The Implant-Induced Foreign Body Response Is Limited by CD13-Dependent Regulation of Ubiquitination of Fusogenic Proteins.

Implanted medical devices, from artificial heart valves and arthroscopic joints to implantable sensors, often induce a foreign body response (FBR), a form of chronic inflammation resulting from the inflammatory reaction to a persistent foreign stimulus. The FBR is characterized by a subset of multinucleated giant cells (MGCs) formed by macrophage fusion, the foreign body giant cells (FBGCs), accompanied by inflammatory cytokines, matrix deposition, and eventually deleterious fibrotic implant encapsulation. Despite efforts to improve biocompatibility, implant-induced FBR persists, compromising the utility of devices and making efforts to control the FBR imperative for long-term function. Controlling macrophage fusion in FBGC formation presents a logical target to prevent implant failure, but the actual contribution of FBGCs to FBR-induced damage is controversial. CD13 is a molecular scaffold, and in vitro induction of CD13KO bone marrow progenitors generates many more MGCs than the wild type, suggesting that CD13 regulates macrophage fusion. In the mesh implant model of FBR, CD13KO mice produced significantly more peri-implant FBGCs with enhanced TGF-ß expression and increased collagen deposition versus the wild type. Prior to fusion, increased protrusion and microprotrusion formation accompanies hyperfusion in the absence of CD13. Expression of fusogenic proteins driving cell-cell fusion was aberrantly sustained at high levels in CD13KO MGCs, which we show is due to a novel CD13 function, to our knowledge, regulating ubiquitin/proteasomal protein degradation. We propose CD13 as a physiologic brake limiting aberrant macrophage fusion and the FBR, and it may be a novel therapeutic target to improve the success of implanted medical devices. Furthermore, our data directly implicate FBGCs in the detrimental fibrosis that characterizes the FBR.

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.

Tuning the immune reaction to manipulate the cell-mediated degradation of a collagen barrier membrane.

In order to elicit a desired barrier function in guided bone regeneration (GBR) or guided tissue regeneration (GTR), a barrier membrane has to maintain its integrity for a certain period of time to guarantee the regeneration of target tissue. Due to the complexity and variety of clinical conditions, the healing time required for tissue regeneration varies from one case to another, which implies the need for tailoring the barrier membranes to diverse conditions via manipulating their degradation property. As a "non-self" biomaterial, a barrier membrane will inevitably trigger host-membrane immune response after implantation, which entails the activation of phagocytic cells. In the degradation process of a barrier membrane, the cell-mediated degradation may play a more vital role than enzymatic and physicochemical dissolution; however, limited studies have been carried out on this topic. In this context, we investigated the cell-mediated degradation and illustrated the possible key cells and mediators for immunomodulation via in vivo and in vitro studies. We discovered that IL-13, a key cytokine mainly released by T helper 2 cells (Th2), induced the formation of foreign body giant cells (FBGCs), thus resulting in membrane degradation. Neutralizing IL-13 could suppress membrane degradation and formation of FBGC. The contributions of this study are (1) unveiling the immune mechanisms underlying the cell-mediated collagen membrane degradation; (2) allowing the formation of an "immunodegradation" strategy to develop an "immune-smart" barrier membrane to manipulate its degradation; (3) providing the key regulatory immune cells and cytokines for the immunomodulation target in collagen membrane degradation. STATEMENT OF SIGNIFICANCE: The significance of this research includes.

Transient Receptor Potential Vanilloid 4 Is Required for Foreign Body Response and Giant Cell Formation.

The presence of biomaterials and devices implanted into soft tissue is associated with development of a foreign body response (FBR), a chronic inflammatory condition that can ultimately lead to implant failure, which may cause harm to or death of the patient. Development of FBR includes activation of macrophages at the tissue-implant interface, generation of destructive foreign body giant cells (FBGCs), and generation of fibrous tissue that encapsulates the implant. However, the mechanisms underlying the FBR remain poorly understood, as neither the materials composing the implants nor their chemical properties can explain triggering of the FBR. Herein, we report that genetic ablation of transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable mechanosensitive cation channel in the transient receptor potential vanilloid family, protects TRPV4 knockout mice from FBR-related events. The mice showed diminished collagen deposition along with reduced macrophage accumulation and FBGC formation compared with wild-type mice in a s.c. implantation model. Analysis of macrophage markers in spleen tissues and peritoneal cavity showed that the TRPV4 deficiency did not impair basal macrophage maturation. Furthermore, genetic deficiency or pharmacologic antagonism of TRPV4 blocked cytokine-induced FBGC formation, which was restored by lentivirus-mediated TRPV4 reintroduction. Taken together, these results suggest an important, previously unknown, role for TRPV4 in FBR.

