Live imaging the foreign body response in zebrafish reveals how dampening inflammation reduces fibrosis.

Implanting biomaterials in tissues leads to inflammation and a foreign body response (FBR), which can result in rejection. Here, we live image the FBR triggered by surgical suture implantation in a translucent zebrafish model and compare with an acute wound response. We observe inflammation extending from the suture margins, correlating with subsequent avascular and fibrotic encapsulation zones: sutures that induce more inflammation result in increased zones of avascularity and fibrosis. Moreover, we capture macrophages as they fuse to become multinucleate foreign body giant cells (FBGCs) adjacent to the most pro-inflammatory sutures. Genetic and pharmacological dampening of the inflammatory response minimises the FBR (including FBGC generation) and normalises the status of the tissue surrounding these sutures. This model of FBR in adult zebrafish allows us to live image the process and to modulate it in ways that may lead us towards new strategies to ameliorate and circumvent FBR in humans.This article has an associated First Person interview with the first author of the paper.

Polytetrafluoroethylene topographies determine the adhesion, activation, and foreign body giant cell formation of macrophages.

Polytetrafluoroethylene (PTFE) is one of the commonly used materials in making various cardiovascular implants. However, the success rates of these implants in several occasions are hindered by unwanted immune responses from immune cells, such as macrophages. In this study, we investigated the response of macrophages with different structures (flat, expanded, and electrospun) of PTFE having varied surface topographies: smooth planar surface (flat PTFE), node-fibrils (ePTFE), and randomly oriented microfibers (electrospun PTFE). The electrospun PTFE showed the least adhesion of macrophages. Also, the morphology of macrophages adhered on electrospun PTFE exhibited minimal activation. The macrophage pro-inflammatory cytokine secretions showed that the lowest level of TNF-α was produced on electrospun PTFE whereas IP-10 was produced in lowest levels on expanded PTFE (ePTFE). The production of IL-6 and MCP-1 cytokines was also dependent on the structure of PTFE that the macrophages interacted with, but in a time-dependent manner. Confocal microscopy images taken at 7, 14, and 21 days showed that the electrospun PTFE resulted in the lowest percentage of macrophage fusion, thus indicating the least possible chance of foreign body giant cell (FBGC) formation. Therefore, this study showed that electrospun PTFE with randomly oriented microfibers can provide reduced adhesion, activation, and FBGC formation of macrophages compared to the smooth and planar surface of flat PTFE and node-fibril structured surface of ePTFE. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2441-2450, 2017.

Phospholipase Cγ1 suppresses foreign body giant cell formation by maintaining RUNX1 expression in macrophages.

Foreign body giant cell (FBGC) formation is associated with the inflammatory response following material implantation. However, the intracellular signaling events that regulate the process remain unclear. Here, we investigated the potential role of phospholipase C (PLC)γ1, a crucial enzyme required for growth factor-induced signaling, on FBGC formation. Knock-down of PLCγ1 using shRNA induced FBGC formation accompanied by increased expression of cathepsin K, DC-STAMP and CD36. Re-addition of PLCγ1 decreased FBGC formation. PLCγ1-deficiency caused a decrease in RUNX1 and subsequent PU.1 upregulation while subsequent rescue of RUNX1 in sh-PLCγ1-transfected cells strongly inhibited FBGC formation. FBGC generated by knock-down of PLCγ1 using shRNA resulted in strongly increased TNF-α production, with augmented activation of ERK, p38 MAPK and JNK, and subsequently NF-κB. Taken together, we suggest that PLCγ1 plays a role in the foreign body response by regulating the RUNX1/PU.1/DC-STAMP axis in macrophages.

Ultrastructural aspects of foreign body giant cells generated on different substrates.

Implantation of biomaterials into the body, e.g. for tissue engineering purposes, induces a material-dependent inflammatory response called the foreign body reaction (FBR). A hallmark feature of this response is the formation of large multinucleated cells: foreign body giant cells (FBGCs). Biomaterials like cross-linked and non-cross-linked collagen often induce the formation of FBGCs. It is unknown whether different biomaterials result in the formation of different FBGCs. To investigate this, we implanted cross-linked and non-cross-linked dermal sheep collagen subcutaneously in mice. After 21 days the implanted material was collected and prepared for ultrastructural analysis. More FBGCs formed on and between implants of cross-linked collagen compared to non-cross-linked material. The ultrastructural aspects of the FBGCs present on the two types of implants proved to be similar. On both materials, they formed long slender protrusions on the basolateral membrane, they were very rich in mitochondria, contained numerous nuclei, and showed signs of the presence of a clear zone facing the implanted material. Similar clear zones, that resemble osteoclastic features, were also seen in FBGCs generated in vitro on bone slices, but these cells did not form a ruffled border. However, similarities in ultrastructure such as the occurrence of slender protrusions and high mitochondrion content were also found in the FBGCs generated in vitro. These data indicate that FBGCs formed on different substrates share many morphological characteristics. The formation of long finger-like protrusions seemed typical for the FBGCs, in vivo as well as in vitro, however the function of these structures needs further analysis.

