Multinucleated Giant Cells: Good Guys or Bad Guys?

Tissue Engineering

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Multi‐Nucleated Giant Cells: Good Guys or Bad Guys? (DOI: 10.1089/ten.TEB.2017.0242) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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DOI: 10.1089/ten.TEB.2017.0242

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Multi‐Nucleated Giant Cells: Good Guys or Bad Guys? Richard J Miron1,2*, Dieter D. Bosshardt3* 1

Department of Periodontics and Oral Medicine, University of Michigan School of

Dentistry, Ann Arbor, Michigan, USA.2 Department of Periodontology, College of Dental Medicine, Nova Southeastern University, Fort Lauderdale, Florida, USA.      3

Head of Oral Histology, Dental School, University of Bern, Bern, Switzerland

* Corresponding authors: Richard J. Miron Department of Periodontics and Oral Medicine University of Michigan School of Dentistry 1011 N. University Ave. Ann Arbor, MI 48109‐1078 USA Email: [email protected] Dieter D. Bosshardt Head of the Robert K. Schenk Laboratory of Oral Histology University of Bern, Bern, Switzerland Email: [email protected]



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2 Abstract: Multi‐nucleated 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 towards 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 towards 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. Keywords: Macrophage, bone regeneration, osteoimmunology, biomaterial integration, multi‐nucleated giant cells, foreign body cells



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3 1. Introduction

Multi‐nucleated giant cells (MNGCs) are an intriguing cell‐type derived from the fusion of monocytes/macrophages. Interestingly, giant cells are most frequently found in the human body as multi‐nucleated osteoclasts located in bone tissues responsible for the resorption of bone, but more recently observed around implanted biomedical devices due to their growing use. In the latter group, MNGCs have frequently been associated with a foreign body reaction whereby a large invasion of accumulated macrophages collected on the implanted biomaterial surface leads to MNGC formation followed by material rejection in many cases (1, 2). In bone tissues, the usage of implanted bone biomaterials not only in the dental, oral and craniofacial field, but also in orthopedics and neurosurgery has continued to rise with major increases in bone biomaterials brought to market (3). While it was once thought in the bone structure context that all giant cells were osteoclasts capable of resorbing and replacing implanted bone biomaterials with newly formed bone, recent evidence now shows that many clinically used bone biomaterials are surrounded by MNGCs virtually incapable of resorbing bone grafts (4). Monocytes and macrophages are some of the most abundant cell types found in the human body and act as key implicators in bone‐biomaterial integration since they represent the first cell‐types in contact with these materials (5). It is noteworthy that macrophages are one of the most‐broad ranging cell‐types capable of polarizing entirely from contributors of tissue inflammation (M1 macrophage) towards contributors of wound‐healing (M2 macrophage) (6). Due to their broad role in regulating tissue homeostasis, they have extensively been studied in innate and adaptive immunity, as modulators of wound healing, hematopoiesis as well as malignancy (6).   Despite their widespread role in regulating tissue homeostasis and integration, a recent systematic review found that in the field of bone‐biomaterial integration, less than 10% of



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4 all studies focused on the osteo‐immunological reaction of cells towards biomaterials with over 90% of research focused primarily on osteoblast/fibroblast behavior to implanted bone biomaterials (7). This discrepancy is difficult to understand given the fact monocytes and macrophages are the first cells to come in contact with implanted biomaterials and the major modulators of tissue biomaterial integration (8). Interestingly, certain classes of bone biomaterials such as high‐temperature sintered xenografts and certain zirconia dental implants are known inducers of MNGC formation, yet these biomaterials fully integrate readily in vivo (4, 9). In certain clinical situations, such bone substitute materials are favorably utilized due to their ‘low‐substitution’ rate meaning that bone volume can be maintained for long periods. Clinician often choose to apply such materials in areas such as the labial surface in the maxillary esthetic zone following dental implant placement whereby such biomaterials can “maintain space” due to low bone turnover and limited material resorption.  While we have been continuously intrigued by the role of MNGCs in bone‐biomaterial integration, recent studies investigating atherosclerotic plaque from the past 3‐5 years have provided evidence that immune cells (macrophages and MNGCs) are the responsible cell‐type controlling ectopic calcification in arterial walls (10‐14). It therefore is of relevance that while MNGCs around atherosclerotic plaque are considered pathological due their role in arterial calcification (a life threatening situation), a similar MNGC around a bone biomaterial may actually be considered a natural turnover mechanism if performing a similar role, thereby creating high interest in the field of bone‐biomaterial research. Therefore, the purpose of this review article is to further summarize knowledge acquired to date on MNGC with the following specific aims: 1) to present and summarize MNGC biology in bone tissues including their fusion from their precursor cells; the osteal macrophage (OsteoMac). 2) We then describe and compare macrophage polarization and MNGC formation around ectopic calcification of arteries. 3) Lastly, this review article discusses the role of MNGCs around A) bone grafts, B) dental implants and more



