OsteoMacs: Key players around bone biomaterials

Biomaterials 82 (2016) 1e19

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Review

OsteoMacs: Key players around bone biomaterials Richard J. Miron*, Dieter D. Bosshardt** Department of Oral Surgery and Stomatology, Department of Periodontology, University of Bern, Freiburgstrasse 7, 3010 Bern, Switzerland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 October 2015 Received in revised form 12 December 2015 Accepted 15 December 2015 Available online 20 December 2015

Osteal macrophages (OsteoMacs) are a special subtype of macrophage residing in bony tissues. Interesting findings from basic research have pointed to their vast and substantial roles in bone biology by demonstrating their key function in bone formation and remodeling. Despite these essential findings, much less information is available concerning their response to a variety of biomaterials used for bone regeneration with the majority of investigation primarily focused on their role during the foreign body reaction. With respect to biomaterials, it is well known that cells derived from the monocyte/macrophage lineage are one of the first cell types in contact with implanted biomaterials. Here they demonstrate extremely plastic phenotypes with the ability to differentiate towards classical M1 or M2 macrophages, or subsequently fuse into osteoclasts or multinucleated giant cells (MNGCs). These MNGCs have previously been characterized as foreign body giant cells and associated with biomaterial rejection, however more recently their phenotypes have been implicated with wound healing and tissue regeneration by studies demonstrating their expression of key M2 markers around biomaterials. With such contrasting hypotheses, it becomes essential to better understand their roles to improve the development of osteo-compatible and osteo-promotive biomaterials. This review article expresses the necessity to further study OsteoMacs and MNGCs to understand their function in bone biomaterial tissue integration including dental/orthopedic implants and bone grafting materials. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Macrophage Bone regeneration Osteoimmunology Biomaterial integration Tissue response Multi-nucleated giant cells OsteoMacs Foreign body cells

1. Introduction Monocytes and macrophages are some of the most abundant cell type found in the bone marrow. Furthermore, they represent the first cell types that interact with foreign pathogens and implanted medical devices. Classical studies have demonstrated that macrophages are rapidly recruited to infectious and injury sites where they play critical roles in innate immunity. Here they demonstrate broad roles and are responsible for regulating tissue homeostasis including innate and adaptive immunity, wound healing, hematopoiesis and malignancy [1]. Based on their crucial and distinct roles in tissue homeostasis and immunity, they are attractive therapeutic targets for a broad range of pathologies. Furthermore, they are key players in tissue integration of various biomaterials across a wide range of tissues. Yet the field of bone-biomaterial biology has largely omitted their

* Corresponding author. University of Bern, Head of Oral Cell Biology, Switzerland. ** Corresponding author. University of Bern, Head of Oral Histology, Switzerland. E-mail addresses: [email protected] (R.J. Miron), dieter.bosshardt@ zmk.unibe.ch (D.D. Bosshardt). http://dx.doi.org/10.1016/j.biomaterials.2015.12.017 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

importance over the years. For instance, a recent systematic review of dental and orthopedic implants found that over 90% of research in this area focused primarily on in vitro behavior of osteoblasts on implant surfaces while only a small percentage (roughly 10%) was dedicated to immune cells including monocytes, macrophages, osteoclasts, leukocytes and multinucleated giant cells (MNGCs) [2]. With the advancements made in the field of osteoimmunology, it becomes vital to better understand the response of these cell types to various bone biomaterials. Immune cells play a pivotal role in determining the in vivo fate of bone biomaterials by either facilitating new bone formation around bone-implanted devices but have also been associated with creating an inflammatory fibrous tissue encapsulation. It is now understood that macrophages are the major effector cell in immune reactions to biomaterials where they are indispensable for osteogenesis. Knockout models have demonstrated that a loss of macrophages around bone grafting materials may entirely abolish their osteoinductive potential, thus confirming their primary role in the immune system modulation later responsible for guiding osteogenesis [3]. Over the years, complex studies from basic research have revealed the dynamic interactions between the skeletal system and immune system [4e6]. It has been shown that a population of

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tissue macrophages named “OsteoMacs” resides within bone as a distinctive canopy structure overlying mature osteoblasts [6]. Although initial bone fracture healing experiments have been characterized by infiltration of inflammatory cells, most of these initial studies focused primarily on the secretion of various cytokines and growth factors important for the inflammatory process including cell recruitment [7e9] and neovascularization [10]. Although macrophages in general have been implicated as key contributors to inflammation, a series of experiments have also revealed their essential roles in bone repair with recent findings demonstrating that even MNGCs may be categorized with a tissue repair phenotype by demonstrating release of M2-related cytokines and growth factors [11]. Thus, the differentiation of monocytes towards M1 or M2 macrophages, as well as their fusion to osteoclasts or MNGCs in response to various biomaterials remains extremely poorly understood. Furthermore, the main factors responsible for directing their phenotypes towards more specialized cell-types in response to biomaterials also remains poorly characterized. Human histological samples from our dental clinic using a variety of bone grafting materials for bone augmentation procedures have consistently shown a substantially high number of MNGCs around bone substitute materials grafts in stable situations harvested years after original surgeries were performed [12]. Furthermore, a select class of bone substitutes grafts consistently associated with higher than average maintenance of bone mass in grafted sites, are routinely found with significantly higher numbers of MNGCs (Fig. 1). This has led our research team to further question the role of MNGCs on biomaterials as these cells were once thought to only contribute to the foreign body reaction [11]. Interestingly, studies investigating atherosclerotic plaque have provided evidence that macrophages very commonly fuse into MNGCs (also termed foam cells) that enhance calcified tissues surrounding arterial walls; an area that otherwise should not produce any mineralized tissues [13e17]. While the production of mineralized tissues from MNGCs in atherosclerosis leads to a pathological state, recently our group has questioned whether this situation might be advantageous around bone biomaterials. Thus, it is clear that a substantial amount of additional work is needed with respect to understanding macrophage and MNGCs function especially as it relates to bone biomaterials. It may be possible that MNGCs in certain situations leading to a pathological state (e.g. calcified tissues around arteries) might be therapeutic in others (bone biomaterials). As part of an overview on the current knowledge regarding immune cells and bone biomaterials, this review article aims to: 1) Characterize and review the key basic science studies involving OsteoMacs that demonstrate their pivotal role in bone biology. 2)

Provide background knowledge on monocytes and the great potential for these cells to differentiate into a variety of cell-types including M1 and M2 macrophages, MNGCs, FBGCs and osteoclasts. 3) Review the current literature on monocyte/macrophage studies with respect to bone biomaterials including bone grafts and dental/orthopedic implants. 4) Provide additional evidence from calcified atherosclerotic plaque that macrophages/MNGCs are potent inducers of mineralization by demonstrating that even in a pathological state, macrophages/MNGCs are the responsible celltype contributing to calcification in arteries. 5) Demonstrate evidence from animal and human histological samples from our research center that MNGCs are routinely found around bone biomaterials in high numbers and commonly associated with the maintenance of high bone volume leading to the hypothesis that these cells may very well be one of the key players responsible for the maintenance of bone homeostasis.

2. OsteoMacs: biological basis and key roles in bone formation The term ‘OsteoMacs’ was originally given by a group of basic researchers in Australia led by Allison Pettit. Original observations described in the mid 1980s sought to characterize the role of osteal macrophages in bone biology [18]. Hume et al. were one of the first to clearly demonstrate that periosteal and endosteal tissues contained a discrete population of resident tissue macrophages in line with traditional bone cell nomenclature [5,6]. OsteoMacs constitute approximately one sixth of all cells residing in bone marrow and display a stellate morphology allowing them to achieve extension coverage of bone surfaces suggesting that they may form a comprehensive communication network [6]. It is clear now that this subset of CD68þ cell-type is derived from a resident population of macrophages like macrophages found in other tissues [19e21]. More recent research has clearly confirmed that macrophages may subdivide and proliferate from resident tissues contrasting original theories expressing that these cells are derived from monocyte precursors from the blood-stream [22e25]. The general role of OsteoMacs has been described as immune surveillance cells in the bone microenvironment. A number of previous studies have demonstrated that this subset of macrophages are able to function as phagocytes [26,27], are capable of detecting bacterial products [28,29], and respond to antigens [27,30]. In vitro cell culture systems have further provided evidence by demonstrating how primary murine osteoblast cultures are able to respond to pathophysiological levels of lipopolysaccharide (LPS), characteristics of the M1 macrophage later discussed in this article [6]. These observations as well as others report the potential cross-

Fig. 1. MNGCs on bone substitute materials including (A, D) Bio-Oss® (HA), (B, E) Straumann® BoneCeramic (BCP), and (C, F) NanoBone® (HA-silica gel). LM (AeC) and TRAP histochemistry (DeF).