Nanoparticle delivery of miR-223 to attenuate macrophage fusion.

The foreign body response (FBR) begins with injury acquired during implantation of a biomaterial (BM) and is detrimental due to the eventual encapsulation of the implant. Fusion of macrophages to form foreign body giant cells (FBGC), a hallmark of the FBR, is the consequence of a multistep mechanism induced by interleukin (IL)-4 that includes the acquisition of a fusion competent state and subsequent cytoskeletal rearrangements. However, the precise mechanism, regulation, and interplay among molecular mediators to generate FBGCs are insufficiently understood. Seeking novel mediators of fusion that might be regulated at the post-transcriptional level, we examined the role of microRNAs (miRs) in this process. A miR microarray was screened and identified miR-223 as a negative regulator of macrophage fusion. In addition, transfection of primary macrophages with a mir-223 mimic attenuated IL-4-induced fusion. Furthermore, miR-223 KO mice and mir-223 deficient cells displayed increased fusion in vivo and in vitro, respectively. Finally, we developed a method for in vivo delivery of miR-223 mimic utilizing PLGA nanoparticles, which inhibited FBGC formation in a biomaterial implant model. Our results identify miR-223 as a negative regulator of fusion and demonstrate miR-223 mimic-loaded nanoparticles as a therapeutic inhibitor of macrophage fusion.

The Foreign Body Giant Cell Cannot Resorb Bone, But Dissolves Hydroxyapatite Like Osteoclasts.

Foreign body multinucleated giant cells (FBGCs) and osteoclasts share several characteristics, like a common myeloid precursor cell, multinuclearity, expression of tartrate-resistant acid phosphatase (TRAcP) and dendritic cell-specific transmembrane protein (DC-STAMP). However, there is an important difference: osteoclasts form and reside in the vicinity of bone, while FBGCs form only under pathological conditions or at the surface of foreign materials, like medical implants. Despite similarities, an important distinction between these cell types is that osteoclasts can resorb bone, but it is unknown whether FBGCs are capable of such an activity. To investigate this, we differentiated FBGCs and osteoclasts in vitro from their common CD14+ monocyte precursor cells, using different sets of cytokines. Both cell types were cultured on bovine bone slices and analyzed for typical osteoclast features, such as bone resorption, presence of actin rings, formation of a ruffled border, and characteristic gene expression over time. Additionally, both cell types were cultured on a biomimetic hydroxyapatite coating to discriminate between bone resorption and mineral dissolution independent of organic matrix proteolysis. Both cell types differentiated into multinucleated cells on bone, but FBGCs were larger and had a higher number of nuclei compared to osteoclasts. FBGCs were not able to resorb bone, yet they were able to dissolve the mineral fraction of bone at the surface. Remarkably, FBGCs also expressed actin rings, podosome belts and sealing zones--cytoskeletal organization that is considered to be osteoclast-specific. However, they did not form a ruffled border. At the gene expression level, FBGCs and osteoclasts expressed similar levels of mRNAs that are associated with the dissolution of mineral (e.g., anion exchange protein 2 (AE2), carbonic anhydrase 2 (CAII), chloride channel 7 (CIC7), and vacuolar-type H+-ATPase (v-ATPase)), in contrast the matrix degrading enzyme cathepsin K, which was hardly expressed by FBGCs. Functionally, the latter cells were able to dissolve a biomimetic hydroxyapatite coating in vitro, which was blocked by inhibiting v-ATPase enzyme activity. These results show that FBGCs have the capacity to dissolve the mineral phase of bone, similar to osteoclasts. However, they are not able to digest the matrix fraction of bone, likely due to the lack of a ruffled border and cathepsin K.

Phenotypic expression in human monocyte-derived interleukin-4-induced foreign body giant cells and macrophages in vitro: dependence on material surface properties.