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5 specifically C) during peri‐implant infection/regeneration as a potential shift between M1‐ MNGC and M2‐MNGC polarization. In summary, the authors express the requisite to no longer refer to MNGCs as ‘good’ or ‘bad’ cells, but instead point towards the necessity to better characterize these giant cells scientifically and appropriately as M1‐MNGC and M2‐ MNGC accordingly. 2.1 MNGC biology in bone tissues including their fusion from precursor cells Over the years, basic science research has revealed the dynamic interactions between the skeletal and immune systems thereby rendering the creation of an entire field termed ‘osteo‐immunology’ to further study bone‐immune interactions (15‐17). With respect to bone tissues, precursor cells of MNGCs are thought to be derived from osteal macrophages (‘OsteoMacs’), a distinct population of macrophages residing within bone actively participating in bone modeling and remodeling throughout the lifespan of mature osteoblasts (17). Initial macrophage experiments identified their ability to polarize towards 2 specific phenotypes, classical M1 pro‐inflammatory macrophages and M2 wound healing macrophages. Classical M1 macrophages are induced in response to LPS expressing pro‐ inflammatory cytokines such as TNF‐alpha (18, 19), IL‐6 (20, 21) and IL‐1β (18, 22) all contributing to tissue inflammation and osteoclastogenesis.  M2 macrophages are induced by IL‐4 and IL‐13 and typically produce TGF‐β and arginase, both factors implicated in the tissue‐repair process (23‐26). Although macrophages were initially and more commonly known as being implicated in the inflammatory process, a series of mechanistic experiments have also revealed their essential roles in bone development, formation and repair by releasing M2‐related cytokines and growth factors (27‐31). Nevertheless, the differentiation and polarization of monocytes towards M1 or M2 macrophages, as well as their fusion to osteoclasts or MNGCs in response to external stimuli and/or bone biomaterials remains to this day, extremely poorly understood.



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6 Previously we reviewed how the monocyte/macrophage lineage polarize and differentiate towards MNGCs and osteoclasts (Figure 1). Due to common precursors, OsteoMacs have been the favored precursor cell of MNGCs (5). Interestingly, differences in surface markers and gene expression between osteoclasts and MNGCs have been reviewed recently (32). To date, the transcriptional regulation responsible for the formation of osteoclasts versus MNGCs from precursor cells in bone tissues is poorly characterized with much greater investigation needed. Emerging findings from our laboratories as well as others have shown that MNGCs are also capable of polarizing much like macrophages towards M1 and M2 phenotypes (27), thereby opening the possibility that they act as key regulators during biomaterial integration and peri‐implant infection/resolution.   2.2 Giant‐cell formation and function While it is known that MNGC formation is the result of cells derived from the monocyte/macrophage lineage, over the years a variety of different names have been given based on their perceived roles in tissues including foreign body cell (FBC), foreign body giant cell (FBGC), multinucleated cell (MNC), multinucleated giant cell (MNGC), giant‐ body foreign cell (GBFC), or ‘foam’ cell. Despite this, it is important to note that these cells are phenotypically derived from the same precursor cells and often confused in terminology. Early in vitro experiments dealt primarily with demonstrating their role in response to pathogens; an obvious inflammatory state. In those studies, large MNGCs with 15 nuclei or more were routinely found in response to high‐virulence mycobacterium thereby inducing a ‘foreign body’ terminology for MNGC described as FBGCs (33). Simply put, these ‘unhappy’ macrophages were shown to routinely fuse in response to foreign pathogens and thus given the working name FBGCs.



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7 More recently, much advancement has been made with respect to MNGCs. Many biological aspects of their behavior have been discovered from biomaterials implanted in soft tissues including their recognition motifs, adhesion molecules, fusion pathways, as well as specific intercellular and intracellular signaling pathways (34, 35). Yet with respect to bone biomaterials, a great deal of information is still lacking regarding their cellular control and since their binding motifs are often presented in response to hydroxyapatite‐ derived surfaces onto bone‐biomaterial surfaces, great differences are expected in comparison to soft tissue biomaterials yet this field of research remains entirely unstudied. It is also interesting to note that MNGCs in response to biomaterials form in response to foreign particles greater in size than permitted for macrophage phagocytosis to occur (36). In response, ‘frustrated’ macrophages fuse into larger MNGCs in an attempt to thereafter phagocytize larger foreign particles (36). In the context of soft‐tissue biomaterial integration, it has been proposed that ‘FBGCs are generated by macrophage fusion and serve the same purpose as osteoclasts, degradation/resorption/removal of the underlying substrate’ (37). While this may be the case in soft‐tissues and the authors do not oppose this view, it is interesting to point out that MNGCs are also found around bone biomaterials, characteristically different than osteoclasts by their inability to resorb and replace bone grafts and dental implants. Therefore, and as previously stated: ‘If these cells serve the same purpose as osteoclasts in bone, one has to ask the question why are these cells found in bone altogether?’ (5). 3. Macrophage Polarization and MNGCs in Atherosclerosis A major pathway for the development of calcifying arteries in atherosclerosis is the critical role and involvement of immune cells/macrophages during ectopic bone formation. While it is known that initially, classically activated M1 macrophages are present within these



