OsteoMacs: Key players around bone biomaterials

Full text

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
article info
Article history:
Received 1 October 2015
Received in revised form
12 December 2015
Accepted 15 December 2015
Available online 20 December 2015
Keywords:
Macrophage
Bone regeneration
Osteoimmunology
Biomaterial integration
Tissue response
Multi-nucleated giant cells
OsteoMacs
Foreign body cells
abstract
Osteal macrophages (OsteoMacs) are a special subtype of macrophage residing in bony tissues. Inter-
esting 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/macro-
phage lineage are one of the first cell types in contact with implanted biomaterials. Here they demon-
strate 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 regen-
eration 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 develop-
ment 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 inte-
gration including dental/orthopedic implants and bone grafting materials.
©2015 Elsevier Ltd. All rights reserved.
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
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 facili-
tating 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
*Corresponding author. University of Bern, Head of Oral Cell Biology,
Switzerland.
** Corresponding author. University of Bern, Head of Oral Histology, Switzerland.
E-mail addresses: richard.miron@zmk.unibe.ch (R.J. Miron), dieter.bosshardt@
zmk.unibe.ch (D.D. Bosshardt).
Contents lists available at ScienceDirect
Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
http://dx.doi.org/10.1016/j.biomaterials.2015.12.017
0142-9612/©2015 Elsevier Ltd. All rights reserved.
Biomaterials 82 (2016) 1e19

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 cyto-
kines 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 cyto-
kines and growth factors [11]. Thus, the differentiation of mono-
cytes 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 special-
ized 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 macro-
phage and MNGCs function especially as it relates to bone bio-
materials. 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 po-
tential for these cells to differentiate into a variety of cell-types
including M1 and M2 macrophages, MNGCs, FBGCs and osteo-
clasts. 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 cell-
type contributing to calcification in arteries. 5) Demonstrate evi-
dence from animal and human histological samples from our
research center that MNGCs are routinely found around bone bio-
materials 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 con-
tained 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 CD68þcell-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 macro-
phages 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).
R.J. Miron, D.D. Bosshardt / Biomaterials 82 (2016) 1e192

lineage plasticity and crosstalk between osteoblasts and hemato-
poietic 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 depo-
sition 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 cul-
tures 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 osteo-
clasts 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 pheno-
types. In vitro research has demonstrated that bone marrow-
derived macrophages [38] and rheumatoid synovium-derived
macrophages [39] can be differentiated into osteoclasts confirm-
ing that mature macrophages can at least in vitro, serve as multi-
nucleated 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. Never-
theless, 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 cyto-
kines 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 demon-
strates their ability to regulate cell fusion of monocytic cells and
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 macrophage-
specific genes (csf1r, CD14) are induced in primary mouse chon-
drocyte 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 trans-
forming growth factor
b
(TGF-
b
)[53], osteopontin [54], 1,25-
dihydroxy-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 microenvi-
ronment. 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 osteo-
blasts [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 canopy-
like 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 Fas-
induced apoptosis (Mafia) transgenic mouse model, which can
induce macrophage depletion via synthetic ligand treatment [59].
In this system, OsteoMac canopy architecture was disrupted lead-
ing to a complete loss of mature osteoblasts and bone modeling at
the bone interface [59].
It was originally proposed that during bone remodeling, osteo-
clasts provide a ‘coupling signal’to promote and coordinate oste-
oblast 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 pro-
posed that OsteoMacs may be the cells responsible for coupling-
like 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
R.J. Miron, D.D. Bosshardt / Biomaterials 82 (2016) 1e19 3

produce TGF-
b
[53] and ephrin B2 [64,65] opening the possibility
that OsteoMacs are also capable of fulfilling such roles. Neverthe-
less, 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 pro-
posed models show that OsteoMacs have as function to both survey
for alteration in the local environment as well as guide bone for-
mation 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 sub-
sequently provide ongoing anabolic signals to the underlying os-
teoblasts [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 under-
lying 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 bal-
ance between bone-resorbing osteoclasts and bone-forming oste-
oblasts [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 postu-
lated 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 dis-
rupted osteoblast function. Furthermore, using 2 separate animal
models to deplete OsteoMacs (Mafia transgenic mice or adminis-
tration of clodronate-loaded liposomes) it was found that 1) there
was a loss of endosteal osteoblasts, 2) there were marked re-
ductions of HSC trophic cytokines at the endosteum and 3) HSC
mobilization into the blood occurred. Furthermore, RT-PCR ana-
lyses on the endosteal RNA from treated Mafia mice revealed a 35-
fold 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 ossifi-
cation [48]. Furthermore, a separate group found that depletion of
macrophages using Mafia mice led to early skeletal growth retar-
dation and progressive osteoporosis (25% reduction in bone min-
eral 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 mac-
rophages 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 osteo-
clasts, since they are derived from the same precursor cells [74].Itis
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 cur-
rent in vivo models could benefit from future refining as consid-
erable cellular plasticity is found between these 2 cell types [38].
Further specific investigation on the role of OsteoMacs versus os-
teoclasts 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 pop-
ulations, MNGCs and osteoclasts.
While the role of monocyte-derived macrophages during
pathological tissue inflammation has recently been comprehen-
sively reviewed [79], recent research has demonstrated that most
R.J. Miron, D.D. Bosshardt / Biomaterials 82 (2016) 1e194