The effects of different material surfaces on phenotypic expression in macrophages and foreign body giant cells (FBGC) were addressed using our in vitro system of interleukin (IL)-4-induced macrophage fusion and FBGC formation. Arginine-glycine-aspartate (RGD)-, vitronectin (VN)-, and chitosan (CH)-adsorbed cell culture polystyrene, carboxylated (C, negatively charged) polystyrene, and unmodified (PS, non-cell culture treated) polystyrene were compared for their abilities to support monocyte/macrophage adhesion and IL-4-induced macrophage fusion. Pooled whole cell lysates from four different donors were evaluated by immunoblotting for expression of selected components in monocytes, macrophages, and FBGC. In addition to RGD and VN as previously shown, we find that CH supports macrophage adhesion and FBGC formation, whereas C or PS support macrophage adhesion but do not permit macrophage fusion under otherwise identical conditions of IL-4 stimulation. Likewise, components related to macrophage fusion (CD206, CD98, CD147, CD13) are strongly expressed on RGD-, VN-, and CH-adsorbed surfaces but are greatly diminished or not detected on C or PS. Importantly, material surfaces also influence the FBGC phenotype itself, as demonstrated by strong differences in patterns of expression of HLA-DR, B7-2, B7-H1, and toll-like receptor (TLR)-2 on RGD, VN, and CH despite morphologic similarities between FBGC on these surfaces. Likewise, we observe differences in the expression of B7-2, α2-macroglobulin, TLR-2, and fascin-1 between mononuclear macrophages on C and PS. Collectively, these findings reveal the extent to which material surface chemistry influences macrophage/FBGC phenotype beyond evident morphological similarities or differences and identify CH as an FBGC-supportive substrate.

In vivo quantitative and qualitative assessment of foreign body giant cell formation on biomaterials in mice deficient in natural killer lymphocyte subsets, mast cells, or the interleukin-4 receptorα and in severe combined immunodeficient mice.

In previous studies that explored the influence of cytokines on foreign body giant cell (FBGC) formation, we focused on interleukin (IL)-4 and IL-13, each of which was discovered to induce macrophage fusion leading to FBGC formation in vitro. Two correlative in vivo studies also confirmed that IL-4 plays a role in FBGC formation on implanted biomaterials, but that T lymphocytes are not the source of IL-4 or other cytokines that support this process. The present study focused on identification of the cellular source of macrophage fusion-inducing cytokines, including natural killer (NK) or NKT lymphocytes and mast cells using mouse models genetically deficient in each of these cell types, as well as IL-4 receptor alpha(IL-4Rα)-deficient and severe combined immunodeficient (SCID) mice. Polyetherurethane (PEU) and polyethylene terephthalate (PET) polymers were subcutaneously implanted and retrieved after 14, 21, or 28 days. FBGC formation was evaluated using quantitative and qualitative data from retrieved polymer surfaces. Both types of data indicate that, compared to normal control mice, neither NK or NKT lymphocytes nor mast cells are required for FBGC formation. Furthermore, FBGC formation on biomaterials can proceed in IL-4Rα-deficient and in SCID mice. Similar conclusions were made regarding FBGC formation on both PEU and PET biomaterials. These data suggest that other sources of IL-4/IL-13 and/or additional macrophage fusion-inducing cytokines can mediate FBGC formation on implanted biomaterials, or that, in the absence of normal primary pathways, FBGC formation is nevertheless supported by redundant innate mechanisms.

Differential expression of chemokines, chemokine receptors and proteinases by foreign body giant cells (FBGCs) and osteoclasts.

Osteoclasts and foreign body giant cells (FBGCs) are both derived from the fusion of macropahges. These cells are seen in close proximity during foreign body reactions, therefore it was assumed that they might interact with each other. The aim was to identify important genes that are expressed by osteoclasts and FBGCs which can be used to understand peri-implantitis and predict the relationship of these cells during foreign body reactions. Bone marrow macrophages (BMM) were treated with receptor activator of nuclear factor kappa B ligand (RANKL) to produce osteoclasts. Quantitative PCR (qPCR) was used to identify the genes that were expressed by osteoclasts and FBGCs compared to macrophage controls. TRAP staining was used to visualise the cells while gelatine zymography and western blots were used for protein expression. Tartrate-resistant acid phosphatase (TRAP), matrix metallo proteinase 9 (MMP9), nuclear factor of activated T cells 1 (NFATc1), cathepsin K (CTSK) and RANK were significantly lower in FBGCs compared to osteoclasts. Inflammation specific chemokines such as monocyte chemotactic protein (MCP1 also called CCL2), macrophage inflammatory protein 1 alpha (MIP1α), MIP1ß and MIP1γ, and their receptors CCR1, CCR3 and CCR5, were highly expressed by FBGCs. FBGCs were negative for osteoclast specific markers (RANK, NFATc1, CTSK). FBGCs expressed chemokines such as CCL2, 3, 5 and 9 while osteoclasts expressed the receptors for these chemokines i.e. CCR1, 2 and 3. Our findings show that osteoclast specific genes are not expressed by FBGCs and that FBGCs interact with osteoclasts during foreign body reaction through chemokines.