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8 tissues expressing high levels of IFN‐gamma (38, 39), it was recently discovered that they later polarize towards M2 wound healing macrophages thereby causing ectopic bone calcification within arterial walls (40). Within the inner most intimal layer of the artery, monocytes have been shown to accumulate and differentiate into macrophages where they ingest modified lipoproteins via scavenger receptors and secrete inflammatory mediators giving rise to lipid‐rich macrophages (foam cells) responsible for the lipid core buildup. For yet an unknown mechanism, a transient shift of polarization of these cells has been observed from M1 to M2 macrophages subsequently causing ectopic bone formation and calcification of arteries (41). Interestingly, it has been demonstrated that the formation of M2 macrophages was primarily activated by endoplasmic reticulum (ER) stress, an ongoing area of research in the cardiovascular field (40). Current research in this field has proposed actually suppressing M2 macrophage phenotypes and attempt to modify/re‐polarize macrophages back to pro‐inflammatory M1 macrophages in order to prevent ectopic mineralization/calcification of arterial walls; a scenario highly dangerous and life‐ threatening (40). Presently, it remains poorly understood why these macrophages shift from M1 to M2 phenotypes in these tissues. Nevertheless, it remains interesting to note how in one clinical scenario, M2‐macrophage accumulation and MNGC ‘foam cells’ in calcified arteries producing ectopic bone are considered pathological and potentially life threatening, whereas that same cell‐type located around certain classes of bone biomaterials would ultimately be considered greatly therapeutic, thus necessitating the need for future research in these fields. 4.1 MNGCs around Bone Biomaterials It was once thought that all MNGCs surrounding bone biomaterials were osteoclasts due to their similarities in histological features between both cell types. More



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9 recent research derived from soft tissue biomaterial integration has however revealed marked differences between MNGC formation and osteoclasts (42, 43). One of the main difficulties encountered to date in the bone biology field has been to characterize giant cells from bone biopsies. MNGCs assessed around dental implant surfaces are routinely embedded in resins (as opposed to paraffin due to the presence of metal or ceramic), thereby preventing the ability to use routine antibody staining and immunohistochemistry or ‐fluorescent techniques commonly utilized in paraffin sections. Interestingly, initial observations noted that MNGCs located around bone grafts appeared to be distinctly different from osteoclasts (44). In this study, it was found that certain synthetic bone particles appeared to recruit MNGCs similar to osteoclasts by their TRAP activity, ruffled borders with calcitonin receptor expression; however an obvious inability to resorb bone particles was observed (44). What was the role of these cells in bone tissues? Some years later, Kelly and Schneider implanted mineralized and demineralized bone, as well as composite (non‐mineralized) materials into the dorsal body wall of young adult rats and found that only a small population of MNGCs was observed on the composite implant whereas MNGCs on demineralized implants demonstrated high TRAP expression similar to osteoclasts (45). Around the same time, Marks and Chambers observed giant cells to mineralized bone particles in subcutaneous tissues that did not share similar functional features and enzymatic activity as osteoclasts (46). Interestingly, in 1995 Dersot et al. renamed these giant cells ‘macrophage polykaryons’ after confirming certain differences between the MNGCs recruited to a synthetic material fabricated of hydroxyapatite revealing that ‘they did not exhibit the morphologic, enzymatic and functional characteristics of osteoclasts’ (47).



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10 Interestingly, a recent review article by our group characterized cell surface marker differences between osteoclasts and MNGCs. Out of 19 potential markers which have been previously utilized to identify osteoclasts from MNGCs, only 5 markers were found to represent ideal candidates whereby expressed in only 1 of the 2 cell types. These included calcitonin receptor and RANK in osteoclasts and CD86 (B7‐2), CD206 and HLA‐DR in MNGCs (32). Of the remaining, 6 were found more highly expressed in one cell type versus the other including TRAP, cathepsin K and MMP9 in osteoclasts and CD68, CD98 and B7‐H1 (PD‐L1) in MNGCs. The remaining 8 markers including CD9, CD13, CD14, CD40, CD44, CD51, CD147 and EMR1 (F4/80) showed no preference for either cell type (32). While these markers have now been identified in giant cells around various bone biomaterials, much further research is needed to fully characterize them. Therefore, research dating back over 20 years has hinted at the fact that giant cells around bone grafts are different from osteoclasts either morphologically, enzymatically or functionally, yet the ability to accurately characterize them accordingly by cell surface markers and receptors did not exist at that time. Furthermore, their role was much more frequently associated with a foreign body reaction as it relates to bone‐biomaterial integration. Here we present evidence that MNGCs may also serve as a bone‐enhancing cell‐type similar to those found around atherosclerotic calcification. 4.2 Evidence that MNGCs induce vascularization and new bone formation The role of macrophages and MNGCs around bone grafting materials has gained tremendous momentum with the advancements made in the field of osteoimmunology (48). Despite the growing trend to study macrophage behavior and polarization around bone biomaterials, this review focuses only on MNGC function as it relates to their ability to induce vascularization and ultimately new bone formation.



