Cellular and microenvironmental cues that promote macrophage fusion and foreign body response.

During the foreign body response (FBR), macrophages fuse to form foreign body giant cells (FBGCs). Modulation of FBGC formation can prevent biomaterial degradation and loss of therapeutic efficacy. However, the microenvironmental cues that dictate FBGC formation are poorly understood with conflicting reports. Here, we identified molecular and cellular factors involved in driving FBGC formation in vitro. Macrophages demonstrated distinct fusion competencies dependent on monocyte differentiation. The transition from a proinflammatory to a reparative microenvironment, characterised by specific cytokine and growth factor programmes, accompanied FBGC formation. Toll-like receptor signalling licensed the formation of FBGCs containing more than 10 nuclei but was not essential for cell-cell fusion to occur. Moreover, the fibroblast-macrophage crosstalk influenced FBGC development, with the fibroblast secretome inducing macrophages to secrete more PDGF, which enhanced large FBGC formation. These findings advance our understanding as to how a specific and timely combination of cellular and microenvironmental factors is required for an effective FBR, with monocyte differentiation and fibroblasts being key players.

Monocyte-Macrophage Lineage Cell Fusion.

Cell fusion (fusogenesis) occurs in natural and pathological conditions in prokaryotes and eukaryotes. Cells of monocyte-macrophage lineage are highly fusogenic. They create syncytial multinucleated giant cells (MGCs) such as osteoclasts (OCs), MGCs associated with the areas of infection/inflammation, and foreign body-induced giant cells (FBGCs). The fusion of monocytes/macrophages with tumor cells may promote cancer metastasis. We describe types and examples of monocyte-macrophage lineage cell fusion and the role of actin-based structures in cell fusion.

Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation.

Multinucleated giant cells are formed by the fusion of macrophages and are a characteristic feature in numerous pathophysiological conditions including the foreign body response (FBR). Foreign body giant cells (FBGCs) are inflammatory and destructive multinucleated macrophages and may cause damage and/or rejection of implants. However, while these features of FBGCs are well established, the molecular mechanisms underlying their formation remain elusive. Improved understanding of the molecular mechanisms underlying the formation of FBGCs may permit the development of novel implants that eliminate or reduce the FBR. Our previous study showed that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel/receptor, is required for FBGC formation and FBR to biomaterials. Here, we have determined that (a) TRPV4 is directly involved in fusogenic cytokine (interleukin-4 plus granulocyte macrophage-colony stimulating factor)-induced activation of Rac1, in bone marrow-derived macrophages; (b) TRPV4 directly interacts with Rac1, and their interaction is further augmented in the presence of fusogenic cytokines; (c) TRPV4-dependent activation of Rac1 is essential for the augmentation of intracellular stiffness and regulation of cytoskeletal remodeling; and (d) TRPV4-Rac1 signaling axis is critical in fusogenic cytokine-induced FBGC formation. Together, these data suggest a novel mechanism whereby a functional interaction between TRPV4 and Rac1 leads to cytoskeletal remodeling and intracellular stiffness generation to modulate FBGC formation.

Biomaterial-induced multinucleated giant cells express proinflammatory signaling molecules: A histological study in humans.

The biomaterials physicochemical characteristics influence their cellular reaction, degradation and regenerative capacities. Macrophages and multinucleated giant cells (MNGCs) are observed in the augmentation area of biomaterials. This study, for the first time, evaluated the polarization pattern of macrophages and MNGCs in response to two different bone substitute materials (synthetic bone substitute material [SBSM] = NanoBone vs. xenogeneic bone substitute material [XBSM] = Bio-Oss) in human bone biopsies compared to non-augmented bone (control). Histomorphometrical analysis of the polarization in proinflammatory (M1) and anti-inflammatory (M2) cells was performed using different immunohistochemical markers: CD-68 = macrophages; CCR-7 and Cox-2 (M1) and CD-206 and CD-163 (M2) and tartrate-resistant acid phosphatase (TRAP). The macrophage polarization pattern in SBSM showed a significantly higher number of M1 cells than did XBSM and non-augmented bone. XBSM induced a significantly higher number of CD-206-positive macrophages than SBSM did. No significant difference was found between XBSM and the non-augmented bone. MNGCs expressed CD-68 and TRAP. In both test-groups, MNGCs showed a high proinflammatory character (CCR-7 and Cox-2-positive) and their number in the SBSM group was significantly higher than that of XBSM. The tissue distribution showed a significantly low percentage of the remaining biomaterial in SBSM compared to XBSM. Within the limitations of this study, these findings show that MNGCs exhibit a rather proinflammatory character and lead to biomaterial degradation, once they are induced in a high number. The premature degradation of bone substitute materials is compensated with a high percentage of connective tissue and not new bone formation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 780-790, 2019.