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lineage plasticity and crosstalk between osteoblasts and hematopoietic cells in vitro [31e33], which adds difficulty to clearly assign the cells responsible for specific functions reported in primary ‘osteoblast’ cultures. This has since best been exemplified in a study by Chang et al. who showed that by removing macrophages from primary osteoblast cultures, a 23-fold reduction in mineral deposition was observed [6]. It was concluded that it was the OsteoMacs, and not the osteoblasts as originally hypothesized, within these in vitro culture systems that responded to pathophysiological concentrations of cytokines and their removal from calvarial cultures significantly decreased in vitro mineralization by osteoblasts [6]. 2.1. Are OsteoMacs precursors to osteoclasts and multinucleated giant cells (MNGCs)? Due to common precursors [34] and anatomical location, an obvious hypothesis would be to assume that OsteoMacs in osteal tissues serve as precursors for osteoclasts and MNGCs. In bone tissues, osteoclasts have historically been characterized by their multinucleated phenotype and ability to resorb bone [35]. More recently however, MNGCs have been found in bone tissues with slightly different histological appearance largely characterized by their inability to quickly resorb bone grafts [36]. Differences in surface markers and gene expression between osteoclasts and MNGCs are discussed later in this article. To date, the differentiation and transcription factors responsible for the formation of osteoclasts versus MNGCs from precursor cells in bone tissues has remained poorly characterized. Although OsteoMacs are clearly not osteoclasts (both confirmed by the lack of multinucleation and lack of expression of common osteoclast markers such as F4/80 antigen) [34,37], their plasticity in situ suggests that these cells can further respond to stimuli by locally differentiating towards various multinucleated cell phenotypes. In vitro research has demonstrated that bone marrowderived macrophages [38] and rheumatoid synovium-derived macrophages [39] can be differentiated into osteoclasts confirming that mature macrophages can at least in vitro, serve as multinucleated cell precursors. Alternatively, it is well known that more undifferentiated cells from the myeloid lineage also available within bone can more efficiently differentiate into multinucleated cells [40]. These findings have certainly argued against OsteoMacs being the ‘preferred’ multinucleated precursor cell in basic bone biology research [41]. Very recent research has demonstrated that MNGCs express many M2 macrophage-like markers implicated in wound healing and tissue regeneration and it was suggested that this specialized cell type resembled predominantly an M2 wound healing OsteoMac [11]. Yet it has also been argued that OsteoMacs may contribute to the osteoclast precursor pool and MNGCs under pathological conditions. Thus, it remains somewhat unclear how each terminal cell type from the monocyte lineage is capable of fully differentiating under various conditions/micro-environments. It is evident that macrophages demonstrate a great phenotypic plasticity in vivo and seem to respond to varying stimuli by differentiating towards various cell types at least in vitro. Nevertheless, there is clearly a lack of knowledge for which factors guide these cells to differentiate and/or fuse into MNGCs under various conditions. Ongoing research in this area is urgently needed. Currently, it is known that activated OsteoMacs are able to produce a wide variety of either pro- or anti-inflammatory cytokines leading to multinucleated cell formation. OsteoMacs have been shown to secrete TNF-alpha [42,43], interleukin IL-6 [44], IL-1 [42,45] and interferon-b [46,47] in response to various in vitro conditions. The number of factors released by OsteoMacs demonstrates their ability to regulate cell fusion of monocytic cells and

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their subsequent differentiation. How OsteoMacs specifically respond to various biomaterials will be discussed later in this article. 2.2. OsteoMacs function in osteoblast mineralization The preliminary finding from primary osteal tissues clearly demonstrated that OsteoMacs play a pronounced role in osteoblast function and differentiation by demonstrating a 23-fold decrease in mineralization potential when OsteoMacs were removed from in vitro culture systems [6]. Interestingly, depletion of OsteoMacs in vivo by various knockout systems has also been shown to markedly reduce bone formation [6,48]. These previous studies are similar to a number of investigations over the years describing osteoclasteosteoblast coupling mechanisms in vivo [49e51]. Interestingly in the mid-2000s, it was shown that macrophagespecific genes (csf1r, CD14) are induced in primary mouse chondrocyte differentiation cultures [52] raising the possibility that macrophages may also be heavily implicated in chondrogenesis, a key step in endochondral ossification. This hypothesis was later confirmed in a recent macrophage knockout system [48]. Previous reports have also shown that macrophages may produce a number of potent bioactive growth factors for osteoblasts including transforming growth factor b (TGF-b) [53], osteopontin [54], 1,25dihydroxy-vitamin D3 [55] and BMP-2 [56]. These factors are known inducers of extracellular matrix deposition and new bone formation and are classical characteristics of the M2 macrophage. The plasticity of macrophages suggests that their trophic role in bone tissues are highly regulated by changes to the microenvironment. OsteoMacs are capable of promoting anabolic function in certain conditions whereas in others they are responsible for creating and directing an inflammatory environment. 2.3. OsteoMacs and bone modeling Bone modeling is an anabolic process involving new bone deposition and is unlike bone remodeling, which involves the careful and coordinated balance between osteoclasts and osteoblasts [51,57]. It has previously been reported that macrophages are found localized at the bone modeling site on cortical diaphyseal endosteal bone surfaces without the presence of osteoclasts in the vicinity [6]. This process has been described as ‘forming a canopylike cell structure’ where OsteoMacs were seen encapsulating the functionally mature osteoblasts suggesting that they are heavily implicated in the bone modeling process [6,58]. Once again, the functional importance of OsteoMacs was demonstrated by knockout systems where macrophages were depleted using a Fasinduced apoptosis (Mafia) transgenic mouse model, which can induce macrophage depletion via synthetic ligand treatment [59]. In this system, OsteoMac canopy architecture was disrupted leading to a complete loss of mature osteoblasts and bone modeling at the bone interface [59]. It was originally proposed that during bone remodeling, osteoclasts provide a ‘coupling signal’ to promote and coordinate osteoblast activity [51]. Interestingly with the numerous advancements made in the field of osteocyte biology, it has recently been proposed that osteocytes are also implicated in the bone remodeling process by dictating both osteoblast and osteoclast activity [60]. Given that the bone modeling animal models lack osteocytes and osteoclasts during the developmental stages of bone modeling, it was proposed that OsteoMacs may be the cells responsible for couplinglike signals dictating osteoblast function. While evidence from the literature has previously suggested that TGF-b and ephrin B2 are implicated as possible coupling factors between osteoclasts and osteoblasts [51,61e63], macrophages have also been shown to

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produce TGF-b [53] and ephrin B2 [64,65] opening the possibility that OsteoMacs are also capable of fulfilling such roles. Nevertheless, a great deal of research is still necessary to further understand the role of OsteoMacs during bone modeling where there is an absence of osteoclasts and osteocytes. It is interesting to note that depletion of OsteoMacs in vivo using the macrophage Fas-induced apoptosis (Mafia) mouse caused a complete loss of osteoblast bone formation at the bone surface demonstrating that OsteoMacs are an integral component of bone tissues and play a pivotal role in bone homeostasis [6]. These proposed models show that OsteoMacs have as function to both survey for alteration in the local environment as well as guide bone formation in vivo. In response to anabolic stimuli, they function to recruit mesenchymal progenitor cells and induce their proliferation and differentiation towards bone forming osteoblasts. They subsequently provide ongoing anabolic signals to the underlying osteoblasts [6]. It has been further proposed that once the stimulus is removed, OsteoMacs within the canopy withdraw and either migrate to a new area, or undergo apoptosis whereas the underlying mature osteoblasts revert to a bone lining cell phenotype, become embedded within bone as osteocytes or undergo apoptosis [6]. 2.4. OsteoMacs and bone remodeling As described earlier, bone remodeling implicates the fine balance between bone-resorbing osteoclasts and bone-forming osteoblasts [51,57]. Resorption signals including RANKL and CSF-1 are expressed by bone lining cells and osteocytes, and are necessary for directing osteoclastogenesis and bone resorption. It was previously proposed that osteoclasts subsequently provide the coupling signal coordinating osteoblast activity to facilitate bone deposition and mineralization [51]. It has been reported that during this process, osteoclasts are only located at the leading edge of the formation phase and have moved or undergone apoptosis before new bone formation is completed [66]. Therefore, certain investigators have posed the question: ‘what cellular/molecular mechanism drives osteoblasts to initiate mineralization and complete the remodelling cycle following osteoclast apoptosis?’ [67]. As previously mentioned, OsteoMacs have been shown to form a cellular canopy structure around osteoblasts during bone modeling. This process was postulated to create an enclosed compartment for local communication and coordination during the complex remodelling process [68]. Although it is proposed that osteoclasts likely have the dominant role in orchestrating the recruitment, proliferation and initial differentiation of pre-osteoblasts during bone remodeling based on the release of cytokines from resorbed bone, the various roles of OsteoMacs in combination with their anatomical location and canopy architecture have recently postulated the idea that they may also be a necessary requirement for optimal mineralization by osteoblasts [6]. Furthermore, due to their close proximity to bone surfaces and well-known ability to detect dying cells [69], OsteoMacs are an obvious candidate to detect and respond to bone damage, a critical event for osteoclast recruitment, thus initiation the bone remodeling phase [70]. More recently, it has been demonstrated that bone marrow macrophages are responsible for maintaining hematopoietic stem cell (HSC) niches and that subsequent depletion facilitates the mobilization of HSCs [23]. Winkler et al. investigated the regulation of endosteal niches, by studying the mobilization of HSCs into the blood-stream in response to granulocyte colony-stimulating factor (G-CSF) [23]. They found that G-CSF rapidly depletes endosteal osteoblasts leading to the suppression of endosteal bone formation and decreases the expression of factors required for HSC retention and self-renewal. G-CSF administration also depleted a population