Foreign body giant cells and osteoclasts are TRAP positive, have podosome-belts and both require OC-STAMP for cell fusion.

Macrophages have the ability to fuse and form multinucleated giant cells such as Osteoclast (OCs) and FBGCs. Osteoclast stimulatory transmembrane protein (OC-STAMP) is an important cell surface protein involved in the formation of OCs. This study sought to determine if OC-STAMP also regulates formation of FBGCs using expression analysis and subsequent inhibition studies. qPCR and Western blot analysis showed that OC-STAMP expression is significantly higher in FBGCs compared to control monocytes (P < 0.05). Four days following cell culture, OCs were positive for TRAP and F-actin ring formation, but FBGCs were not. In contrast, FBGCs were positive for TRAP and showed podosome belts comprised of F-actin on Day 8. FBGCs were subsequently plated onto dentine, but despite presenting some morphologic features of OCs (OC-STAMP expression, TRAP reactivity, and podosome belts) they failed to resorb bone. To evaluate a role for OC-STAMP in FBGCs, we inhibited this cell surface protein with anti-OC-STAMP antibody and observed that cell fusion and podosome belt formation was inhibited in both OCs and FBGCs. Our data support the hypothesis that OC-STAMP is a regulatory molecule for FBGCs; and that they are functionally distinct from OCs, despite similarities in gene expression profile, podosome belt formation, and TRAP expression.

An essential role for STAT6-STAT1 protein signaling in promoting macrophage cell-cell fusion.

Macrophage lineage cells such as osteoclasts and foreign body giant cells (FBGCs) form multinuclear cells by cell-cell fusion of mononuclear cells. Recently, we reported that two seven-transmembrane molecules, osteoclast stimulatory transmembrane protein (OC-STAMP) and dendritic cell-specific transmembrane protein (DC-STAMP), were essential for osteoclast and FBGC cell-cell fusion in vivo and in vitro. However, signaling required to regulate FBGC fusion remained largely unknown. Here, we show that signal transducer and activator of transcription 1 (STAT1) deficiency in macrophages enhanced cell-cell fusion and elevated DC-STAMP expression in FBGCs. By contrast, lack of STAT6 increased STAT1 activation, significantly inhibiting cell-cell fusion and decreasing OC-STAMP and DC-STAMP expression in IL-4-induced FBGCs. Furthermore, either STAT1 loss or co-expression of OC-STAMP/DC-STAMP was sufficient to induce cell-cell fusion of FBGCs without IL-4. We conclude that the STAT6-STAT1 axis regulates OC-STAMP and DC-STAMP expression and governs fusogenic mechanisms in FBGCs.

Biocompatibility of an experimental MTA sealer implanted in the rat subcutaneous: quantitative and immunohistochemical evaluation.

The tissue reaction promoted by an experimental mineral trioxide aggregate sealer (MTAS) in the rat subcutaneous was evaluated by morphological and morphometric analyses. In the animals from each group (n = 20), polyethylene tubes filled with MTAS, Portland cement (PC) or MTA were implanted in the dorsal subcutaneous. In the control group, empty tubes were implanted. After 7, 14, 30, and 60 days, the specimens were fixed and embedded in paraffin. In the HE-stained sections, the numerical density of inflammatory cells (IC) in the capsule was evaluated and statistical analyses performed (p ≤ 0.05). The expression of osteopontin (OPN) was evaluated by immunohistochemistry. The von Kossa method for detection of calcified structures was also performed. A moderate inflammatory process in the capsule was seen in all groups, at 7 and 14 days. At 60 days, significant reduction in the number of IC was verified in comparison to initial periods; however, significant differences were not verified among the groups. OPN immunolabeling was observed in the fibroblasts cytoplasm of the capsule next to the implants. Structures von Kossa-positive were observed in the capsule adjacent to all materials implanted at 7, 14, and 30 days. The results strongly indicate that MTAS presents biocompatibility similarly to MTA and PC.