Tumor necrosis factor receptor-associated factor 6 is required to inhibit foreign body giant cell formation and activate osteoclasts under inflammatory and infectious conditions.

Osteoclasts and foreign body giant cells (FBGCs) are derived from common progenitors and share properties such as multi-nucleation capacity induced by cell-cell fusion; however, mechanisms underlying lineage determination between these cells remain unclear. Here we show that, under inflammatory conditions, osteoclasts are stimulated in a manner similar to M1 macrophages, while formation of FBGCs, which exhibit M2-like phenotypes, is inhibited in a manner similar to that seen in M1/M2 macrophage polarization. FBGC/osteoclast polarization was inhibited by conditional knockout of tumor necrosis factor receptor associated factor 6 (Traf6) in adults in vivo and in vitro. Traf6-null mice were previously reported to die soon after birth, but we found that Traf6 deletion in adults did not cause lethality but rather inhibited osteoclast activation and prevented FBGC inhibition under inflammatory conditions. Accordingly, basal osteoclastogenesis was significantly inhibited by Traf6 deletion in vivo and in vitro and accompanied by increased bone mass. Lipopolysaccharide-induced osteoclast formation and osteolysis were significantly inhibited in Traf6 conditional knockout mice. Our results suggest that Traf6 plays a crucial role in regulating M1 osteoclast and M2 FBGC polarization and is a potential therapeutic target in blocking FBGC inhibition, antagonizing osteolysis in inflammatory conditions, and increasing bone mass without adverse effects in adults.

In vivo response to decellularized mesothelium scaffolds.

Biological surgical scaffolds are used in plastic and reconstructive surgery to support structural reinforcement and regeneration of soft tissue defects. Macrophage and fibroblast cell populations heavily regulate scaffold integration into host tissue following implantation. In the present study, the biological host response to a commercially available surgical scaffold (Meso BioMatrix Surgical Mesh (MBM)) was investigated for up to 9 weeks after subcutaneous implantation; this scaffold promoted superior cell migration and infiltration previously in in vitro studies relative to other commercially available scaffolds. Infiltrating macrophages and fibroblasts phenotypes were assessed for evidence of inflammation and remodeling. At week 1, macrophages were the dominant cell population, but fibroblasts were most abundant at subsequent time points. At week 4, the scaffold supported inflammation modulation as indicated by M1 to M2 macrophage polarization; the foreign body giant cell response resolved by week 9. Unexpectedly, a fibroblast subpopulation expressed macrophage phenotypic markers, following a similar trend in transitioning from a proinflammatory to anti-inflammatory phenotype. Also, α-smooth muscle actin-expressing myofibroblasts were abundant at weeks 4 and 9, mirroring collagen expression and remodeling activity. MBM supported physiologic responses observed during normal wound healing, including cellular infiltration, host tissue ingrowth, remodeling of matrix proteins, and immune modulation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 716-725, 2018.

Tumefactive foreign body giant cell reaction following high-pressure paint injection injury: A case report and review of literature.

High-pressure paint injection injury is an uncommon but well-described injury. The histologic features of long-term paint injection injury with retained material are less recognized. A 46-year-old male presented clinically as "recurrent giant cell tumor of tendon sheath." The right index finger demonstrated fusiform enlargement by a pigmented mass with diffuse infiltration into the soft tissue of the hand. Histologically the tumor showed multiple giant cells in a fibrotic stroma extending into the dermis. There were multiple types of foreign material including diffuse brown black pigment, weakly optically polarizing foreign material and white inclusions with a "train track" appearance. The cells were positive for CD68 and negative for S100 antigen. Further investigation revealed that the patient had a history of high-pressure paint injection injury to his digit 6 years prior. Foreign material injected under high pressure into tissues may result in a pseudo-neoplastic foreign body granulomatous reaction that can mimic giant cell tumor of tendon sheath. Our case demonstrates that this reaction can be florid and can have slow growth over years. A high index of suspicion, a good clinical history and careful examination can distinguish these 2 entities.

The Neglected Villain of Bioprosthetic Degeneration: Inflammatory and Immune Processes.