of OsteoMacs found in endosteal tissues, which subsequently disrupted osteoblast function. Furthermore, using 2 separate animal models to deplete OsteoMacs (Mafia transgenic mice or administration of clodronate-loaded liposomes) it was found that 1) there was a loss of endosteal osteoblasts, 2) there were marked reductions of HSC trophic cytokines at the endosteum and 3) HSC mobilization into the blood occurred. Furthermore, RT-PCR analyses on the endosteal RNA from treated Mafia mice revealed a 35fold decrease in osteocalcin expression when compared to controls. Taken together these results demonstrate the pivotal role of maintaining endosteal HSC niches that subsequently leads to downstream OsteoMac function and osteoblast bone formation [23]. In a similar study, Alexander et al. demonstrated also that osteal macrophages promote in vivo intramembranous bone healing in a mouse tibial model [71]. The authors used a very similar approach to the previous study by knocking out macrophage populations using Mafia mice and clodronate liposome delivery. Following tibial injury, they demonstrated that the depletion of OsteoMacs led to significantly reduced intramembranous ossification bone healing whereas administration of CSF-1 in the animal models led to an increase in OsteoMac number at the injury site, which concurrently increased new matrix deposition and mineralization [71]. A study with a similar animal model also demonstrated that fracture healing via periosteal callus formation also requires OsteoMacs for both the initiation and progression of early endochondral ossification [48]. Furthermore, a separate group found that depletion of macrophages using Mafia mice led to early skeletal growth retardation and progressive osteoporosis (25% reduction in bone mineral density, 60% reduction in number of mesenchymal progenitor cells) by 3 months [72]. Of particular interest, animals that were treated with anabolic factors such as PTH showed a significantly higher level of OsteoMacs further suggesting their important role in bone remodeling [73]. It is difficult to assess technically whether osteoclasts or macrophages are more important for bone remodeling and regulating osteoblast activity. The main reason is that most of the mutations to date that affect macrophages also have a large impact on osteoclasts, since they are derived from the same precursor cells [74]. It is therefore extremely difficult to knock down just macrophages without compromising osteoclast activity [74]. In contrast, it is possible to abolish osteoclasts by specifically targeting OPG by blocking the actions of RANKL [75]. Since there is a close lineage relationship between macrophages and osteoclasts [34], the current in vivo models could benefit from future refining as considerable cellular plasticity is found between these 2 cell types [38]. Further specific investigation on the role of OsteoMacs versus osteoclasts and their contribution to bone remodeling is needed to clearly delineate all the cellular participants and molecular factors in osteoblast coupling. 3. Monocyte/macrophage characterization and differentiation The goal of the present review article is not to fully detail monocyte differentiation towards their numerous downstream cell types. For an excellent summary on these events/topic, the reader is kindly directed to the following extensive review articles [1,76e78]. Instead we focus on presenting a broad overview on the current differentiation parameters seen in monocytes and specifically look at key markers and gene expression patterns of macrophage populations, MNGCs and osteoclasts. While the role of monocyte-derived macrophages during pathological tissue inflammation has recently been comprehensively reviewed [79], recent research has demonstrated that most

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monocytes do not substantially contribute to macrophage populations in the steady state. Until recently, a fundamental dogma in immunology was reversed whereby it was originally hypothesized that all tissue-resident macrophages were derived from a constant recruitment of blood monocytes, as originally described by van Furth and Cohn [80]. More recently however, data from a number of studies have challenged these original findings and shown that the majority of tissue resident macrophages including OsteoMacs are self-renewable within resident tissues [22e25]. Since macrophages have many functions in development, tissue homeostasis and the resolution of inflammation, accordingly there is much interest in potentially manipulating these cells for therapeutic benefit [76]. For this to occur, better understanding over their broad plasticity and key parameters inducing their differentiation towards certain subsets is absolutely necessary. Colony-stimulating factor 1 (CSF1, also known as M-CSF) is the growth factor by which monocyte development is completely dependent. Mice that are deficient in CSF1 or its receptor CSF1R (CD115) exhibit severe malformations and monocytopenia [81e83]. Therefore, perhaps the single most important gene to the monocytic lineage is CSF1, as macrophages, osteoclasts and MNGCs are all dependent on its expression for survival. The existence of monocyte subsets in human blood in the late 1980s described 2 main subpopulations defined as CD14þ monocytes (which can be further subdivided into distinct populations of CD14þCD16þ and CD14þCD16 monocytes) and CD14lowCD16þ monocytes [84]. Cell differences in the monocyte lineages have been observed between species and most notably between humans and mice. In mice, LY6Clow cells are the equivalent of human CD14lowCD16þ and may therefore be terminally differentiated towards resident macrophages [85,86]. By contrast, mice LY6Chi monocytes are the equivalent of CD14þ monocytes in humans representing the classical monocytes recruited to sites of inflammation [87]. Both of these subsets of monocytes may give rise to macrophages. Early studies have long considered macrophages as long-lived, terminally differentiated cells with little capacity to proliferate [88,89], however, recent findings that macrophages can self-renew in resident tissues such as bone contradicted these original findings. In certain settings, IL-4 has been identified as the major factor inducing macrophage proliferation [90] and CSF-1 has also been shown to promote proliferation as well as monocyte recruitment [91]. Since the survival, proliferation and differentiation of macrophages is dependent on CSF-1 [92], it is also known to be more highly expressed during infection, inflammation and tissue injury leading to the rapid recruitment of monocytes to induce their differentiation towards macrophages that drive innate and adaptive immune responses [19]. Macrophages and bone resorbing osteoclasts are closely related however can be distinguished by the presence of additional nuclei in osteoclasts as well as morphological features and expression of marker proteins later discussed [19,93]. Macrophages have a large capacity to secrete a wide range of cytokines and regulatory molecules in response to their microenvironment [81,94,95]. The most widely used antibody to identify macrophages in murine tissues has been the F4/80 monoclonal antibody [37]. This marker is also used to distinguish them from osteoclasts as F4/80 is rapidly down-regulated during osteoclastogenesis. Initial macrophage experiments identified macrophages into 2 specific cell types, classical M1 pro-inflammatory macrophages and M2 tissue resolution/wound healing macrophages (Fig. 2). Classical pro-inflammatory stimuli in response to LPS include TNF-alpha [42,43], IL-6 [96,97] and IL-1b [42,45] all contributing to tissue

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inflammation and osteoclastogenesis. M2 macrophages typically produce TGF-b and arginase, both factors implicated in tissuerepair processes [98e101]. Table 1 presents a general overview of differences observed between M1 and M2 macrophages. Fig. 2 presents an overview of general cell-types derived from the monocyte lineage. In vitro differentiation of macrophage towards the M1 phenotype can best be produced with IFN-gþ þ LPS and TNF-a, whereas M2 macrophages are typically produced with either IL-4 or IL-13 [78]. The in vitro culture with IL-4 causes upregulation of 2 key M2 markers including TGF-b and arginase largely assumed to participate in tissue regeneration [78,98e101]. It has also been shown that IL-4 increases the expression of the mannose receptor CD206. Whereby once M2 macrophages included a wide variety of characteristics as originally defined, more recent research has subdivided their classification in M2a/b/c to further express the differences found between certain M2 macrophages [102] (Fig. 2). Briefly, the M2a phenotype is produced by exposure to IL-4 þ Il-13 acting through IL-4Ra to increase the expression of CD206, arginase, and TGF-b [99,103e106]. The M2b phenotype has been described following exposure to a combination of IgG-immune complexes and IL-1R ligands in turn increasing IL10 production and decreasing IL-12 largely contributing to antiinflammatory properties [107,108]. Cell culture with IL-10 or glucocorticoids produces the M2c phenotype characterized by high IL10 and low IL-12 production [108], as well as increased surface receptor CD163 [109,110]. While the aim of this review article is not to give a background on the specificity of the various M2 macrophage subgroups, it remains important to note that various cell culture models have distinct M2 macrophage characteristics. For an excellent overview on this topic, the reader is kindly directed to a recent review article on M1 and M2 paradigm of macrophage activation [1]. Phenotyping macrophages has typically been carried out with cell surface markers including CD11b, CD68, macrophage antigen2, and F4/80. Interestingly, intensive research over the last decade has provided new and improved ways for further phenotyping macrophages based on their gene-expression profiles in response to various stimuli. As such, early functional studies of macrophage phenotyping noted that gene-expression plasticity existed when macrophages were ‘polarized’ to either the M1 or M2 phenotypes [108,111e113]. Gene expression makes sense in these scenarios as macrophages are likely more responsive to various stimuli such as infection, cell death, implanted biomaterials yet in general seem to maintain the plasticity to subsequently return to more polarized states based on signals from their micro-environment. Despite the research that has been performed on this topic over the past decade, one common reported difficulty in the literature with respect to macrophage phenotyping is the fact that major differences between human and mice macrophages exist complicating the overall progression of the field of osteoimmunology as discussed below. 3.1. Macrophage phenotyping - mouse versus human macrophages; how different are they? One of the most controversial aspects of macrophage biology that has certainly limited research progress is the concern over perceived differences between mouse and human macrophages. Some researchers have gone to such lengths as to suggest that human macrophages are ‘fundamentally’ different from their mouse counterparts and thus should be studied as entirely separate entities [114,115]. An example of this is the fact that mouse M2a macrophages have been phenotypically characterized by their expression of arginase, an important component of tissue regeneration [99,116].

