but had no effect on anti-inflammatory IL-10 production indicating lymphocytes promote a pro-inflammatory response to biomaterials. Lymphocytes also did not significantly influence MMP-9, TIMP-1, and TIMP-2 production. Interactions through indirect (paracrine) signaling showed a significant effect in enhancing adherent macrophage/FBGC activation at early time points whereas interactions via direct (juxtacrine) mechanisms dominated at later time points. Biomaterial surface chemistries differentially affected the observed responses as hydrophilic/neutral and hydrophilic/anionic surfaces, evoked the highest levels of activation relative to the other surfaces but did not facilitate lymphocyte enhancement of adherent macrophage/FBGC activation.     

Adsorbed serum proteins responsible for surface dependent human macrophage behavior

Substrate specific cellular responses are the result of a complex biological system that includes protein adsorption, receptor-ligand binding, and signal transduction. This investigation attempted to identify specific proteins adsorbed from human serum that may be responsible for the previously reported in vitro surface dependent behavior of human macrophages and foreign body giant cells (FBGCs). The adsorption of human albumin, alpha(2)-macroglobulin, complement factor 3b, fibronectin, IgG, thrombospondin, vitronectin (VN), and von Willebrand factor (vWF) from a 25% serum solution was quantified with (125)I-labeled protein. Adsorption substrates included clean glass, alkyl-silane modified glass, amino-silane modified glass, poly(ethylene oxide) (PEO)-coupled glass, and the reference biomaterials poly(etherurethane urea), Silastic(R), and poly(tetrafluoroethylene) (PTFE). Following quantification of 2-h adsorption, surfaces were treated with sodium dodecyl sulfate (SDS) and the level of adsorbed proteins remaining was quantified. The pre- and post-SDS adsorption were both compared to previously reported surface dependent in vitro macrophage and FBGC behavior on the same surfaces; however, no correlations could be made. Adsorption strength, defined as the percentage of initially adsorbed protein that remained adsorbed after SDS treatment, correlated well with previously reported in vitro cellular behavior indicating that adsorbed vWF, IgG, and VN may be involved in the modulation of adherent macrophage and FBGC behavior. Those surfaces that strongly adsorbed vWF also inhibited long-term macrophage adhesion, while those surfaces that strongly adsorbed IgG promoted long-term macrophage adhesion. In addition, the highest levels of FBGC formation had been observed only on those surfaces that strongly adsorbed VN. Subsequent human monocyte cultures on protein preadsorbed substrates confirmed the inhibitory effect of adsorbed vWF and the promoting effect of IgG on longterm macrophage adhesion as predicted by adsorption strength correlations. However, preadsorbed VN was not observed to modulate FBGC formation, which is in contrast to the conclusions of the adsorption correlations.     

Effects of surface-coupled polyethylene oxide on human macrophage adhesion and foreign body giant cell formation in vitro.

Surface immobilized polyethylene oxide (PEO) has been shown to efficiently reduce protein adsorption and cellular adhesion, resulting in a biologically passive surface. To explore the in vitro effects of surface immobilized PEO on the human inflammatory cells, macrophages, and foreign body giant cells (FBGCs), we developed a diisocyanate-based method for coupling PEO to amine-modified glass, a surface previously shown to enhance macrophage adhesion and FBGC formation. Contact angle analysis and X-ray photoelectron spectroscopy confirmed the presence of PEO molecules bound to the surface and revealed that PEO molecular weight significantly influenced the efficiency of PEO coupling. We used a 10-day human monocyte culture protocol to demonstrate that the presence of surface coupled PEO molecules does not significantly decrease initial monocyte density or monocyte-derived macrophage density after 3 days. However, PEO-coupled surfaces significantly reduced long-term monocyte-derived macrophage density and virtually eliminated interleukin-4-induced FBGC formation observed at day 10. The cellular response to these PEO-coupled surfaces was related to the molecular weight of the PEO chains, which was varied between 200 Da and 18.5 kDa. These results suggest that an optimized PEO surface treatment may be effective in reducing inflammatory cell adhesion and possible degradation during the inflammatory response to an implanted biomedical device.     