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11 Abshagen and colleagues were one of the first to show the implications of MNGCs in vascularization (49). Owing to the importance of adequate vascularization during bone augmentation procedures, they studied the effects of NanoBone, a fully synthetic nanocrystalline bone graft, in a murine dorsal skinfold model. It was found that NanoBone markedly increased ingrowth of vascularized fibrous tissue associated with an increase in MNGC formation (49). In 2011, Ghanaati et al. found that implanting fibroin scaffolds with previously harvested osteoblasts served to instruct host endothelial cells to migrate, proliferate, and initiate the process of scaffold vascularization (50). It was concluded that the robust effect on scaffold vascularization at least appeared at the time to occur in concert with the pro‐angiogenic stimuli arising from host immune cells, most notably macrophages and MNGCs (50). It was later shown histologically by this same group in a split‐mouth design in 8 oral cancer patients, that both NanoBone as well as DBBM led to MNGC formation with similar new bone volume after a 6 month healing period (51). This study confirmed the presence of MNGCs in stable bone even in oral cancer patients (51). In 2014, Tour et al. showed that bone marrow stromal cells (BMSCs) enhanced the osteogenic properties of hydroxyapatite scaffolds by modulating the foreign body reaction (52). They loaded (BMSC)‐incorporated biomimetic constructs composed of hydroxyapatite into rat calvarial critical‐sized defects (8 mm) (52). Therefore, BMSC‐loaded biomimetic constructs significantly enhanced bone repair by modulating the foreign body reaction and MNGC formation (52), confirming the previous report by Ghanaati and co‐ workers (51). Others have also shown that cell incorporation into bone grafts leads to an increase in MNGC formation and subsequent angiogenesis (53, 54).



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12 An interesting study revealed that when zinc was added to tricalcium phosphate (TCP) bone grafts, RAW264.7 cells cultured with different dosages of zinc supplements demonstrated that zinc could influence both the activity and the formation of MNGCs (55). After a 12‐week implantation period in the paraspinal muscle of canines, de novo bone formation and bone incidence increased with increasing zinc content (and MNGC formation), indicating that zinc incorporated in TCP can modulate bone metabolism and render TCP osteoinductive through MNGC formation (55). This group later showed that by using liposomal clodronate to inhibit the formation of macrophage/MNGC/osteoclast progenitors, no ectopic bone formation could take place around osteoinductive bone grafts at least partially implicating immune cells in the formation of ectopic bone (56). Later, Davison et al. demonstrated that the scale of TCP biomaterial surface architecture could further affect MNGC/osteoclast cellular resorption (57).

Jensen et al. showed in both a human and animal study that the presence of

MNGCs was frequently found around DBBM bone grafts (4, 58). In a human study, it was found that long‐term stability of bone augmentation was observed histologically in 12 human biopsies between 14 and 80 months after augmentation surrounded by many MNGCs displaying little to no bone resorption of such bone particles (4). In a minipig study it was shown that two types of low‐resorbing bone grafting materials formed MNGCs on the surface of bone grafts (58). While MNGCs formed sealing zones and ruffled borders, both features of mature osteoclasts, MNGCs also demonstrated distinctly different histological features depending on the bone substitute material used with no evidence of particle resorption (58). In a human study the material‐specific tissue response to the synthetic, hydroxyapatite‐based bone substitute material NanoBone was compared with that of DBBM (59). In sinus cavities of 14 patients augmented with NanoBone and DBBM (split‐ mouth design), MNGCs were found on both bone graft particles with a significantly higher



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13 number of MNGCs and blood vessels in the NanoBone group compared to DBBM group (59). The influence of granule size of 2 biphasic bone substitutes (BCP, 400‐700 μm and 500‐ 1000 μm) was evaluated on the induction of MNGCs and implant bed vascularization in a subcutaneous implantation model in rats (60). Higher numbers of MNGCs were detected in the group with small granules starting on day 30, with higher vascularization. Furthermore, the results revealed that a synthetic bone substitute material could induce tissue reactions similar to those of some xenogeneic materials, thus pointing to a need to elucidate their "ideal" physical characteristics (60). Another study showed that addition of monocytes to bone grafts led to their being involved in the tissue response to a biomaterial, however without marked changes in the overall tissue reaction (61). Lastly, high‐temperature sintering of xenogeneic bone substitutes was found to increase MNGC formation associated with enhanced vascularization (62). These results revealed that high heat treatment of xenografts led to an elevation in the inflammatory tissue response to the biomaterial, and a combined enhancement in MNGC formation (62). In that study, it was concluded that: “further clarification of the differentiation of the multi‐nucleated giant cells toward so‐called osteoclast‐like cells or foreign‐body giant cells is needed to relate these cells to the physicochemical composition of the material” (62).

