Adsorbed fibrinogen enhances production of bone- and angiogenic-related factors by monocytes/macrophages.

Macrophages are phagocytic cells with great importance in guiding multiple stages of inflammation and tissue repair. By producing a large number of biologically active molecules, they can affect the behavior of other cells and events, such as the foreign body response and angiogenesis. Since protein adsorption to biomaterials is crucial for the inflammatory process, we addressed the ability of the pro-inflammatory molecule fibrinogen (Fg) to modulate macrophage behavior toward tissue repair/regeneration. For this purpose, we used chitosan (Ch) as a substrate for Fg adsorption. Freshly isolated human monocytes were seeded on Ch substrates alone or previously adsorbed with Fg, and allowed to differentiate into macrophages for 10 days. Cell adhesion and morphology, formation of foreign body giant cells (FBGC), and secretion of a total of 80 cytokines and growth factors were evaluated. Both substrates showed similar numbers of adherent macrophages along differentiation as compared with RGD-coated surfaces, which were used as positive controls. Fg did not potentiate FBGC formation. In addition, actin cytoskeleton staining revealed the presence of punctuate F-actin with more elongated and interconnecting cells on Ch substrates. Antibody array screening and quantification of inflammation- and wound-healing-related factors indicated an overall reduction in Ch-based substrates versus RGD-coated surfaces. At late times, most inflammatory agents were down-regulated in the presence of Fg, in contrast to growth factor production, which was stimulated by Fg. Importantly, on Ch+Fg substrates, fully differentiated macrophages produced significant amounts of macrophage inflammatory protein-1delta (MIP-1δ), platelet-derived growth factor-BB, bone morphogenetic protein (BMP)-5, and BMP-7 compared with Ch alone. In addition, other important factors involved in bone homeostasis and wound healing, such as growth hormone, transforming growth factor-ß3, and insulin-like growth factor-binding proteins, as well as several angiogenic mediators, including endocrine gland-derived vascular endothelial factor, fibroblast growth factor-7, and placental growth factor, were significantly promoted by Fg. This work provides a new perspective on the inflammatory response in the context of bone repair/regeneration mediated by a pro-inflammatory protein (Fg) adsorbed onto a biomaterial (Ch) that does not otherwise exhibit osteogenic properties.

Gene expression during S. epidermidis biofilm formation on biomaterials.

Biomaterial-centered infections are initiated by adhesion of bacteria to an implant, followed by colonization and mature biofilm formation. Staphylococcus epidermidis is commonly identified as the cause of these device-centered infections. This study used an in vitro model to evaluate temporal changes in the expression of genes-icaADBC, agrBDCA, aap, and atle-that have been identified to play a role in the pathogenesis of S. epidermidis infections. Real-time reverse transcription-polymerase chain reaction was used to determine changes in gene expression from S epidermidis biofilm grown on polyurethanes (Elasthane 80A, hydrophobic) modified with polyethylene oxide (Elasthane 80A-6PEO, hydrophilic) and fluorocarbon (Elasthane 80A-6F, hydrophobic). In vitro expression of the ica locus, which is involved in initial adhesion and intracellular aggregation, increased up to 100-fold from 2 to 48 h, whereas gene expression for autolysin AtlE decreased slightly from 2 to 12 h, followed by a 10-fold increase by 48 h. Upregulation of the aap gene associated with bacterial accumulation and the agr quorum-sensing system was observed during biofilm formation over 48 h. In addition, no correlation was observed between S. epidermidis gene expression and biomaterial surface chemistry. This study used an in vitro model to demonstrate that enhanced expression of the atle, aap, agr, and ica genes plays an important role in initial foreign body colonization and potentially in the establishment of a device-associated infection.

Lymphocyte adhesion and interactions with biomaterial adherent macrophages and foreign body giant cells.

To characterize the effects of adherent macrophages and biomaterial surface chemistries on lymphocyte adhesion and activation, lymphocytes were co-cultured with monocytes alone and together, directly and separated by a porous membrane transwell on hydrophobic, hydrophilic/neutral, hydrophilic/anionic, and hydrophilic/cationic biomaterial surfaces. Surface adherent cells were quantitatively analyzed after 3 days utilizing immunofluorescence and phase contrast imaging. After periods of 3, 7, and 10 days, secreted interferon-gamma (IFN-gamma) was quantified by ELISA. Limited direct biomaterial-adherent lymphocytes were identified regardless of the presence of macrophages or foreign body giant cells (FBGC). The majority of adherent lymphocytes, which were T cells (>95%) rather than natural killer cells, predominantly interacted with adherent macrophages and FBGCs; greater than 90% were interacting on surfaces with higher levels of adherent macrophages and FBGCs and greater than 55% were interacting on surfaces with lower levels of macrophages and FBGCs. The hydrophilic/anionic surface promoted higher levels of macrophage- and FBGC-adherent lymphocytes but was nonselective for lymphocyte subtype interactions. The hydrophilic/neutral surface was selective for CD4+ T lymphocyte interactions while the hydrophobic surface was selective for CD8+ T lymphocyte interactions. IFN-gamma was produced in direct and indirect co-cultures but not in lymphocyte- and monocyte-only cultures suggesting that lymphocytes are activated via macrophage-derived cytokines rather than direct biomaterial contact. Direct lymphocyte interactions with adherent macrophages/FBGCs enhanced IFN-gamma production relative to indirect co-cultures. These results suggest that lymphocytes prefer interactions with adherent macrophages and FBGCs, resulting in lymphocyte activation, and these interactions can be influenced by biomaterial surface chemistries.