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.

Multinucleated Giant Cells: Good Guys or Bad Guys?

Multinucleated giant cells (MNGCs) are a special class of giant cell formed by the fusion of monocytes/macrophages abundantly found in human tissues. While historically their role around certain classes of biomaterials have been directly linked to a foreign body reaction leading to material rejection, recent accumulating evidence has put into question their role around certain classes of bone biomaterials. It was once thought that specifically in bone tissues, all giant cells were considered osteoclasts characterized by their ability to resorb and replace bone grafts with newly formed native bone. More recently, however, a special subclass of bone biomaterials has been found bordered by large MNGCs virtually incapable of resorbing bone substitutes even years after their implantation yet surrounded by stable bone. Interestingly, research from the field of cardiovascular disease has further shown how a shift in macrophage polarization from M1 "tissue-inflammatory" macrophages toward M2 "wound-healing" macrophages in atherosclerotic plaque may lead to MNGC formation and ectopic calcification of arteries. Despite the growing observation that MNGC formation occurs around certain bone biomaterials, their role in these tissues remains extremely poorly understood and characterized. In summary, four central aspects of this review are discussed with a focus on (1) the role of MNGCs in bone/tissue biology, and their ability to induce vascularization/new bone formation, their role around, (2) bone substitutes for bone augmentation, (3) dental implants, as well as (4) during peri-implant infection. The authors express the necessity to no longer refer to MNGCs as "good" or "bad" cells, but instead point toward the necessity to more specifically characterize them scientifically and appropriately as M1-MNGC and M2-MNGC accordingly. Future research investigating the factors influencing their polarization as a "center of control" is also likely to act as a key factor in the progression/resolution of various diseases.

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.

Multinucleated giant cells in the implant bed of bone substitutes are foreign body giant cells-New insights into the material-mediated healing process.

In addition to macrophages, multinucleated giant cells (MNGCs) are involved in the tissue reaction to a variety of biomaterials. Especially in the case of bone substitute materials it has been assumed that the MNGCs are osteoclasts, based on the chemical and physical similarity of many materials to the calcified matrix and the bony environment in which they are used. However, many studies indicate that these cells belong to the cell line of the foreign body giant cells (FBGCs), which are of "inflammatory origin", although they have been shown to possess both a pro- and also anti-inflammatory phenotype. Moreover, no information is available about their role in the tissue reaction to bone substitute materials. The present study was conducted to analyze the origin of MNGCs in the implant beds of a synthetic and a xenogeneic bone substitute and focused on the application of immunohistochemical methods. Two antibodies against integrin molecules specific for osteoclasts (ß-3 integrin) or FBGCs (ß-2 integrin) were used to distinguish both giant cell types. The results of the present study indicate that the MNGCs induced by both kinds of bone substitutes are FBGCs, as they express only ß-2 integrin in contrast to the osteoclasts outside of the immediate implantation areas, which only demonstrate ß-3 integrin expression. These data give new insight into the tissue reaction to both xenogeneic and synthetic bone substitutes. Based on this new knowledge further research concerning the proteomic profile of the FBGCs especially based on the different physicochemical properties of bone substitutes is necessary. This may show that specific characteristics of bone substitutes may exhibit a substantial influence on the regeneration process via the expression of anti-inflammatory molecules by FBGCs. Based on this information it may be possible to formulate and choose bone substitutes that can guide the process of bone tissue regeneration on the molecular level. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1105-1111, 2017.

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.

Diversity of multinucleated giant cells by microstructures of hydroxyapatite and plasma components in extraskeletal implantation model.