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Fig. 2. Monocyte differentiation including expression markers into Osteoclasts, M1, M2a, M2b, M2c macrophages and MNGCs.

Table 1 Summary of in vitro culture conditions of M1 and M2 macrophages.

Activator Proinflammatory cytokines iNOS Anti-inflammatory cytokines CD206 Dectin-1 Ym1 Phagocytosis/Endocytosis Matrix proteins Markers Human: Mouse: Transcription factors Human: Mouse: Cytokines Human: Mouse: Chemokines Human: Mouse:

M1 macrophage

M2 macrophage

IFN-gamma, TNF-alpha, LPS IL-1B, TNF-alpha, IL6, IL12 High (in rodents only) Low Low Low Low High MMP9

IL-4, IL-13 Low Low TGF-B high, IL-10 Low High High High Decreased phagocytosis of implanted particles FN, TGFB1, MMP1, MMP12, TG, F13A1

CD64, IDO, SOCS1, CXCL10 CXCL9, CXCL10, CXCL11, NOS2

MRC1, TGM2, CD23, CCL22 Mrc1, tgm2, FizzI, Ym1/2, Arg1

pSTAT1, IRF5 pSTAT1, pSTAT6-ve, Socs1

IRF4, SOCS1*, GAT3* SOCS3 pSTAT6, pSTAT1-, Soc2

TNF, IL6, IL1b, IL12A, IL12b, IL23A TNF, IL-6, IL-27, Tnf23a

IL-10 IL-10, IL-6

CXCL10, IL8, CCL5, CXCL9, CXCL11, CCL18-ve

CCL4, CCL13, CCL17, CCL18, CCL17 CCL24, CXCL13, CCL1, CCL22, CCL20

Interestingly however is the fact that M2a macrophages in humans do not express arginase at all [117,118]. In general, arginase is highly expressed during injury [119e122] and is thought to contribute to collagen production during wound healing [119,123,124]. The differences between human and mouse macrophages extends beyond the scope of this review article, however a brief list of reported differences is compiled in Fig. 3 [125]. In summary, it is evident that there exists a lack of information that has thus far prevented progress in macrophage biology. Furthermore, evidence that demonstrates quite significant differences between human and mouse cell surface markers adds to the complexity encountered studying their phenotypes [108,126e129]. Ideally knockout animals are utilized to determine the role of

specific genes in biology. However, based on the perceived differences between species, some have questioned this line of research is even valid or necessary at all with respect to macrophage biology. Although in general many studies demonstrate an M1 macrophages phenotype by demonstrating the expression of IL-1B, IL-6, IL-8, TNF-alpha, and IFN-gamma to promote inflammation, or M2 macrophages by producing IL1Ra, IL-4, IL-10 and arginase to promote inflammatory resolution, future research is necessary to determine the differences between human and mouse phenotypes as there are perceived differences from the literature [99,102,130e133].

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Fig. 3. Framework for Describing Activated Macrophages (reprinted with permission from Ref. [125]), (A) Examples of widely used macrophage preparations. CSF-1-grown mouse adherent macrophages from bone marrow (BM) or CD14þ monocytes are used as the exemplars for marker evaluation and standardized activation conditions. Macrophages can also be generated with GM-CSF, where a CD11cþ dendritic cell (DC) population is also present depending on the culture conditions. In mice, thioglycollate injection followed by peritoneal lavages is used for generating macrophage populations with differing yields and properties, whereas many organ systems in mice and humans are sources of tissueinfiltrating macrophages., (B) Marker systems for activated macrophages. Shown are functional subdivisions according to stimulation of mouse CSF-1 macrophages or human monocyte-derived CSF-1 macrophages with the existing M1-M2 spectrum concept [1,99,228]. Stimulation conditions are IL-4, immune complexes (Ic), IL-10, glucocorticoids (GC) with transforming growth factor b (TGF-b), glucocorticoids alone, LPS, LPS and IFN-g, and IFN-g alone. Marker data were drawn from a wide range of published and unpublished data from the authors' laboratories and represent a starting consensus [229e238]. An asterisk indicates corroboration of human IL-4 genes by deep sequencing., (C) Using genetics to aid in macrophage-activation studies. Mutations in Akt1 and Klf4 cause a “switch” to M(LPS)- and M(IFN-g)-associated gene expression, whereas mutations in Akt2 and Klf6 show the reverse phenotype. Mutations in Stat6, Ppard, Pparg, and Irf4 and IRF5 depletion are involved in the maintenance and/or amplitude of activation.

3.2. Giant-cell formation and function It is well recognized that multinucleated giant-cell (MNGC) formation is the result of cells derived from the monocyte/macrophage lineage. While various names have been given to these cell types over the years (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), it is

important to note that very early experiments dealt primarily with their characterization in response to pathogens. In vitro models demonstrated that large MNGCs with more than 15 nuclei were found in response to high-virulence mycobacterium whereas lowvirulence mycobacterium consistently produced low numbers of nuclei per cell (less than 7) [134]. These studies point to the fact that monocyte/macrophage stimulation in response to pathogens formed MNGCs, which were at the time described as FBGCs. These

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‘unhappy’ macrophages seemed to fuse in response to foreign pathogens and this has been the basis over the last several decades for terming these cells as FBGCs. Today, much has been learned with respect to multinucleated giant cells. Many aspects of their behavior have been discovered including their recognition motifs, adhesion, fusion and activation molecules as well as specific intercellular and intracellular signaling pathways [135,136]. Yet a great deal of information is still lacking regarding their cellular control which is vital to further utilize macrophages as therapeutic targets for the treatment of various pathologies associated with their misbalance. Below we describe osteoclast differentiation, and later contrast their differences with MNGCs (Table 2).

3.3. Osteoclast formation and function Simply put, osteoclasts are the multinucleated counterpart to osteoblasts responsible for bone resorption. Along with osteoblasts, they play a pivotal role in bone homeostasis and bone remodeling by continuously maintaining a steady state between bone formation and bone resorption. Disruption in their activity has the potential to cause major systemic conditions such as osteoporosis characterized by low bone mass typically found in postmenopausal women [137]. Interestingly, osteoclast precursors are also derived from the bone marrow like early monocytes and macrophages that circulate through blood and bind to the surface of bone. Although the exact molecular mechanism involved in the recruitment and targeting of osteoclasts to bone surfaces remains largely unknown, it has been shown that integrin [alpha]v[beta]3 is the dominant osteoclast binding domain and one of the typical markers used to identify the osteoclast phenotype but is absent on

macrophage precursors however progressively induced by RANKL [138]. These integrins recognize a number of ECM molecules found in bone including osteopontin, fibronectin, vitronectin and fibrinogen which typically bind through an RGD peptide domain [139,140]. Following adhesion to bone, monocyte precursors and macrophage precursors absolutely require 2 main cytokines for their differentiation to osteoclasts, CSF1 and RANKL. Animals lacking CSF1 are osteopetrotic and have severe growth retardation [67,141,142]. Furthermore, mutation in CSF1 receptor has a similar phenotype [82]. While CSF1 is also required for macrophage differentiation, RANKL is typically considered the master gene for osteoclast differentiation and regulation [143]. OPG is another peptide synthesized by osteoblasts and osteocytes known to recognize RANKL and thus acts as a decoy receptor competing with RANK [75]. It has been shown that overproduction of RANKL can lead to osteoporosis, whereas osteopetrosis is caused by increased OPG production. One question that remained following these key studies was how/what factors were responsible for the fusion of multinucleated cells. Yagi et al. showed in a knockout animal model that dendritic cell-specific trans-membrane protein (DC-STAMP) was required for the fusion of both osteoclasts and MNGCs [144]. It was observed that osteoclasts found in DC-STAMP knockout animals were mononuclear, maintained their bone-resorbing activity, expressed osteoclast markers and cytoskeletal structure but were not capable of cell fusion [144]. Further experiments using retroviral introduction of DC-STAMP in mononuclear osteoclast precursors showed the re-establishment of the multinucleation found in cells derived from DC-STAMP knockout animals further confirming the

Table 2 Contributing factors and differences between osteoclasts and multi-nucleated giant cell activation, adhesion and fusion (adapted from Brodbeck and Anderson 2009). Contributing factor

Osteoclasts

Multinucleated giant cell

Adhesion substrate

Bone Dentin Osteopontin Vitronectin Fibrin(ogen) Bone sialoprotein

Implanted biomaterial

aVb3 a5b1, a3b1

aMb2, aXb2 a5b1, aVb1

Adsorbed surface proteins

Adhesion receptors CD47

Soluble fusion mediators

CCL-2 RANKL M-CSF TNF-alpha IL-1

Cell surface fusion mediator Cell fusion receptors MFR CD48

DC-STAMP

Phenotypic expression

Cathepsin K Acid CD13þ, CD14þ, CD68þ, CD56GrB-, Ki67-

MFR Mannose receptor (CD26) AvB3 RANKL E-Cadherin CD44, CD81, CD9 Connexin 43

Complement component (iC3b) Vitronectin Fibrin(ogen)