Interestingly, in 2015 Katsuyama et al. demonstrated specifically that MNGCs do

not resorb bone but rather express M2 macrophage markers (63). They report the critical findings that strongly suggest that implant failure due to bone loss likely resulted as a direct activity from osteoclasts and not MNGCs since they were unable to resorb bone (63). This report highlights the fact that MNGCs can polarize towards M2 phenotypes with much further research necessary to characterize their behavior around bone biomaterials.



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14 4.3 Role of MNGCs around dental implants It remains of interest to note that to this day, very few studies beyond bone grafting materials have focused on MNGC formation surrounding dental implants. This may be due to the fact that along metal implant surfaces, observable differences between osteoclasts and MNGCs are not as obvious as around bone grafts where MNGCs can be distinguished based on their inability to resorb grafts. Since neither MNGCs nor osteoclasts can resorb metals, samples around dental implants are typically embedded in paraffin where surface marker characterization can be performed.  As such, very little research has investigated MNGC versus osteoclasts with respect to dental implant biomaterials.

It remains interesting to note that to this day, some dental implants and other

bone biomaterials are lost every year due to unknown reasons. It is hypothesized that   such failures could be due to humoral immunity in the case of biomaterials, a field of research that continues to grow rapidly  (5, 64). This area of investigation has been one of the main topics of focus by a prominent research group in Sweden working in implant dentistry for many decades studying the foreign body reaction of host tissues to dental implants (65, 66). These concepts will be discussed later in the section: “Role of MNGCs in peri‐implant infection.” Based on questions emanating from this work, new parameters used to quantify the number and formation of MNGCs around dental implant surfaces termed MNGC‐to‐ implant contact (MIC) and quantified the data in relation to bone‐to‐implant contact (BIC) and peri‐implant bone density (BD) have been established (67). It was found that significant differences in MNGCs were present on all tested implant surfaces, yet this was not associated with an inflammatory cell infiltrate, fibrous encapsulation, or implant osseointegration in the miniature pig defect model (67). Based on these results, it is difficult to assess the role of MNGCs/osteoclasts around dental implants as to date, there



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15 has been no attempt to characterize these cells using cell surface markers. While other studies have shown that implant surface topography/composition has an influence on MNGC formation (68, 69), more studies remain necessary.



4.4 Role of MNGCs in peri‐implant infections   Most of the research to date investigating MNGCs around dental implants has dealt with the clinical condition of peri‐implantitis (65, 66, 70). A group of researchers from Sweden has revealed the marked impact of MNGCs around dental implants during peri‐implant‐ bone destruction. In a publication reviewing the foreign body reaction to biomaterials, a hypothetical view on the mechanisms leading to buildup and breakdown of osseointegrated dental implant interfaces was proposed (71). While the authors provide a view of biomaterial integration as it relates to a “foreign body reaction” of biomaterials into host tissues, it is difficult to gather a clear description/understanding of the role of MNGCs or FBGCs. In their proposed model (Fig. 3E, reprinted with permission), they theorize the mechanisms whereby invading bacterial pathogens induce the fusion of macrophages to FBGCs which then degrade alveolar bone tissues surrounding implants (71). While such a pathway might be possible, it has more recently been shown that fusing macrophages into MNGCs (or FBGCs) are incapable of resorbing bone (Fig. 4) (42), thereby refuting the hypothesis that MNGCs or FBGCs are responsible for resorption of bone following pathogenic infection. It has further been shown that MNGCs have minimal capability to degrade bone when compared to osteoclasts with a 40‐fold decrease in efficiency (42). It is therefore not plausible that these cells are directly responsible for alveolar bone degradation and are more likely to interact with other cell types during this process. This topic has been very superficially studied to date.



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16 It goes without saying the complex nature of MNGC biology would greatly benefit from a more accurate characterization as well as description of their role in bone tissue hemostasis and disease. It is most likely these cells are reversibly capable of shifting polarization in a similar manner to macrophages in response to changes in cytokines and various pathogens within their micro‐environment (72).  Interestingly, recent findings have demonstrated that MNGCs are able to polarize towards wound healing M2‐MNGCs thereby contributing to tissue wound healing and regeneration (27). As it relates to dental implant biology, this field of research has thus far been left entirely unstudied. 5. Discussion and Future Research Outlooks In summary, this review integrates current knowledge to date as it relates to MNGC behavior associated with bone biomaterials. While the role of MNGCs has largely been associated with a foreign body reaction over the years, a more contemporary view is that MNGCs do not prevent osseointegration of dental implants (67) and are capable of expressing M2‐macrophage markers (27). Therefore, based on MNGC fusion from M1 and M2‐macrophages, we propose that MNGCs be assessed more specifically in future research endeavors as M1‐MNGCs and M2‐MNGCs. In this way, their polarization and subsequent fusion from macrophages can be better characterized in health and disease.