UNLABELLED: Foreign body giant cells (FBGCs) and osteoclasts are multinucleated giant cells (MNGCs), both of which are formed by the fusion of macrophage-derived mononuclear cells. Osteoclasts are distinct from FBGCs due to their bone resorption ability; however, not only morphological, but also functional similarities may exist between these cells. The characterization and diversity of FBGCs that appear in an in vivo foreign body reaction currently remain incomplete. In the present study, we investigated an in vivo foreign body reaction using an extraskeletal implantation model of hydroxyapatite (HA) with different microstructures. The implantation of HA granules in rat subcutaneous tissue induced a foreign body reaction that was accompanied by various MNGCs. HA granules composed of rod-shaped particles predominantly induced cathepsin K (CTSK)-positive FBGCs, whereas HA granules composed of globular-shaped particles predominantly induced CTSK-negative FBGCs. Plasma, which was used as the binder of ceramic granules, stimulated the induction of CTSK-positive FBGCs more strongly than purified fibrin. Furthermore, the implantation of HA composed of rod-shaped particles with plasma induced tartrate-resistant acid phosphatase (TRAP)-positive MNGCs in contrast to HA composed of globular-shaped particles with purified fibrin, which predominantly induced CTSK-negative and TRAP-negative typical FBGCs. These results suggest that CTSK-positive, TRAP-positive, and CTSK- and TRAP-negative MNGCs are induced in this subcutaneous implantation model in a manner that is dependent on the microstructure of HA and presence or absence of plasma. STATEMENT OF SIGNIFICANCE: We attempted to elucidate the mechanisms responsible for the foreign body reaction induced by the implantation of hydroxyapatite granules with different microstructures in rat subcutaneous tissue with or without plasma components as the binder of ceramic granules. By analyzing the expression of two reliable osteoclast markers, we detected tartrate-resistant acid phosphatase-positive multinucleated giant cells, cathepsin K-positive multinucleated giant cells, and tartrate-resistant acid phosphatase- and cathepsin K-negative multinucleated giant cells. The induction of tartrate-resistant acid phosphatase-positive multinucleated giant cells was plasma component-dependent while the induction of cathepsin K-positive multinucleated giant cells was influenced by the microstructure of hydroxyapatite. This is the first study to show the conditions dividing the three kinds of multinucleated giant cells in the foreign body reaction.

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.

Heterogeneity of biomaterial-induced multinucleated giant cells: Possible importance for the regeneration process?

Biomaterial-associated multinucleated giant cells (BMGCs) have been found within the implantation beds of many different biomaterials. However, their exact differentiation and their involvement in the inflammatory and healing events of the foreign body response still remain mostly unclear. Silk fibroin (SF) scaffolds, which induces a tissue reaction involving both macrophages and BMGCs, was implanted in the subcutaneous connective tissue of four CD-1 mice for 15 days using an established subcutaneous implantation model. Analysis of macrophage polarization and BMGCs was performed by immunohistochemcial detection of pro- (cyclooxygenase-2 (COX-2), C-C chemokine receptor type 7 (CCR7), nuclear factor "kappa-light-chain-enhancer" (NF-κB)) and anti-(heme oxygenase-1 (HO-1) and mannose receptor (MR, also known as CD206)). Furthermore, histochemical detection of tartrate-resistant acid phosphatase (TRAP) was conducted to test its predictive efficiency for the pro-inflammatory differentiation of cells. An established system for histomorphometrical analysis was used for counting of BMGCs expressing these molecules. The results show that BMGCs express both pro- and anti-inflammatory molecules within the implantation beds of SF scaffolds in comparable numbers, while only statistically significantly lower numbers of TRAP-positive BMGCs were measured in comparison to the BMGCs expressing the above-mentioned molecules. As these data substantiate the heterogeneity of BMGCs, the question arises to what extent BMGCs can "support" the process of tissue regeneration. Furthermore, the data prompt the question to what extent TRAP-expression within a biomaterial implantation bed can be seen as a predictive marker for an inflammatory condition, as in this study no obvious correlation between TRAP-expression and other pro-inflammatory markers could be observed.

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.

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.

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.