CD44 ICAM-1 CCL-2 IL-4 IL-13 INF-g IL-3 Con A PHA MMP9 DC-STAMP CD44, CD47, CD200, signal regulatory protein 1a, IL-4r, E-cadherin, mannore receptor CD13 (aminopeptidase N) Galectin-3 E-Cadherin CD44, CD81, CD9 Connexin 43 P2X7 receptor Presenilin 2 Phagocytosis (frustrated) Acid Reactive oxygen intermediates Lymphocyte co-stimulators (HLA-DR, B7-2, B7eH1, CD98) CD44 (HCAM) CD13þ, CD14þ, CD68þ, CD56GrBþ, Ki67þ

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essential role of DC-STAMP on cell fusion. Since then, forced osteoclast differentiation using CSF1, RANKL and IL-4 (a known inducer of cellecell fusion) were incapable of initiating cell fusion in DCSTAMP knockout cells in vitro [145]. Since these findings, DCSTAMP has been investigated as a therapeutic option to minimize MNGC formation both as a surface protein expressed by macrophages or as a released soluble factor. Research in this area is ongoing. 3.4. Multinucleated giant cell (MNGC) formation and function To date, most of the literature available on MNGCs involves their role around tissue/biomaterial interfaces of implanted medical devices [136]. From this point of view, numerous attempts have been made to characterize these cells as FBGCs, as part of a foreign body reaction. While these cells are not found abundantly in normal physiological tissues, they are found in great number around bone biomaterials with their roles highly left unstudied. Fig. 4 presents a summary of the events taking place between the interactions of MNGC, biomaterials, and exudate/tissue inflammation. Interestingly, MNGCs have also been seen in several tissues where the size of the foreign particle is greater than permitted for macrophage phagocytosis to occur [146]. Thereafter, macrophages have been suggested to fuse in response to this ‘larger than average’ particle size [146]; a ‘frustrated’ macrophage. In the context of biomaterial integration, it has been accepted that ‘FBGCs are generated by macrophage fusion and serve the same purpose as osteoclasts, degradation/resorption/removal of the underlying substrate’ [147]. While we do not oppose this view, it is interesting to point out that MNGCs/FBGCs are also found in response to implanted bone grafting materials and bone-implant titanium

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biomaterials. Therefore, if these cells serve the same purpose as osteoclasts in bone, why are these cells found in bone altogether? A great deal of information on MNGCs originates from implanted medical devices in soft tissues. It has been shown that beta1 and beta2 integrin receptors are the predominant binding domains during monocyte to macrophage development and that IL-4 induces cellecell fusion during FBGC formation [148]. Surface receptors during fusion include CD44, CD47, CD200, signal regulatory protein 1a, IL-4r, E-cadherin, and mannose receptor [136]. Other alpha integrin-binding partners have also been identified in expression prolife studies including [alpha]M[beta]2, [alpha]X[beta]2, [alpha]5[beta]1 more than [alpha]V[beta]1 more than [alpha]3[beta]1, and [alpha]2[beta]1 [140]. MNGCs have been shown to adhere to complement components and fibrinogen and at later time points vitronectin [149]. Other molecules/proteins have been found implicated in MNGC fusion, function and survival including STAT6, P2X7 receptor, and Connexin 43 [150e153]. IL-4 and IL-13 are two important cytokines for MNGC fusion and formation and are thought to be produced mainly by T lymphocytes [154]. Furthermore, macrophage fusion to form giant cells has been shown to also require MMP9 [155]. It has been shown that macrophages/FBGC strongly express HLA-DR, CD98, B7-2 (CD86), and B7eH1 (PD-L1), but not B7-1 (CD80) or B7eH2 (B7RP-1). Molecules expressed on osteoclasts including calcitonin receptor, tartrateresistant acid phosphatase and RANK or dendritic cells including CD1a, CD40, CD83, CD95/fas are found undetectable in MNGCs [156]. In contrast, it has been shown that fusing macrophages/ FBGCs strongly express aX integrin (CD11c), CD68, and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), whereas CD14 is completely down-regulated with IL-4-induced macrophage fusion [156]. Table 2 summarizes previous reports demonstrating differences in factors important to osteoclast and FBGCs formation and function. 4. Monocytes, macrophages, MNGCs and biomaterials

Fig. 4. Proposed foreign body giant cell formation: Inflammatory and wound healing responses to implanted medical devices, prostheses and biomaterials (adapted from Brodbeck and Anderson (2009).

To date, most of our understanding of macrophages/MNGCs around biomaterials has been driven by key studies performed outside the bone biology field [157,158]. Current strategies adapted from soft tissue integration of biomaterials have since been demonstrated as effective strategies in bone biology by mainly focusing on reducing the possibility of a foreign body reaction by reducing either protein adsorption, initial cell adhesion, inflammatory cytokine secretion and/or FBGC fusion around biomaterials [159]. Pioneering research conducted in large part by James Anderson's group revealed that hydrophilic and anionic substrate surfaces caused less macrophage adherence and increased their apoptosis and reduced foreign body giant cell formation [160]. In vitro studies have demonstrated that MNGCs produce a large array of pro-inflammatory cytokines including IL-1b, TNF-alpha, IL6, IL-8, and macrophage inflammatory protein (MIP)-1[beta] [161,162]. Interestingly, polymer biomaterial surface topography and chemistry has been shown to affect the release of these cytokines with preference for hydrophilic/neutral and hydrophilic/ anionic surfaces [161e163]. Furthermore, it has been shown that hydrophilic surfaces markedly reduced the number of adherent macrophages and MNGCs on the surface when compared to hydrophobic surfaces. These studies clearly showed that material surface/chemistry is a strong regulator of induced MNGCs on polymer surfaces for soft tissue integration [162,164]. It has been suggested that unlike osteoclasts, MNGCs adhere to markedly different synthetic surfaces [147], however while the field of MNGCs adherence and expression of integrin protein domains has been largely studied on polymers, very little information is available on a wide array of very frequently used bone biomaterials such

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as dental/orthopedic implants and bone grafting materials. In addition, it was also shown that topographical effects had an influence on TNF-alpha and VEGF secretion; characteristics of the M2 macrophage [159]. More recently, phenotypic expression of human monocyte-derived IL-4 induced FBGCs and macrophages in vitro has revealed the strong dependence on material surface properties with certain substrates being more prone to form FBGCs [165]. These key findings generated over the past 2 decades have largely been adopted to bone biomaterials without much further study. Below we summarize the current literature involving searches for macrophages, monocytes, FBGCs and MNGCs around bone grafting materials and dental/orthopedic implants. 4.1. Macrophages, MNGCs and dental/orthopedic implants The role of macrophages around bone implants has been a topic of study both in vitro and in vivo over several decades. While it became clear several years ago that cells from the monocytic lineage were one of the first to come in contact with titanium surfaces, their exact role in implant dentistry was largely left unanswered In fact, a recent in vitro systematic review relating to the topic of cells and dental implant surfaces revealed that approximately 90% of all published papers on the topic focused mainly on osteoblast behavior on a variety of implant surfaces with either varying surface topography, surface material compositions or surface hydrophilicity [2]. This finding grossly demonstrates the lack of study with immune-modulation of implant surface; i.e. the entire field of osteoimmunology. It remains interesting to note that to this day, a small percentage of implants are lost every year for completely unknown causes likely dealing with humoral immunity [166]. Recently, a prominent group in Sweden working in the field of dental implants has been largely implicated and interested in the foreign body reaction around dental implants [167,168]. While their key research topic relates primarily to how cells derived from the monocytic lineage are able to fuse and form MNGCs (also termed GFBCs by these authors), their main research focus is on how these cells are implicated in longeterm equilibrium to avoid material rejection citing their role in biomaterial failure [167e169]. However, interesting recent findings have demonstrated clearly through a variety of experimental designs that MNGCs express a large variety of markers associated with M2 macrophages in vivo [11]. These surprising findings have led to the hypothesis that these giant cells found around implanted biomaterials may in fact contribute more so to tissue integration as opposed to material rejection. Interested by these findings, our group recently established new parameters used to quantify these cell types around 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) (Fig. 5) [170e172]. Despite MNGCs being present on all tested implant surfaces, MNGCs were not associated with an inflammatory cell infiltrate or with fibrous encapsulation in osseointegration of this defect model in miniature pigs. In the paper by Chappuis et al. (2015), MNGCs were less numerous on the Ti implants (range: 3.9e5.2%) compared to the two types of ceramic implants (range: 17.6e30.3%, p < 0.0001) (Fig. 6). However, no correlation could be found between the newly formed peri-implant bone density, defined as the percentage of new bone area inside the screw threads (nBD), and the presence of MNGCs [172]. Therefore, it becomes very difficult to assess the role of these cells as almost no information has characterized these cells in vivo or investigated the release of factors implicated in the differentiation of cells leading to hard tissue formation around bone biomaterials. Below we summarize studies that have focused on the roles of cells derived from the monocytic lineage on bone implant surfaces (Table 3).