In an attempt to avoid the destructive process of bioprosthetic heart-valve calcification associated with the use of glutaraldehyde, valves are today prepared using low concentrations of the crosslinking reagent. In this review, we summarize our findings and those of others that confirm that the immunogenicity of such tissue is not sufficiently masked and that a defined humoral response is indeed mounted against a repertoire of antigens unrelated to those associated with vascularized and non—cross-linked xenograft organs. We demonstrate the need for increased cross-linking of tissue to satisfactorily mitigate that response; furthermore, we examine the impact of increased cross-link density on the macrophage as antigen presenting cell with respect to its involvement in both tissue erosion and pannus overgrowth. Finally we present evidence for a role of circulating antibodies in bioprosthesis calcification.

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.

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.

Reducing the inflammatory responses of biomaterials by surface modification with glycosaminoglycan multilayers.

Chronic inflammatory responses after implantation of biomaterials can lead to fibrotic encapsulation and failure of implants. The present study was designed to reduce the inflammatory responses to biomaterials by assembling polyelectrolyte multilayers (PEMs) composed of glycosaminoglycans (GAGs) and chitosan (Chi) on glass as model surfaces through layer-by-layer (LBL) technique. Surface plasmon resonance (SPR) and water contact angle (WCA) investigations confirmed the multilayer build-up with alternating deposition of GAGs and Chi layers, while zeta potential measurements showed significant negative charges after multilayer deposition, which further proved the PEM formation. Macrophage adhesion, macrophage spreading morphology, foreign body giant cell (FBGC) formation, as well as ß1 integrin expression and interleukin-1ß (IL-1ß) production were all significantly decreased by GAG-Chi multilayer deposition in comparison to the primary poly (ethylene imine) (PEI) layer. Thereby, the type of GAGs played a pivotal role in inhibiting the inflammatory responses to various extents. Especially heparin (Hep)-Chi multilayers hindered all inflammatory responses to a significantly higher extent in comparison to hyaluronic acid (HA)-Chi and chondroitin sulfate (CS)-Chi multilayer systems. Overall, the present study suggests a great potential of GAG-Chi multilayer coating on implants, particularly the Hep-Chi based systems, to reduce the inflammatory responses.

Interleukin-1 receptor-associated kinase-4 (IRAK4) promotes inflammatory osteolysis by activating osteoclasts and inhibiting formation of foreign body giant cells.

Formation of foreign body giant cells (FBGCs) occurs following implantation of medical devices such as artificial joints and is implicated in implant failure associated with inflammation or microbial infection. Two major macrophage subpopulations, M1 and M2, play different roles in inflammation and wound healing, respectively. Therefore, M1/M2 polarization is crucial for the development of various inflammation-related diseases. Here, we show that FBGCs do not resorb bone but rather express M2 macrophage-like wound healing and inflammation-terminating molecules in vitro. We also found that FBGC formation was significantly inhibited by inflammatory cytokines or infection mimetics in vitro. Interleukin-1 receptor-associated kinase-4 (IRAK4) deficiency did not alter osteoclast formation in vitro, and IRAK4-deficient mice showed normal bone mineral density in vivo. However, IRAK4-deficient mice were protected from excessive osteoclastogenesis induced by IL-1ß in vitro or by LPS, an infection mimetic of Gram-negative bacteria, in vivo. Furthermore, IRAK4 deficiency restored FBGC formation and expression of M2 macrophage markers inhibited by inflammatory cytokines in vitro or by LPS in vivo. Our results demonstrate that osteoclasts and FBGCs are reciprocally regulated and identify IRAK4 as a potential therapeutic target to inhibit stimulated osteoclastogenesis and rescue inhibited FBGC formation under inflammatory and infectious conditions without altering physiological bone resorption.

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.

Back-side wear in HexLoc cups clinico-radiological, immunohistopathological, finite element, and retrieval analysis studies.

The HexLoc locking system was designed to prevent back-side wear of the polyethylene liner in the modular cementless metal-backed acetabular cup, but failed. Back-side wear was analyzed using clinico-radiological data, immunohistopathology, finite element modeling (FEM, and retrieval analysis. Screw holes allowed entry of titanium oxide and exit of polyethylene particles. Birefringent polyethylene wear particles were found behind the metal cup in macrophages containing pro-inflammatory tumor necrosis factor-α and interleukin-1ß, whereas fibroblast-like cells stained for osteoclastogenic receptor activator of nuclear factor kappa B ligand (RANKL). Computerized tomography revealed granulomas (83% versus 17 %) and cortical destruction (50% versus 5%) better than radiographs. In FEM, a change of the abduction angle from 45 to 60 deg, and liner thickness from 4.8 mm to 2.5 mm, increased the back-side wear by 90% and 120%, respectively. Screw holes were stress concentration areas; their removal decreased wear by 40%. Modeling results were validated in retrieved implants, which demonstrated extensive back-side wear damage of liners with a high abduction angle. Combined clinico-radiological, immunohistopathological, FEM, and retrieval analysis disclosed that back-side wear in the HexLoc design is sensitive to the abduction angle, liner thickness, and presence of screw holes.