Modulation of cellular responses on engineered polyurethane implants.

An in vivo rat cage implant system was used to study the effect of polyurethane surface chemistries on protein adsorption, macrophage adhesion, foreign-body giant cell formation (FBGCs), cellular apoptosis, and cytokine response. Polyurethanes with zwitterionic, anionic, and cationic chemistries were developed. The changes in the surface topography of the materials were determined using atomic force microscopy and the wettability by dynamic contact angle measurements. The in vitro protein adsorption studies revealed higher protein adsorption on cationic surfaces when compared with the base, while adsorption was significantly reduced on zwitterionic (**p < 0.01) and anionic (*p < 0.05) polyurethanes. Analysis of the exudates surrounding the materials revealed no differences between surfaces in the types or levels of cells present. Conversely, the proportion of adherent cells undergoing apoptosis, as determined by annexin V-FITC staining, increased significantly on anionic followed by zwitterionic surfaces (60 + 5.0 and 38 + 3.7%) when compared with the base. Additionally, zwitterionic and anionic substrates provided decreased rates of macrophage adhesion and fusion into FBGCs, whereas cationic surfaces promoted macrophage adhesion and FBGC formation. Visualization of the F-actin cytoskeleton by Alexa Fluor 488 phalloidin showed a significant delay in the cytoskeletal fusion response on zwitterionic and the anionic surfaces. The real-time polymerase chain reaction (PCR) analysis of proinflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-10) and pro-wound healing cytokines (IL-4 and TGF-ß) revealed differential cytokine responses. Cationic substrates that triggered stimulation of TNF-α and IL-4 were associated with more spread cells and higher FBGCs, whereas zwitterionic and anionic substrates that suppressed these cytokines levels were associated with less spread cells and few FBGCs. These studies have revealed that zwitterionic and anionic polyurethane surface chemistries can not only reduce nonspecific adhesion, fusion, and inflammatory events but also effectively promote cellular apoptosis in vivo.

Osteoclast stimulatory transmembrane protein and dendritic cell­specific transmembrane protein cooperatively modulate cell­cell fusion to form osteoclasts and foreign body giant cells.

Cell­cell fusion is a dynamic phenomenon promoting cytoskeletal reorganization and phenotypic changes. To characterize factors essential for fusion of macrophage lineage cells, we identified the multitransmembrane protein, osteoclast stimulatory transmembrane protein (OC-STAMP), and analyzed its function. OC-STAMP­deficient mice exhibited a complete lack of cell­cell fusion of osteoclasts and foreign body giant cells (FBGCs), both of which are macrophage-lineage multinuclear cells, although expression of dendritic cell specific transmembrane protein (DC-STAMP), which is also essential for osteoclast/FBGC fusion, was normal. Crossing OC-STAMP­overexpressing transgenic mice with OC-STAMP­deficient mice restored inhibited osteoclast and FBGC cell­cell fusion seen in OC-STAMP­deficient mice. Thus, fusogenic mechanisms in macrophage-lineage cells are regulated via OC-STAMP and DC-STAMP.

Foreign body-type multinucleated giant cells induced by interleukin-4 express select lymphocyte co-stimulatory molecules and are phenotypically distinct from osteoclasts and dendritic cells.

Foreign body-type multinucleated giant cells (FBGC), formed by macrophage fusion, are a prominent cell type on implanted biomaterials, although the roles they play at these and other sites of chronic inflammation are not understood. Why lymphocytes are present in this scenario and the effects of fusing macrophages/FBGC on subsequent lymphocyte responses are also unclear. To address the physiological significance of FBGC in this regard, we employed our in vitro system of interleukin (IL)-4-induced human monocyte-derived macrophage fusion/FBGC formation. Initially, we pursued the identities of lymphocyte co-stimulatory molecules on fusing macrophages/FBGC. In addition, we further compared the FBGC phenotype to that currently associated with osteoclasts and dendritic cells using recognized markers. Immunoblotting of cell lysates and immunochemistry of macrophages/FBGC in situ, revealed that IL-4-induced macrophages/FBGC strongly express HLA-DR, CD98, B7-2 (CD86), and B7-H1 (PD-L1), but not B7-1 (CD80) or B7-H2 (B7RP-1). Furthermore, molecules currently recognized to be expressed on osteoclasts (calcitonin receptor, tartrate-resistant acid phosphatase, RANK) or dendritic cells (CD1a, CD40, CD83, CD95/fas) are undetectable. In contrast, fusing macrophages/FBGC strongly express the macrophage markers αX integrin (CD11c), CD68, and dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), whereas CD14 is completely down-modulated with IL-4-induced macrophage fusion. These novel data demonstrate that IL-4-induction of macrophage multinucleation/FBGC formation features the acquisition of a CD14-negative phenotypic profile which is distinguishable from that of dendritic cells and osteoclasts, yet potentially exhibits multiple capacities for lymphocyte interactions with resultant lymphocyte down-modulation.