Over 10 years ago, Takabe et al. investigated the effect of commercially pure titanium substrate topography on adherent macrophage osteogenic and osteoinductive cytokine expression [173]. The J774A.1 murine macrophage was investigated for cell adhesion and TGF-b1 and BMP-2 gene expression onto polished, machined, and grit-blasted cpTi surfaces. Macrophage adhesion increased with time on all surfaces and spreading increased with increasing surface roughness (polished < machined < grit-blasted). BMP-2 expression was also further enhanced on roughened surfaces [173]. Refai et al. further showed that surface roughness increased secretion of pro-inflammatory markers including IL-1B, IL-6, and TNF-alpha, and chemokines including monocyte chemoattractant protein 1 and macrophage inflammatory protein 1alpha with and without culture containing LPS [174]. In a second study investigating the role of titanium surface topography on J774A.1 macrophage inflammatory cytokines and nitric oxide production, it was once again confirmed via real-time PCR that proinflammatory mediators were more highly expressed on gritblasted/acid etched rough surfaces than on smoother ones [175]. Ghrebi et al. later showed that macrophage shape was significantly altered on roughened surfaces activating early ERK1/2 signaling as well as vinculin and pFAK expression in macrophages [176]. In 2007, Makihira et al. was one of the first to show that titanium surface roughness accelerated RANKL-dependent differentiation of RAW264.7 macrophages into osteoclasts [177]. They found that surface roughness promoted the expression of TRAP and Cathepsin K [177]. In 2008, one of the first studies investigated the cross-talk between macrophages and osteoblasts on titanium-based particles [178]. Within their experimental design it was found that exposure of co-cultured macrophages to sub-cytotoxic doses of titanium particles did not change the osteoblastic expression of RANKL or OPG into media, however both IL6 and PGE2 levels increased to a similar extent after treatment with Ti particles [178]. The results from that study indicate that interactions of osteoblasts and macrophages respond to micro-particles of titanium and their crosstalk seems to play an important role in guiding new bone formation [178]. In 2010, an animal study confirmed that new bone formation in vivo is preceded by macrophage accumulation [179]. It was found that although roughened SLA surfaces displayed higher levels of mineralization, the analysis of immunohistochemistry demonstrated that the predominant cell type at 1 week prior to mineralization was the macrophage, whereas polished surfaces demonstrated less adhered macrophages found on the surfaces with little to no signs of mineralization suggesting for one of the first times an important role of macrophages on guiding new bone formation [179]. These results were later confirmed in a clinical study assessing the early molecular changes of osseointegration of dental implants in humans via Affymetrix gene analysis [180]. It was shown that an abundant upregulation of several differential markers of alternative activated macrophages was observed including MRC1, MSR1, MS4A4A, SLC38A6 and CCL18 [180]. In 2010, Hefti et al. compared osteoclast resorption pits on bone with titanium and zirconia surfaces [181]. Results from this study showed that osteoclasts resorb bone in similar roughness and pit sizes as titanium surfaces which may additional provide some insight into structural requirements for bone remodeling on implanted biomaterials [181]. Miller et al. investigated for the first time the in vitro effects of hydrophilic roughened titanium surfaces on blood clot formation, platelet activation and activation of the complement system [182]. They found that untreated Ti surfaces displayed thin blood clots whereas alkali treatment of roughened surfaces enhanced the ability for nucleated cells to adhere from whole blood [182]. Hamlet et al. then showed that surface hydrophilicity down-regulated key pro-inflammatory cytokines including TNF-alpha, IL-1alpha and IL-

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Fig. 5. MNGCs on dental implants made of Ti, TiZr, TAV, ZrO2, and ZrO2/Al2O3. Undecalcified ground sections (reprinted with permission from Ref. [172]).

Fig. 6. Influence of ER stress pathways in macrophage differentiation (reprinted with permission from Ref. [132])., ER stress pathways include the following: c-Jun N-terminal kinase (JNK), PPARg, scavenger receptor CD36, and SR-A1. Macrophage M1 receptors include CCR7 and CD86; M2 receptors include MR and CD163.

1B and chemokine CCL2, whereas hydrophobic rough surfaces tended to increase them [183]. In 2014, Alfari et al. further showed that hydrophilic SLA surfaces down-regulated the expression of 10 key pro-inflammatory genes namely TNF, IL-1a, IL-1B, CCL-1, -3, -19 and 20, CXCL-1 and 8 and IL-1 receptor type 1 when compared to SLA [184]. In 2012, ceramic modifications of porous titanium surfaces were investigated for their effect on macrophage activation [185]. Titanium surfaces were covered with bioactive hydroxyapatite (HA), bioglass and calcium silicate. RAW264.7 macrophage adhesion, morphology and activation were assessed. It was found that calcium silicate decreased the macrophage adherence and upregulated pro-inflammatory mediators such as TNF-alpha, IL-6 and IL12. HA decreased cell adherence with little change in mediators [185]. This study proposed for the first time that bioactive materials such as HA and BG may improve osseointegration via macrophage activation [185]. Brinkmann et al. investigated the effects of surface roughness of titanium implants on osteoclast behavior in vitro

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Table 3 Studies investigating monocytes, macrophages, osteoclasts or MNGCs on dental/orthopedic implant surfaces. Author

Year Main finding

Takebe et al. Refai et al. Tan et al. Makihira et al. Valles et al. Chehroudi et al. Hefti et al. Milleret et al. Hamlet Scislowska-Czarnecka et al. Brinkmann et al. Barth et al. Ghrebi et al. Alfarsi et al. Thalji et al.

2003 2004 2006 2007 2008 2009 2010 2011 2011 2012

Ma et al. Lu et al.

Surface roughness increased J774A.1 macrophage adhesion and release of BMP2. Surface roughness increases RAW 264.7 macrophage secretion of pro-inflammatory cytokines. LPS further increases this finding. Surface roughness increased pro-inflammatory cytokine production in J774A.1 macrophages Surface roughness increased osteoclast differentiation of RAW 264.7 macrophages cultured with RANKL. Titanium particles influence macrophage cross-talk with osteoblasts and modulate IL6 and PGE2 expression. Bone formation on rough surfaces is preceded by macrophage accumulation on rough titanium surfaces in vivo Osteoclasts resorb bone in similar shapes and sizes to titanium implant surfaces Alkaline treatment and hyperhydrophilic surfaces improve nucleated cell adhesion and blood clot thickness Hydrophilic titanium surfaces reduces pro-inflammatory cytokine gene expression in RAW 264.7 macrophages Ceramic modifications to porous titanium can modulate gene expression of RAW 264.7 macrophages.

2012 2012 2013 2014 2014

Surface roughness increases osteoclast differentiation and actin rings on titanium surfaces Surface roughness promotes macrophage differentiation towards the M2 phenotype responsible for enhanced wound repair Surface topography modulates cell spreading and increases vinculin distribution and ERK1/2 signaling in macrophages. Hydrophilic SLA significantly downregulated 10 pro-inflammatory cytokines in macrophages. Clinical study demonstrating that early osseointegration of human dental implants involve an abundance of upregulated activated macrophage genes 2014 Anodization and UV irradiation can be used to generate hydrophilic titanium surfaces able to modulate pro M2 polarization of macrophages both in vitro and in vivo 2015 Nano-micron rough titanium surfaces may also further reduce a pro-inflammatory macrophage phenotype

Table 4 Studies investigating monocytes, macrophages, osteoclasts or MNGCs on bone grafting materials. Author

Year

Main finding

Yamada et al. Benahmed et al. Benahmed et al. Silva et al. Rice et al. Curran et al. Xia et al. Fellah et al. Fellah et al. Egli et al. Gamblin et al. Davison et al. Davison et al. Davison et al. Kweon et al.

1996 1996

Osteoclasts capable of resorbing BCP bone grafts First in vitro model testing monocyte/macrophage degradation of bone grafts including BCP and HA. It was found that macrophages could degrade BCP faster than HA. LPS increases biomaterial degradation by human monocytes/macrophages in vitro

1997 2003 2003 2005 2006 2007 2010 2011 2014 2014a 2014b 2014c 2014

Macrophages seeded on BCP release higher levels of calcium when compared to neighboring cells. The percentage of TCP within the BCP granules was a governing factor in macrophage cellular response. The percentage content of TCP was a more significant factor than granule size on macrophage pro-inflammatory release. Established a model to investigate cell-mediated cement degradation in vitro The BCP micro particles <20 micro m initiated an inflammatory response which might play an important role in osteogenesis. The smallest microparticles decreased the viability of macrophages and enhanced the secretion of pro-inflammatory cytokines (IL-6 and TNF-alpha) Thermal treatment of calcium phosphates affect osteoclast activity in vitro BCP bone grafts with MSCs increased osteoblast gene expression and macrophages at 2 and 4 weeks, osteoclastogenesis at 4 and 8 weeks. Liposomal clodronate inhibition of macrophage/osteoclastogenesis inhibits osteoinduction in beta-TCP scaffolds Osteoclast activation occurs more favorably on submicron topographies On submicrostructured TCPs, osteoclasts survived, fused, differentiated, and extensively resorbed TCP whereas FBGC had no ability to resorb TCP. Inhibition of foreign body giant cell formation on silk fibroin scaffolds can be achieved with 4-hexylresorcinol through suppression of diacylglycerol kinase delta gene expression Chen et al. 2014 B-TCP extracts were able to switch macrophage phenotype towards the M2 extreme and increase the expression of BMP2 Chen et al. 2014b Magnesium is able to improve the osteoimmunomodulatory properties of macrophages which enhances osteogenic differentiation and inhibits osteoclastogenesis Chen et al. 2015 Cobalt switched mcrophage phenotype towards the M1 extreme, releasing pro-inflammatory cytokines and bone catabolic factors Davison et al. 2015 Material parameters - namely, surface microstructure, macrostructure, and surface chemistry e are critical in promoting osteoclastogenesis and triggering ectopic bone formation Shiwaku et al. 2015 Osteoclast differentiation is partially impaired by increased HA content, but not by the presence of micropores within BCP scaffolds, thus favouring osteoblast crosstalk

[186]. It was found that osteoclasts show similar characteristics on rough titanium surfaces when compared to bone, whereas reduced activity was observed on smooth titanium surfaces [186]. As the field of osteoimmunology was further advanced, Barth et al. showed that surface roughness promoted an M2 macrophage phenotype suggesting that these cells may additionally contribute to enhanced wound healing [187]. Interestingly, Ma et al. demonstrated in an in vivo study that anodization at 5 and 20 V as well as UV irradiation used to generate hydrophilic titanium surfaces could also be utilized as strategies to increase M2 macrophage polarization [188]. Furthermore, Lu et al. further showed that a reduction in immune response was observed on nano- and submicron rough titanium demonstrating for the first time that nanofeatures on implant surfaces may also be controlled to reduce proinflammatory mediators and induce an M2 macrophage

phenotype onto bone biomaterials [189].