The physiological behavior of MNGCs, especially as it relates to dental implants is a

growing area of research. While MNGCs are known to exist on various dental implant surfaces (at least during the osseointegration phase), one of the key differences between these tissues from others are that they are trans‐mucosal and thus bear the risk of being invaded by bacterial pathogens found in the oral cavity (Fig. 6). If bacterial accumulation occurs on an implant surface, it is likely that both macrophages and/or MNGCs will polarize towards M1‐macrophage and M1‐MNGCs respectively, creating an environment



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17 dictating tissue inflammation and further tissue breakdown. While but a hypothesis, it is now known that these cells are completely unable to resorb bone and therefore their interaction with other cells such as osteoclasts (direct interaction vs. indirect interaction via. e.g. T‐cells, se before) is highly probably. How cell‐cell communication occurs (via direct cell contact or paracrine activity) during this process also remains completely unstudied.

At the same time, it is known that MNGCs can also exist on the surface of fully

integrated biomaterials even years after their implantation. These MNGCs are often seen surrounded by stable bone and our research team has now shown that these cells express M2‐related phenotypes (unpublished data). Therefore, evidence that MNGCs are both capable of acting as M1 tissue inflammatory and/or M2 tissue wound healing MNGCs in various bone environments should therefore be characterized more appropriately as M1‐ MNGCs and M2‐MNGCs according to their role in tissue homeostasis. It is also interesting to point out that very similar to macrophage biology, MNGCs lining a dental implant surface could potentially co‐exist as M1‐MNGCs at the coronal portion of the implant surface during periodontal bacterial invasion, yet also exist as M2‐MNGCs at the protected apical portion of the implant not yet contaminated by bacterial invasion. While this hypothesis requires much further investigation, interestingly it has been shown by Spiller et al. that macrophages proficiently polarize from M1 to M2 and vice versa in as little as 3 days (Figure 6) (72). It therefore remains of interest if MNGCs are capable of also shifting their polarization as readily with similar efficiencies. If so, one future strategy to resolve peri‐implant infection might be to subsequently focus on macrophage/MNGC polarization which may further improve/enhance current strategies attempting to resolve peri‐ implantitis focused solely on strategies stimulating bone formation and suppressing bone resorption via growth factors and bioactive materials. While only a hypothesis, more evidence now points to the marked impact of immune cells in the field of bone biomaterial integration and homestasis.



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18 Another aspect that remains of interest in the dental field is the influence of macrophage/MNGC interactions to multiple biomaterials in small spaces. For example, tooth loss in the esthetic zone is commonly replaced by dental implants fabricated from titanium often requiring contour augmentation procedures with bovine‐derived bone grafting materials and porcine collagen membranes (73). Therefore, within this small regenerated area (often 1mm in thickness), macrophages and MNGCs will be in contact with 1) a titanium dental implant, 2) a bovine bone graft and 3) a porcine collagen membrane. It therefore remains unknown how polarization of macrophages/MNGCs is affected by the response of these cells to various materials simultaneously, nor has their effect on cell‐cell interactions within this micro‐environment including osteoblasts, osteoclasts, osteocytes, fibroblasts, endothelial cells, and leukocytes been investigated. What would happen if one class of biomaterial favors M2‐MNGC formation whereas another favors M1‐MNGC? How then would tissue integration occur in such a limited‐sized environment? Furthermore, there are many patient‐related risk factors such as (poly)‐ medications that may further increase susceptibility in some patients. The current influence of the large array of medications currently utilized on immune cells and their influence on bone‐biomaterial tissue integration is another field of research completely unstudied. Interestingly, a category of bone diseases including giant cell tumors, cherubism, and Noonan's syndrome include large populations of MNGCs. These cells are associated with significant areas of osteolysis and their activities have been "reversed" with a disparate group of medications e.g. interferon, denosumab, and calcitonin. Very little is known to date regarding the specific characterization of these MNGCs as a potential third population of giant cells or M1‐/M2‐MNGCs. Therefore, it remains of interest to further investigate MNGCs found in the above‐mentioned bone diseases with the appropriate markers comparing osteoclasts versus MNGCs and M1 versus M2 macrophage markers to better characterize their differences. A great deal of new information could be learned which may also further lead to new therapeutic strategies.

In summary, it is clear that much future research is needed to better understand

MNGC formation and behavior in bone tissue homeostasis. Within the present article, we



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19 introduce the notion that MNGCs should no longer be referred to as ‘good’ MNGCs or ‘bad’ FGBCs and instead propose the necessity to better 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 such as peri‐implantitis. Conflict of Interest The authors report no conflict of interests or financial disclosures. Acknowledgements The authors would like to thank Professor William Giannobile, University of Michigan, for critically reviewing the manuscript.