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.

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.

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.

Vitronectin is a critical protein adhesion substrate for IL-4-induced foreign body giant cell formation.

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 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 h), 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 microg/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.

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.

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.

Effects of adsorbed heat labile serum proteins and fibrinogen on adhesion and apoptosis of monocytes/macrophages on biomaterials.

A previously established human monocyte culture protocol was used to determine the effects of varying adsorbed proteins on monocyte/macrophage adhesion and survival on dimethyl-silane (DM) or RGD modified glass coverslips. Cells were allowed to adhere for 2 h in the absence of protein or in the presence of serum, fibrinogen (Fg), heat inactivated serum (HIS), serum supplemented with Fg or HIS with Fg. Cell adhesion and apoptosis rates were determined on days 0 (2 h), 3, 7 and 10 of culture. The presence of serum alone in the initial culture was sufficient to optimize monocyte/macrophage adhesion and survival rates. Adding Fg to serum did not increase adhesion nor decrease apoptotic rates. No protein or the addition of HIS during the initial incubation period significantly decreased monocyte/macrophage adhesion and survival on both surfaces, however, the addition of Fg to HIS restored adhesion and survival rates to those seen with in the presence of serum alone on RGD surfaces. These studies demonstrate that monocyte/macrophage adhesion and survival on biomaterial surfaces are optimized by adsorbed heat labile serum proteins while adsorbed Fg plays a surface property-dependent role.

Interleukin-4 inhibits tumor necrosis factor-alpha-induced and spontaneous apoptosis of biomaterial-adherent macrophages.

Biocompatibility of implanted materials is determined by the host foreign-body response, which is comprised of cellular (adherent monocytes and macrophages) and soluble (secreted cytokines) components. Modulating the presence, activity or both of adherent macrophages may increase or decrease the biocompatibility of implants because these cells remain adherent to the implant surface and fuse to form foreign-body giant cells (FBGCs), leading to failure of the implant. An attractive mechanism of eliminating these cells is through the induction of apoptosis; therefore ways of inducing or inhibiting apoptosis of biomaterial-adherent inflammatory cells are being investigated. We hypothesized that interleukin-4 (IL-4) promotes macrophage survival by inhibiting tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. We found that TNF-alpha induces apoptosis in a time- and dose-dependent manner, whereas IL-4 inhibits TNF-alpha-induced and spontaneous apoptosis of biomaterial-adherent macrophages. Blocking experiments and evaluation of shedding of soluble TNF receptor type I (TNF-RI) demonstrated that endogenous TNF-alpha production is responsible for spontaneous apoptosis of biomaterial-adherent cells and that IL-4 inhibits this apoptosis by increasing levels of shedding of soluble TNF-RI. These findings suggest that TNF-alpha and IL-4 play key roles in determining the fate of biomaterial-adherent cells and that fusion of macrophages into FBGCs is a mechanism for promoting inflammatory-cell survival on implanted materials.

Shear stress and material surface effects on adherent human monocyte apoptosis.

Monocytes play a critical role as both phagocytes and mediators of inflammatory responses in the prevention of cardiovascular device-related infections. However, persistent infection of these devices still occurs and may be attributed to deleterious cellular alterations resulting from monocyte interactions with a foreign material in an environment of dynamic flow. Thus, the effects of both shear stress and adhesion to material surfaces on human monocyte apoptosis were investigated. A rotating disk system generated physiologically relevant shear stress levels (0-14 dyn/cm(2)), and shear-related apoptosis occurring in adherent monocytes was characterized. Using annexin V analysis, apoptosis of polyurethane-adherent monocytes under shear for 4 h increased to levels >70% with increasing shear in a near-linear fashion (r2 = 0.713). It was qualitatively confirmed using confocal microscopy that filamentous (F)-actin distribution was altered, that DNA fragmentation occurred, and that activated caspases were involved in shear-induced apoptosis. Static studies determined that spontaneous apoptosis was material-dependent over 72 h by demonstrating marked differences between apoptosis of monocytes adherent to a polyurethane compared to an alkyl-modified glass. Treatment with TNF-alpha augmented this material dependency in a dose-dependent fashion over time. F-actin content of TNF-alpha-treated cells decreased to <62% of untreated cells. We conclude that concomitant effects from both material surfaces and dynamic flow mediate human monocyte apoptosis and may have serious implications in the context of implanted cardiovascular device infection.