4.2. Macrophages, MNGCs and bone grafting materials The role of macrophages around bone grafting materials has been studied much earlier than on titanium implants and in recent years has gained tremendous momentum with the advancements made in the field of osteoimmunology (Table 4). Despite recent trends studying the role of monocyte-derived cells in vitro, the difficulty in culturing cells on 3-dimensional bone grafts with varying particle sizes significantly increases the degree of difficulty to perform such in vitro research. Therefore, large gaps can be found in the literature with little investigation in the early 2000s studying the role of macrophages and osteoclasts on these bone grafting materials. In recent years, the paradigm has shifted from

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the osteopromotive potential of bone substitute materials towards their immunomodulatory behavior thus emphasizing the importance of initial immune cell responses to biomaterial interactions and subsequent effects of factors released by immune cells on osteoblastic cells. It has previously been reported that macrophages are the major effector cells in immune reaction to implants and that they are indispensable for osteogenesis and that their heterogeneity and plasticity render macrophages a primer target for immune system modulation [190]. In one of the first studies, Yamada et al. studied the effects of osteoclast resorption on biphasic calcium phosphate (BCP) ceramic bone grafts and observed after 4 days by SEM that degraded calcium phosphate crystals inside the resorption lacunae appeared to have been dissolved by acids [191]. Furthermore, Behnamed et al. investigated the influence of monocyte and macrophage phagocytosis of calcium phosphate particles and their possible involvement in their degradation due to their sensitivity to secreted cytokines [192]. They tested the behavior of human monocytes placed on the surface of HA and BCP tablets in the presence of vitamin D3 and INF-gamma. After short-term culture (6 days), morphological events were observed by histological and SEM analysis characterizing resorption lacunae demonstrating for the first time the ability for cells to degrade bone grafts of various compositions (HA versus BCP) [192]. In 1997, these same authors found that addition of LPS into their culture system increased biomaterial degradation [193]. In 2003, Silva et al. investigated the effects of BCP on macrophage function in vitro [194]. They described an in vitro phenomenon regarding the effect of surface reactivity of BCP granules on human macrophage locomotion, secretion and allowed further macrophage adhesion [194]. Furthermore in 2003, Rice et al. investigated various bone grafts ranging in tricalcium phosphate (TCP) and HA composition from 20, 50, 80, and 100% TCP [195]. Grafts were also classified into two distinct size ranges, small 2e4 mm in diameter and large 4e6 mm in diameter, and their potential as bone grafting materials was assessed using biocompatibility cell culture systems of primary-derived peripheral human blood monocytes and human osteoblasts isolated from bone. It was found that the higher content TCP materials, 80% and 100% TCP, had a detrimental effect on viable cell adhesion after day 1 whereas low % of TCP granules decreased a pro-inflammatory response [195]. Furthermore in 2005, Curran et al. investigated the inflammatory potential of BCP granules in an osteoblast/macrophage co-culture system with different HA/TCP ratios [196]. Again it was found that higher content of beta-TCP materials, (80% and 100% TCP) did not support viable cell adhesion after 1 day, whereas lower content TCP materials, (20% and 50% TCP) granules supported viable cell adhesion throughout the observation period [196]. In 2006, Xia et al. investigated the in vitro biodegradation of 3 brushite calcium phosphate cements by RAW264.7 macrophages [197]. The results demonstrated that RAW264.7 cells formed multinucleated TRAP-positive osteoclast-like cells capable of ruffled border formation and lacunar resorption. This first study of its kind set up a useful model to investigate the cell-mediated brushite calcium phosphate cement degradation in vitro by macrophages and osteoclasts [197]. Fellah et al. investigated the inflammatory reaction in rat muscle after implantation of BCP micro particles [198]. Three fractions of BCP micro particles <20, 40e80, and 80e200 mm were sieved and the micro particles were carefully characterized by using X-ray diffraction (XRD), SEM and laser scattering. A fibrous tissue encapsulation of the BCP micro particles was observed for all 3 groups of micro particles. The comparison of the cellular response indicated that the total number of cells was significantly higher for BCP <20 mm than for 40e80 and 80e200 mm. The number of macrophages was relatively higher for the smallest than for the

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intermediate and largest fractions, whereas the relative percentage of giant cells was higher for the intermediate and largest size of particles [198]. These authors later performed a second study investigating the effect of micro particles on macrophage viability and release of pro-inflammatory factors [199]. It was found that the smallest micro particles decreased the viability of macrophages and enhanced the secretion of pro-inflammatory cytokines (IL-6 and TNF-alpha) by macrophages [199]. Based on these observations, it may be concluded that particle size seems to greatly influence both macrophage activation as well as their fusion to MNGCs. Egli et al. demonstrated the effect of thermal treatments of calcium phosphate biomaterials to fine tune the physico-chemical properties on osteoclast resorption [200]. In that study, investigation of alpha-TCP, beta-TCP and HA demonstrated that a simple thermal treatment at temperatures of 450e600 Celcius favored osteoclast behavior [200]. In 2014, an interesting study sought to investigate the role of macrophages and osteoclasts in an ectopic bone model [201]. It was found that bone grafts fabricated from BCP implanted with human mesenchymal stem cells (hMSCs) demonstrated more signs of ectopic bone formation by increasing macrophage recruitment at 2 and 4 weeks, osteoclastogenesis and osteogenesis at 4 and 8 weeks [201]. Implantation of bone grafts and hMSCs with an anti-RANKL treatment significantly impaired bone formation thus confirming the necessity of monocyte-derived cells to induce new bone formation in this model [201]. In a similar study, Davison et al. used liposomal clodronate inhibition of macrophage/osteoclast progenitors and demonstrated that without this cell source, osteoinductive beta-TCP bone grafts were no longer capable of forming ectopic bone formation [3]. Further work by this group showed that osteoclast activation occurs more favorably on submicron topographies which thereafter affects ectopic bone formation. These authors proposed that ectopic bone formation in intramuscular sites is controlled by monocyte-derived cells [202]. Later in 2014, Davison et al. differentiated monocyte-derived cells into either osteoclasts or FBGCs and thereafter seeded the cells on various bone grafting materials [203]. It was found that by changing the scale of surface architecture of TCP, cellular resorption could be influenced [203]. On submicrostructured TCPs, osteoclasts survived, fused, differentiated, and extensively resorbed the substrate; however, on microstructured TCP, osteoclast survival, TRAP activation, and fusion were significantly decreased. Interestingly, it was found that FBGC could not resorb either TCP material, suggesting that osteoclast-specific machinery is necessary to resorb TCP [203]. In 2015, Davison et al. hypothesized that surface structural dimensions of 1 mm may be responsible for triggering osteoinduction and osteoclast formation irrespective of macrostructure (e.g., concavities, interconnected macropores, interparticle space) or surface chemistry [204]. Their results indicate that of the material parameters tested - namely, surface microstructure, macrostructure, and surface chemistry e microstructural dimensions are critical in promoting osteoclastogenesis and triggering ectopic bone formation [204]. Shiwaku et al. showed that osteoclast differentiation is partially impaired by increased HA content, but not by the presence of micropores within BCP scaffolds, thus favoring osteoblast crosstalk [205]. The combination of the above mentioned studies have provided rational that 1) surface micro and nanotopography of bone grafting materials are highly responsible for dictating osteoclast-osteoblast crosstalk affecting osteoinduction; 2) Surface material composition also affects osteoinduction and 3) FBGCs are not responsible for material resorption as only osteoclasts are capable of resorbing bone grafting particles at least in vitro. Future research investigating the role of macrophages in the above-mentioned scenarios is ongoing. Kweon et al. performed an interesting study investigating the role of FBGC formation through a knock down system [206].