Macrophage fusion and multinucleated giant cells of inflammation.

Macrophages undergo fusion with other macrophages to form the hallmark multinucleated giant cells of chronic inflammation. However, neither the existence of distinct morphological types of giant cells, the signaling pathways that induce their formation, the molecular mechanism(s) of macrophage fusion, nor the significance of macrophage multinucleation at chronic inflammatory sites are well understood. Our efforts have been focused on these unknowns, particularly as they relate to the foreign body-type giant cells that form on implanted biomaterials and biomedical devices. We have pursued the discoveries of human macrophage fusion factors (interleukin-4, interleukin-13, α-tocopherol) with emphasis on foreign body giant cells, and identified adhesion receptors and signaling intermediates, as well as an adhesion protein substrate (vitronectin) that supports macrophage fusion. Studies on the molecular mechanism of macrophage fusion have revealed it to be a mannose receptor-mediated phagocytic process with participation of the endoplasmic reticulum. Further phenotypic and functional investigations will foster new perspectives on these remarkable multinucleated cells and their physiological significances in multiple inflammatory processes.

Biomimetic coatings for bone tissue engineering of critical-sized defects.

The repair of critical-sized bone defects is still challenging in the fields of implantology, maxillofacial surgery and orthopaedics. Current therapies such as autografts and allografts are associated with various limitations. Cytokine-based bone tissue engineering has been attracting increasing attention. Bone-inducing agents have been locally injected to stimulate the native bone-formation activity, but without much success. The reason is that these drugs must be delivered slowly and at a low concentration to be effective. This then mimics the natural method of cytokine release. For this purpose, a suitable vehicle was developed, the so-called biomimetic coating, which can be deposited on metal implants as well as on biomaterials. Materials that are currently used to fill bony defects cannot by themselves trigger bone formation. Therefore, biological functionalization of such materials by the biomimetic method resulted in a novel biomimetic coating onto different biomaterials. Bone morphogenetic protein 2 (BMP-2)-incorporated biomimetic coating can be a solution for a large bone defect repair in the fields of dental implantology, maxillofacial surgery and orthopaedics. Here, we review the performance of the biomimetic coating both in vitro and in vivo.

Macrophage fusion leading to foreign body giant cell formation persists under phagocytic stimulation by microspheres in vitro and in vivo in mouse models.

The formation of surface-damaging foreign body giant cells (FBGC) from the fusion of macrophages is considered a hallmark of the foreign body response. Experimental evidence indicates that when macrophages are unable to internalize foreign bodies via phagocytosis due to their large size, they acquire a fusogenic phenotype. The mechanism behind this transformation is unclear, and questions, such as which phenotype takes precedence for co-stimulated macrophages engaged in the foreign body response and whether or not such phenotypic alteration is graded, remain unanswered. By recapitulating fusion in vitro using cell lines and primary mouse bone marrow-derived macrophages, we investigated whether concurrent exposure of macrophages to phagocytic and fusogenic stimuli would limit fusion. Induction of phagocytosis by addition of 3.0 mum-diameter polystyrene microspheres to cells under fusogenic conditions, at ratios of 1:10, 1:1, and 10:1 did not prevent fusion. To determine the effect of microsphere phagocytosis on fusion in vivo, we first determined the kinetics of monocyte recruitment, surface adhesion, and fusion following intraperitoneal implantation of a foreign body in a mouse model. Concomitant or subsequent injection of microspheres resulted in their significant accumulation at the biomaterial surface at 2 weeks, but FBGC were still detected. Our findings indicate that despite increasing the abundance of a phagocytic stimulus (microspheres), significant FBGC formation occurs.

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.