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20 References 1.  Konttinen, Y.T., Ma, J., Lappalainen, R., Laine, P., Kitti, U., Santavirta, S., andTeronen, O. Immunohistochemical evaluation of inflammatory mediators in failing implants. International Journal of Periodontics & Restorative Dentistry 262006. 2.  Olmedo, D., Paparella, M., Brandizzi, D., andCabrini, R. Reactive lesions of peri‐implant mucosa associated with titanium dental implants: a report of 2 cases. International journal of oral and maxillofacial surgery 39, 503, 2010. 3.  Major, M.R., Wong, V.W., Nelson, E.R., Longaker, M.T., andGurtner, G.C. The foreign body response: at the interface of surgery and bioengineering. Plastic and reconstructive surgery 135, 1489, 2015. 4. Jensen, S.S., Bosshardt, D.D., Gruber, R., andBuser, D. Long‐term stability of contour augmentation in the esthetic zone: histologic and histomorphometric evaluation of 12 human biopsies 14 to 80 months after augmentation. Journal of periodontology 85, 1549, 2014. 5. Miron, R.J., andBosshardt, D.D. OsteoMacs: Key players around bone biomaterials. Biomaterials 82, 1, 2016. 6.  Martinez, F.O., andGordon, S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000prime reports 6, 13, 2014. 7.  Thalji, G., andCooper, L.F. Molecular assessment of osseointegration in vitro: a review of current literature. The International journal of oral & maxillofacial implants 29, e171, 2014. 8.  Chehroudi, B., Ghrebi, S., Murakami, H., Waterfield, J.D., Owen, G., andBrunette, D.M. Bone formation on rough, but not polished, subcutaneously implanted Ti surfaces is preceded by macrophage accumulation. Journal of Biomedical Materials Research Part A 93, 724, 2010. 9. Kohal, R.J., Weng, D., Bächle, M., andStrub, J.R. Loaded custom‐made zirconia and



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27 62. Barbeck, M., Udeabor, S., Lorenz, J., Schlee, M., Holthaus, M.G., Raetscho, N., Choukroun, J., Sader, R., Kirkpatrick, C.J., andGhanaati, S. High‐Temperature Sintering of Xenogeneic Bone Substitutes Leads to Increased Multinucleated Giant Cell Formation: In Vivo and Preliminary Clinical Results. The Journal of oral implantology 41, e212, 2015. 63.  Katsuyama, E., Miyamoto, H., Kobayashi, T., Sato, Y., Hao, W., Kanagawa, H., Fujie, A., Tando, T., Watanabe, R., Morita, M., Miyamoto, K., Niki, Y., Morioka, H., Matsumoto, M., Toyama, Y., andMiyamoto, T. Interleukin‐1 receptor‐associated kinase‐4 (IRAK4) promotes inflammatory osteolysis by activating osteoclasts and inhibiting formation of foreign body giant cells. The Journal of biological chemistry 290, 716, 2015. 64. Kronström, M., Svensson, B., Erickson, E., Houston, L., Braham, P., andPersson, G.R. Humoral immunity host factors in subjects with failing or successful titanium dental implants. Journal of clinical periodontology 27, 875, 2000. 65. Albrektsson, T., Dahlin, C., Jemt, T., Sennerby, L., Turri, A., andWennerberg, A. Is marginal bone loss around oral implants the result of a provoked foreign body reaction? Clinical implant dentistry and related research 16, 155, 2014. 66.  Trindade, R., Albrektsson, T., Tengvall, P., andWennerberg, A. Foreign Body Reaction to Biomaterials: On Mechanisms for Buildup and Breakdown of Osseointegration. Clinical implant dentistry and related research 2014. 67. Chappuis, V., Cavusoglu, Y., Gruber, R., Kuchler, U., Buser, D., andBosshardt, D.D. Osseointegration of Zirconia in the Presence of Multinucleated Giant Cells. Clinical implant dentistry and related research 18, 686, 2016. 68.  Saulacic, N., Bosshardt, D.D., Bornstein, M.M., Berner, S., andBuser, D. Bone apposition to a titanium‐zirconium alloy implant, as compared to two other titanium‐containing implants. European cells & materials 23, 273, 2012. 69. Saulacic, N., Erdosi, R., Bosshardt, D.D., Gruber, R., andBuser, D. Acid and alkaline etching of sandblasted zirconia implants: a histomorphometric study in miniature pigs. Clinical implant dentistry and related research 16, 313, 2014.