monocytes do not substantially contribute to macrophage pop-
ulations 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 mono-
cytic 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 CD14
þ
mono-
cytes (which can be further subdivided into distinct populations of
CD14
þ
CD16
þ
and CD14
þ
CD16

monocytes) and CD14
low
CD16
þ
monocytes [84]. Cell differences in the monocyte lineages have
been observed between species and most notably between humans
and mice. In mice, LY6C
low
cells are the equivalent of human
CD14
low
CD16
þ
and may therefore be terminally differentiated to-
wards resident macrophages [85,86]. By contrast, mice LY6C
hi
monocytes are the equivalent of CD14
þ
monocytes in humans
representing the classical monocytes recruited to sites of inflam-
mation [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 find-
ings. 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 macro-
phages 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 dif-
ferentiation towards macrophages that drive innate and adaptive
immune responses [19]. Macrophages and bone resorbing osteo-
clasts are closely related however can be distinguished by the
presence of additional nuclei in osteoclasts as well as morpholog-
ical 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 micro-
environment [81,94,95]. The most widely used antibody to iden-
tify 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-1
b
[42,45] all contributing to tissue
inflammation and osteoclastogenesis. M2 macrophages typically
produce TGF-
b
and arginase, both factors implicated in tissue-
repair 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 to-
wards the M1 phenotype can best be produced with IFN-
g
þþLPS
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 up-
regulation 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 þIl-13 acting through IL-4R
a
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 IL-
10 production and decreasing IL-12 largely contributing to anti-
inflammatory properties [107,108]. Cell culture with IL-10 or glu-
cocorticoids produces the M2c phenotype characterized by high IL-
10 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 macro-
phage 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 antigen-
2, 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 differ-
ences between human and mice macrophages exist complicating
the overall progression of the field of osteoimmunology as dis-
cussed 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].
R.J. Miron, D.D. Bosshardt / Biomaterials 82 (2016) 1e19 5

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 dif-
ferences 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 differ-
ences 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 differ-
ences 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 pro-
mote 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].
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.
M1 macrophage M2 macrophage
Activator IFN-gamma, TNF-alpha, LPS IL-4, IL-13
Proinflammatory cytokines IL-1B, TNF-alpha, IL6, IL12 Low
iNOS High (in rodents only) Low
Anti-inflammatory cytokines Low TGF-B high, IL-10 Low
CD206 Low High
Dectin-1 Low High
Ym1 Low High
Phagocytosis/Endocytosis High Decreased phagocytosis of implanted particles
Matrix proteins MMP9 FN, TGFB1, MMP1, MMP12, TG, F13A1
Markers
Human: CD64, IDO, SOCS1, CXCL10 MRC1, TGM2, CD23, CCL22
Mouse: CXCL9, CXCL10, CXCL11, NOS2 Mrc1, tgm2, FizzI, Ym1/2, Arg1
Transcription factors
Human: pSTAT1, IRF5 IRF4, SOCS1*, GAT3* SOCS3
Mouse: pSTAT1, pSTAT6-ve, Socs1 pSTAT6, pSTAT1-, Soc2
Cytokines
Human: TNF, IL6, IL1b, IL12A, IL12b, IL23A IL-10
Mouse: TNF, IL-6, IL-27, Tnf23a IL-10, IL-6
Chemokines
Human: CXCL10, IL8, CCL5, CXCL9, CCL4, CCL13, CCL17, CCL18,
Mouse: CXCL11, CCL18-ve CCL17 CCL24, CXCL13, CCL1, CCL22, CCL20
R.J. Miron, D.D. Bosshardt / Biomaterials 82 (2016) 1e196