Beta1 and beta2 integrins mediate adhesion during macrophage fusion and multinucleated foreign body giant cell formation

An in vitro system of interleukin (IL)-4-induced human monocyte-derived macrophage fusion was used to investigate the cell/substrate adhesive mechanisms that support multinucleated foreign body giant cell (FBGC) formation. Monocytes were cultured for 3 days and IL-4 was added to induce macrophage fusion and FBGC formation by day 7. Functionally defined anti-integrin antibodies demonstrated that initial monocyte adhesion is mediated by beta2 integrins, whereas during the induction of macrophage fusion by IL-4, an additional dependence on beta1 integrins is acquired. The combination of anti-beta1 plus anti-beta2 was most effective, reducing macrophage/FBGC adhesion to 10% of controls. Consistent with integrin-mediated signaling, the tyrosine kinase inhibitor genistein and the phosphatidylinositol-3-kinase inhibitors wortmannin and LY294002 also attenuated macrophage/FBGC adhesion. Confocal microscopic analysis revealed that beta2 integrins are present on monocytes after initial adhesion and are strongly expressed on fusing macrophages, particularly in peripheral cell areas, and on FBGCs. In contrast, beta1 integrins are not detected on monocytes but begin to appear during macrophage development and are strongly expressed on fusing macrophages and FBGCs. For the first time, these results demonstrate the IL-4-induced acquisition of cooperation between beta1 and beta2 integrins in the cell/substrate adhesive interactions that are required for multinucleated FBGC formation.     

Foreign body-type multinucleated giant cell formation is potently induced by alpha-tocopherol and prevented by the diacylglycerol kinase inhibitor R59022

Multinucleated foreign body giant cells (FBGCs) form by monocyte-derived macrophage fusion on implanted biomedical devices and are believed to mediate oxidative damage to biomaterial surfaces. Our in vitro system of human macrophage culture and interleukin (IL)-4-induced FBGC formation was developed to study the macrophage fusion mechanism and the physiological significance of FBGCs on implanted biomaterials and at other sites of chronic inflammation. Here, we demonstrate that the antioxidant vitamin E (90% alpha-tocopherol) moderately induces macrophage fusion and increases IL-4-induced FBGC formation. Moreover, purified alpha-tocopherol, but not beta-, gamma-, or delta-tocopherol, most remarkably induces macrophage fusion, leading to cultures of confluent FBGCs below normal plasma concentrations. This is not observed with the similar antioxidants probucol or Trolox, suggesting that the alpha-tocopherol effects on FBGC formation are independent of its antioxidant activity. Consistent with the reported activation of diacylglycerol kinase by alpha-tocopherol, the diacylglycerol kinase inhibitor R59022 completely abrogates FBGC formation. R59022 inhibition of IL-4-induced FBGC formation is reversed by alpha-tocopherol, suggesting that FBGC formation involves diacylglycerol kinase activation. This study suggests a novel role for diacylglycerol kinase in the mechanism of macrophage fusion/FBGC formation at sites of chronic inflammation and reveals that the pleiotropic lipophilic compound, alpha-tocopherol, is a highly potent macrophage fusion factor.     

Instability of self-assembled monolayers as a model material system for macrophage/FBGC cellular behavior