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29 Table 1: Supporting Evidence that multi‐nucleated giant cells (MNGCs) are associated with increased vascularization and/or new bone formation when around bone biomaterials. Main Findings NanoBone, a fully synthetic nanocrystalline bone graft was

Author

Year

Abshagen

2009

Ghanaati

2011

Ghanaati

2013

Tour

2014

Tuo

2014

Davison

2014a

Davison

2014b

An adaptive immune response in compromised nude mice

Bouvet‐

2014

inhibits ectopic mature bone formation induced by

Gerbettaz

found to markedly increased ingrowth of vascularized fibrous tissue associated with an increase in MNGC formation in a mouse dorsal skinfold model A robust effect on scaffold vascularization occurred in concert with the pro‐angiogenic stimuli arising from host immune cells, most notably macrophage and MNGCs, when silk fibrin scaffolds were pre‐loaded with osteoblasts. The host immune system (macrophages and MNGCs) were thought to be key players in the angiogenic findings Both NanoBone and DBBM formed MNGCs in stable bone after 6 months of healing in patients whereby host bone could be maintained stable with MNGCs Bone marrow stromal cells enhance the osteogenic properties of hydroxyapatite scaffolds by modulating the foreign body reaction Zinc‐incorporated TCP bone grafts induced MNGC formation later leading to the osteoinductivity of these bone grafts Liposomal clodronate to inhibit of macrophage/MNGC progenitor cells inhibited the osteoinductive potential of bone graft confirming the role of macrophage/MNGCs in bone biology The scale of bone grafting material surface architecture of TCP affects MNGC/osteoclast formation and ability for graft resorption



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30 BMSCs/BCP/plasma composite which was suppressed when MNGC formation was lost BMSCs loaded onto BCP bone grafts induce a foreign reaction Gambin

2014

thereby attracting circulating haematopoietic stem cells and inducing their differentiation into macrophages and MNGCs in nude mice, thus favouring new bone formation In a human study, it was found that long‐term stability of

Jensen

2014

Jensen

2015

Lorenz

2015

Barbeck

2015

Barbeck

2015

Katsuyama

2015

Barbeck

2016

bone augmentation was observed histologically in 12 human biopsies between 14 and 80 months after augmentation surrounded by MNGCs years after augmentation procedures   Osteoclast‐like cells (MNGCs) found around DBBM and BCP particles in 3 minipigs. Shown not capable of resorbing bone yet MNGCs demonstrated distinctly different histological features depending on the bone substitute material used MNGCs observed within the NanoBone implantation bed were characteristics more similar to those of foreign body giant cells when compared to DBBM yet also demonstrated higher blood vessel formation investigated the influence of granule size of 2 biphasic bone substitutes (BoneCeramic® 400‐700 μm and 500‐1000 μm) on the induction of multinucleated giant cells (MNGCs) and implant bed vascularization in a subcutaneous implantation model in rats Addition of monocytes to bone biomaterials leads to their being involved in the tissue response to a biomaterial, however without marked changes in the overall reaction. MNGC formation still occurred in relatively similar numbers Demonstrated that MNGCs do not resorb bone and are capable of expressing M2 macrophage markers High‐temperature sintering of xenogeneic bone substitutes



Page 31 of 37

31

leads to an increase in MNGC formation associated with enhanced vascularization



Page 32 of 37

32

Figure Legends

Figure 1: Monocyte differentiation including expression markers into Osteoclasts, M1,

M2a, M2b, M2c macrophages and MNGCs. Reprinted with permission form Miron and

Bosshardt 2016.



Page 33 of 37

33

Figure 2: MNGC on the surface of a bone grafting material deproteinized bovine bone mineral (DBBM) stained for DAPI (top left), HLA‐DR (top right), combined (bottom left) and H&E (bottom right). HLA‐DR is a marker only expressed in MNGCs and non‐expressed in

osteoclasts confirming the presence of MNGC‐specific cells around bone biomaterials.



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34

Figure 3: Proposed peri‐implant defect progression from Trindade et al. (2016). Notice the

illustration that foreign body giant cells are resorbing bone. “A–E, Osseointegration

breakdown, with the loss of the foreign body reaction balance and consequent peri‐

implant bone loss. FBGC = foreign body giant cell; L = lymphocyte; MΦ = macrophage;

O = osteoblast; Oc = osteoclast; Ost = osteocyte; Green bodies = bacteria.”. Reprinted with

permission (71).



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35

Figure 4: Bone resorption by osteoclasts and foreign body giant cells (FBGCs) by ten Harkel et al. (2015). “After 25 days, cells were stained with Richardson’s staining solution (a‐e) and resorption pits were visualized (c‐f) and quantified (g) using Coomassie brilliant blue (CBB). Osteoclasts created resorption pits (Howship’s lacunae) (a; black arrow) and formed a ruffled border (white arrow). No resorption pits nor ruffled borders were visible in the FBGC cultures (d, e). In the resorption pits, collagen fibrils were visible (b; black arrow). Numerous resorption pits were seen in the osteoclast culture (c; black arrow), but no signs of resorption were apparent in the FBGC cultures. Osteoclasts resorbed more than 20% of the bone surface (g). The percent bone resorption graph represents the mean area ± S.D. per 0.25 cm2 bone surface. Scale bar is 10 μm for panels a, b, d, e. Scale bar is 100 μm for panels c and f.” Red asterisk = bone. *p

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