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.

Biocompatibility and osteoconduction of macroporous silk fibroin implants in cortical defects in sheep.

The goal of the presented study was to compare the biocompatibility and cellular responses to porous silk fibroin (SF) scaffolds produced in a water-based (UPW) or a solvent based process (HFIP) using two different SF sources. For that reason, four different SF scaffolds were implanted (n=6) into drill hole defects in the cancellous bone of the sheep tibia and humerus. The scaffolds were evaluated histologically for biocompatibility, cell-material interaction, and cellular ingrowth. New bone formation was observed macroscopically and histologically at 8 weeks after implantation. For semiquantitative evaluation, the investigated parameters were scored and statistically analyzed (factorial ANOVA). All implants showed good biocompatibility as evident by low infiltration of inflammatory cells and the absent encapsulation of the scaffolds in connective tissue. Multinuclear foreign body giant cells (MFGCs) and macrophages were present in all parts of the scaffold at the material surface and actively degrading the SF material. Cell ingrowth and vascularization were uniform across the scaffold. However, in HFIP scaffolds, local regions of void pores were present throughout the scaffold, probably due to the low pore interconnectivity in this scaffold type in contrast to UPW scaffolds. The amount of newly formed bone was very low in both scaffold types but was more abundant in the periphery than in the center of the scaffolds and for HFIP scaffolds mainly restricted to single pores.

In vivo degradation and tissue response to poly(5-ethylene ketal ε-caprolactone-co-D,L-lactide).

Low molecular weight poly(5-ethylene ketal ε-caprolactone-co-D,L-lactide) (PEKCDLLA) is being considered as a viscous liquid, injectable depot for localized drug delivery. This polymer degrades in vitro via surface erosion, which is potentially advantageous for the proposed application. However, the in vivo degradation rate and mechanism, and tissue response, to polymers based on 5-ethylene ketal ε-caprolactone have not yet been reported. The purpose of this study was to measure the in vivo weight loss and change in polymer properties and assess the tissue response to PEKCDLLA after subcutaneous injection in rats. The tissue response was assessed histologically using Masson's trichrome staining and immunohistochemically by staining for CD68 positive cells. The polymer lost weight with time in a nearly linear fashion but did not exhibit significant changes in number average molecular weight, polydispersity index, and glass transition temperature or monomer ratio, consistent with a surface erosion process. The tissue response to the polymer was moderate and comparable to that reported in the literature for other degradable polymers used in clinical applications. These findings indicate that PEKCDLLA is a promising candidate for injectable drug delivery.

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.

Frequent upregulation of cyclin D1 and p16 expression with low Ki-67 scores in multinucleated giant cells.

BACKGROUND/AIMS: Multinucleated giant cells are formed from the fusion of macrophages and are classified into foreign body-type giant cells (FBGCs), osteoclast-type giant cells (OCGCs) and Langhans-type giant cells (LHGCs). OCGCs display upregulated cyclin D1 expression with low Ki-67 activity. However, little is known about the expression of cell cycle regulators in the other types of multinucleated giant cells. We aimed to investigate the cell cycle status of multinucleated giant cells. METHODS: The immunohistochemical expressions of cyclin D1, p16(INK4a) and Ki-67 were analyzed in a total of 127 cases showing multinucleated giant cells. RESULTS: Cyclin D1 was overexpressed in 45 (88%) of 51 FBGC cases, 25 (86%) of 29 OCGC cases and 22 (47%) of 47 LHGC cases. p16(INK4a) showed diffuse nuclear and/or cytoplasmic overexpression in 45 (88%) of 51 FBGC cases, 27 (93%) of 29 OCGC cases and 24 (51%) of 47 LHGC cases. Ki-67 immunostaining was negative in almost all FBGC, OCGC and LHGC cases. CONCLUSION: This study demonstrates that FBGCs and OCGCs frequently show upregulation of cyclin D1 and p16(INK4a) expression with low Ki-67 scores. This suggests that multinucleated giant cells are arrested in the G1/S cell cycle transition.

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.