Novel self-assembled monolayers (SAMs) designed to present homogenous surface chemistries were utilized to further investigate the material surface chemistry dependent macrophage and foreign-body giant cell (FBGC) behaviors, including macrophage adhesion, fusion, and apoptosis. Contact angle analysis revealed instabilities in the --CH(3) and --COOH terminated SAM surfaces upon incubation in serum-free media (SFM) at 37 degrees C or under dry, room temperature conditions. Further analysis indicated that the --CH(3) terminated SAM surface degraded rapidly within 2 h and loss of sufficient SAM units to be comparable to the gold (Au) control surface, within 24 h of incubation in SFM at 37 degrees C. After 5 days of incubation in SFM at 37 degrees C, the contact angles for the --COOH terminated SAM surfaces increased markedly. AFM analysis confirmed the desorption of --CH(3) terminated SAM molecules from the surface with increased roughness and marked appearance of peaks and valleys within 2 h. A decrease in the thickness of the --COOH terminated SAM surface also suggests molecular desorption over time. No significant changes in contact angle or AFM analyses were observed on the --OH terminated SAM surfaces. Cellular adhesion decreased more rapidly on the Au control and --CH(3) terminated SAM surfaces in comparison to the other surfaces. However by day 10, cellular adhesion, fusion, and apoptosis were comparable on all SAM surfaces and the Au control. These studies suggest that SAM surfaces may not be suitable for long-term studies where material dependent properties are investigated.     

Lymphocytes and the foreign body response: lymphocyte enhancement of macrophage adhesion and fusion

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.     

Spatial regulation and surface chemistry control of monocyte/macrophage adhesion and foreign body giant cell formation by photochemically micropatterned surfaces.

A long-standing goal of biomedical device development has been the generation of specific, desired host blood and tissue responses. An approach to meeting this design criteria is precise surface modification that creates micropatterns of distinct physicochemical character to direct cell adhesion and behavior. For this study, poly(ethylene terephthalate) films were coated with poly(benzyl N, N-diethyldithiocarbamate-co-styrene) and sequentially exposed to monomer solutions for photoirradiation. A photomask was placed over different regions to generate micropatterned surfaces with graft polymer stripes of three distinct ionic characters. Human monocytes were cultured on these surfaces to ascertain whether adhesion and fusion of monocytes/macrophages could be controlled. Nonionic polyacrylamide greatly inhibited adhesion and induced clumping of the few monocytes that did adhere. Macrophage adhesion and spreading led to high degrees of interleukin-13 induced foreign body giant cell formation on both the anionic poly(acrylic acid), sodium salt, and benzyl N,N-diethyldithiocarbamate portions of the culture surface. In spite of the highest observed levels of monocyte/macrophage adhesion on cationic poly(dimethylaminopropylacrylamide), methiodide, the adherent cells were not competent to undergo fusion to form foreign body giant cells. These results suggest that inflammatory cell responses may be spatially controlled in a manner that may be ultimately exploited to improve the biocompatibility of medical devices.     

Theoretical analysis on cell size distribution and kinetics of foreign-body giant cell formation in vivo on polyurethane elastomers.

The nature of in vivo leukocyte adhesion and foreign-body giant cell (FBGC) formation on polyurethanes was studied through theoretical and statistical analyses in terms of cell size distribution, density changes, and kinetics of FBGC formation. The results showed that the size distribution of FBGCs followed a "most probable" distribution. During FBGC formation, the densities of FBGCs changed with time. At an early stage, the number of FBGCs increased with time to a maximum at the expense of macrophages. As more FBGCs were formed and less macrophages were present, the fusion of FBGCs among themselves became significant. This, in turn, caused a gradual decrease of FBGC density with time. The rate of FBGC formation was characterized by a rate constant that represented certain characteristics of cell fusion and FBGC formation and the density of initial FBGC-forming macrophages that were a small fraction of leukocytes adhering to the surface. The direct correlations of surface cracking and pitting and adherent FBGCs demonstrated the influence of phagocytic actions of FBGCs on the biostability of implanted polyurethanes. While the cracking was thought to be caused by oxidative degradation facilitated by oxygen ion/radical release of FBGCs, the pitting appeared to result from the Methacrol 2138F aggregates diffusing out of the polymer in an acidic microenvironment under FBGCs, which in turn could be enhanced by the surface degradation and cell phagocytosis. The added Santowhite powder in polyurethane had a significant influence on FBGC formation: It reduced FBGC density and rate of FBGC formation by reducing leukocyte adhesion and the number of macrophages participating in FBGC formation.     

Surface chemistry mediates adhesive structure, cytoskeletal organization, and fusion of macrophages

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.