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Inhibition of foreign body giant cell formation could be achieved by 4-hexylresorcinol (4HR) through suppression of diacylglycerol kinase delta gene expression [206]. Thereafter, silk fibroin scaffolds were grafted into bone defects with 4HR and displayed a significant reduction of granuloma formation and increases in the extent of new bone formation in a rabbit calvarial defect model [206]. Also in 2014, Chen et al. investigated the osteogenic differentiation of bone marrow MSCs by beta-TCP stimulating macrophages via BMP2 signaling pathway [190]. In this study, they used beta-TCP as a model biomaterial to investigate the role of macrophages on biomaterial-stimulated osteogenesis. The extracts from beta-TCP were able to direct macrophage phenotype towards the M2 extreme, which was related to activation of calcium-sensing receptor (CaSR) pathway. It was also found that these macrophages significantly upregulated BMP2 following stimulation with betaTCP, indicating that macrophages may participate in osteogenesis [190]. Thereafter, this same group performed 2 studies demonstrating that the osteoimmunomodulatory properties of magnesium scaffolds significantly enhanced osteogenic differentiation of BMSCs in vitro, whereas osteoclastogenesis was inhibited [207]. In a very recent article, Chen et al. demonstrated that cobalt incorporation into beta-TCP bone grafts switched the macrophage phenotype towards the M1 extreme, releasing pro-inflammatory cytokines and bone catabolic factors in vitro [208]. Therefore, these recent studies also point to the ability for incorporation of various trace elements such as cobalt or magnesium to affect the osteoimmunological behavior of bone grafts. 5. Macrophages and atherosclerosis One of the main features in the development of atherosclerosis is the critical role and involvement of macrophages. It has been reported that atherosclerotic plaque contains high levels of IFNgamma, a T-helper1 cytokine that is a known inducer of the classically activated M1 macrophage (1e3). Interestingly, tissue macrophages found in arteries are known to induce ectopic bone formation in and around vascular tissues, an area where bone should otherwise not form [209]. Two key players known to regulate calcification in vascular tissues are TNF-alpha [209,210] and oncostatin M [210]. In combination with these findings, it has also been shown that macrophage depletion reduces osteophyte formation in osteoarthritic models [211e213] and macrophages have been key players in various other bone loss disorders [214,215]. The combination of these findings have strongly suggested that macrophages play critical roles in bone formation even long before key basic research experiments pointed to their vast roles using transgenic knockout mouse models. In atherosclerosis, an accumulation of LY6Chi monocytes has been the characteristic cell type known to affect both human and experimental animal models [216,217]. Monocytes have been shown to differentiate into macrophages in the artery intima (the most internal layer) and ingest modified lipoproteins via scavenger receptors and secrete inflammatory mediators. Over time, these macrophages give rise to lipid-rich macrophages, which have been given the name ‘foam cells’, and thereby become the key contributors of lipid core buildup followed by subsequent ectopic bone formation [218]. Two key findings recently changed our understanding of tissue macrophages in atherosclerosis. First, it was originally thought that foam-cells were continuously formed from the contributions from blood-bound monocytes requiring continual recruitment. However, it was recently shown that these ‘foam-cells’ are also able to selfrenew by local proliferation [219]. Secondly, it was originally thought that all macrophages involved in atherosclerotic plaque were classical M1 phenotype macrophages. In 2012, Oh et al.

demonstrated that alternatively it was stimulated M2 macrophages that lead to an increase in foam cell formation inducing scavenger receptor CD36 and SR-A1 expression [132]. The formation of M2 macrophages was primarily activated by endoplasmic reticulum (ER) stress (Fig. 5). Interestingly, these authors proposed that suppression of ER stress can shift M2 macrophages towards an M1 phenotype and subsequently suppress foam cell formation, thus a potential therapeutic option for resolution of atherosclerosis [132]. Thus, it demonstrates the extreme plasticity of these cell types and also how therapeutic options are greatly altered depending on the region. In atherosclerotic plaque, it would be of therapeutic benefit to reduce M2 (ectopic bone forming multinucleated giant ‘foamcells’) into more of an M1 phenotype. In contrast, implanted bone biomaterials would greatly benefit from M2 macrophage activation and their potential to form ectopic bone formation. The research outlined by the field of atherosclerosis suggests that the potential activation of macrophages via ER stress might be a potential therapeutic option for bone biomaterials. This hypothesis, however, requires a great deal of further investigation. 6. Future research outlooks In summary, this review article summarized the currently available literature on OsteoMacs, their fusion to MNGCs and their role in bone biomaterial integration. It has been previously shown that depletion of macrophages from primary calvarial osteoblast cultures led to a 23-fold decrease in osteogenic differentiation and mineralization [6,41]. Furthermore, depletion of OsteoMacs from knockout animals led to markedly reduced endochondral and intramembranous bone formation [6,41,72,73], and knockout systems around bone grafting materials completely abolished their osteoinductive activity [3]. Taken together, these findings strongly confirm the essential roles of OsteoMacs in normal bone development, bone remodeling and bone formation. Despite this, there exists a great lack of information surrounding their important roles around bone biomaterials. To date, most of the literature has either focused on osteoblast behavior on various bone biomaterials or on FBGCs around biomaterials implanted in soft tissues. Therefore, there remains a great deal of missing knowledge required to further improve bone biomaterials in the future. Furthermore, additional studies characterizing the differences between mouse and human monocytes/macrophages is pivotal in order to implement therapeutic strategies aimed at manipulating these cells during biomaterial tissue integration. Another area of research that might be valuable to investigators is to determine the differences between MNGCs from varying tissues. How different are MNGCs observed around biomaterial implants fabricated from PLA scaffolds from MNGCs observed around bone grafting materials and titanium implant surfaces? Do they differ substantially from ‘foam-cells’ derived in calcified tissues? Future investigation spanning several fields of research would be greatly beneficial to further improve our understanding of these cell types. Some have defined foreign body reactions as “the presence of macrophages and foreign body giant cells on the surface of the biomaterial, which is an end-stage event in the tissue response continuum but remains for the duration of the implant” [220]. Surprisingly, very little is known about the turnover of macrophages and MNGCs at the implant/biomaterial interface. This question is even more intriguing around bone biomaterials where recruitment of cells to bone-biomaterial surfaces is greatly limited due to bone encapsulation. Data from our laboratory thus far has indicated that these MNGCs remain on the surfaces of bone grafting materials even 10 years after implantation in a completely stable and healthy environment.

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Another aspect that remains largely untouched is the effect of multiple biomaterials in small dimensional bone areas. For example, tooth loss in dentistry is commonly replaced by dental implants fabricated from titanium with the great majority of these procedures requiring contour augmentation procedures utilizing bone grafts [221]. In such cases, the facial wall thickness of bone in normal tissues has been estimated less than 1 mm in the majority of patients [222]. During routine augmentation procedures following tooth extraction, a titanium implant is placed and routinely augmented with a sandwich approach combining 2 bone grafts including an autograft followed by a second bone grafting material. Thereafter, a barrier membrane commonly fabricated from porcine derived collagen membrane is utilized to prevent soft tissue infiltration into the slowly growing bony tissue [221]. Therefore, within this 1 mm region, typical cells are in contact with 3 biomaterials including 1) a titanium dental implant, 2) a bone grafting material from both autogenous source as well as either an allograft or xenograft, and 3) a collagen barrier membrane [221]. It is unknown at present what the influence of having so many different biomaterials might have on such a confined limited space. It may be that certain of these biomaterials favor macrophage homeostasis and tissue integration whereas other biomaterials might cause an inflammatory reaction. If this is the case, it is completely unknown how macrophages situated in such a confined space interact with one another in response to the various signaling molecules secreted in response to the various biomaterials. This area of research is one that is specifically highly relevant to the dental field where a combination of biomaterials, barrier membranes, bone grafts, growth factors and titanium implants are routinely utilized. Furthermore, although the effects of macrophage polarization has thus far been studied individually on various biomaterials such as metals, bioceramics and biopolymers, rarely have these materials been compared directly in well-conducted studies. Instead most studies to date have focused on individual classes of biomaterials making it difficult to assess which class of biomaterial is better suited as an osteo-compatible material versus another. Therefore, the effects of various classes of biomaterials on OsteoMac polarization in relation to their different physical and chemical properties is necessary to further pursue osteo-immunological materials capable of minimizing a foreign body reaction and material rejection and at the same time enhancing bone regeneration. While a recent article has compared MNGC formation on zirconia, alumina-toughened zirconia and titanium implant surfaces (Chappuis et al., 2015), very few studies have compared this highly relevant topic in bone grafting material research where a much greater number of biomaterials exists requiring much further investigation. The effect of various cell types such as MNGCs in bone homeostasis in response to biomaterials is another aspect that is highly relevant and should therefore be studied in the future. Perhaps the best evidence that MNGCs contribute to bone homeostasis came from a recent study conducted by Katsuyama et al., in 2015 [223]. They show specifically that MNGCs do not resorb bone but rather express M2 macrophage-like wound healing and inflammation-terminating molecules [223]. They report critical findings that strongly suggest that implant failure due to bone loss likely results from the activity of osteoclasts and not MNGCs and that MNGCs are unable to resorb bone but rather express wound healing and inflammation terminating molecules such as Ym1 and Alox15 [223,224]. Nevertheless, it must also be taken into consideration that other studies demonstrated that revisited joint replacements extracted following failure consistently found FBGCs predominantly expressing inflammatory M1 factors [146,225e227]. Therefore, it remains a valuable therapeutic option

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