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/macro-
phage 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 low-
virulence 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
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 CD14
þ
monocytes are used as the exemplars for marker evaluation and standardized activation conditions. Macrophagescan also
be generated with GM-CSF, where a CD11c
þ
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 tissue-
infiltrating 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.
R.J. Miron, D.D. Bosshardt / Biomaterials 82 (2016) 1e19 7

‘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 forma-
tion and bone resorption. Disruption in their activity has the po-
tential to cause major systemic conditions such as osteoporosis
characterized by low bone mass typically found in post-
menopausal 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 fibrin-
ogen which typically bind through an RGD peptide domain
[139,140].
Following adhesion to bone, monocyte precursors and macro-
phage 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 dif-
ferentiation, 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 intro-
duction 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
Implanted biomaterial
Adsorbed surface proteins Osteopontin
Vitronectin
Fibrin(ogen)
Bone sialoprotein
Complement component (iC3b)
Vitronectin
Fibrin(ogen)
Adhesion receptors
a
V
b
3
a
M
b
2
,
a
X
b
2
CD47
a
5
b
1,
a
3
b
1
a
5
b
1
,
a
V
b
1
CD44
ICAM-1
Soluble fusion mediators CCL-2
RANKL
M-CSF
TNF-alpha
IL-1
CCL-2
IL-4
IL-13
INF-
g
IL-3
Con A
PHA
MMP9
Cell surface fusion mediator DC-STAMP DC-STAMP
Cell fusion receptors CD44, CD47, CD200, signal regulatory protein 1a, IL-4r, E-cadherin, mannore receptor
MFR MFR
CD48 Mannose receptor (CD26)
AvB3
RANKL
E-Cadherin
CD44, CD81, CD9
Connexin 43
CD13 (aminopeptidase N)
Galectin-3
E-Cadherin
CD44, CD81, CD9
Connexin 43
P2X7 receptor
Presenilin 2
Phenotypic expression Cathepsin K
Acid
CD13þ, CD14þ, CD68þ, CD56-
GrB-, Ki67-
Phagocytosis (frustrated)
Acid
Reactive oxygen intermediates
Lymphocyte co-stimulators
(HLA-DR, B7-2, B7eH1, CD98) CD44 (HCAM)
CD13þ, CD14þ, CD68þ, CD56-
GrBþ, Ki67þ
R.J. Miron, D.D. Bosshardt / Biomaterials 82 (2016) 1e198

essential role of DC-STAMP on cell fusion. Since then, forced oste-
oclast differentiation using CSF1, RANKL and IL-4 (a known inducer
of cellecell fusion) were incapable of initiating cell fusion in DC-
STAMP knockout cells in vitro [145]. Since these findings, DC-
STAMP has been investigated as a therapeutic option to minimize
MNGC formation both as a surface protein expressed by macro-
phages 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 inflamma-
tion. 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
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 implan-
ted 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 in-
duces cellecell fusion during FBGC formation [148]. Surface re-
ceptors 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 mac-
rophages/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, tartrate-
resistant 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
a
X integrin (CD11c), CD68, and dendritic
cell-specific intercellular adhesion molecule-3-grabbing non-
integrin (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
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, inflam-
matory 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-1
b
, TNF-alpha, IL-
6, 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 cyto-
kines 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 hy-
drophobic 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 avail-
able on a wide array of very frequently used bone biomaterials such
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).
R.J. Miron, D.D. Bosshardt / Biomaterials 82 (2016) 1e19 9

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, inter-
esting 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 sur-
prising 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 inflamma-
tory 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 be-
comes 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 sum-
marize 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-
b
1 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 sur-
faces [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 chemo-
attractant 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 pro-
duction, it was once again confirmed via real-time PCR that pro-
inflammatory mediators were more highly expressed on grit-
blasted/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 mac-
rophages respond to micro-particles of titanium and their cross-
talk seems to play an important role in guiding new bone forma-
tion [178]. In 2010, an animal study confirmed that new bone for-
mation 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-
R.J. Miron, D.D. Bosshardt / Biomaterials 82 (2016) 1e1910

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]. Tita-
nium surfaces were covered with bioactive hydroxyapatite (HA),
bioglass and calcium silicate. RAW264.7 macrophage adhesion,
morphology and activation were assessed. It was found that cal-
cium silicate decreased the macrophage adherence and upregu-
lated pro-inflammatory mediators such as TNF-alpha, IL-6 and IL-
12. 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
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-ter-
minal kinase (JNK), PPAR
g
, scavenger receptor CD36, and SR-A1. Macrophage M1 re-
ceptors include CCR7 and CD86; M2 receptors include MR and CD163.
R.J. Miron, D.D. Bosshardt / Biomaterials 82 (2016) 1e19 11

[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. demon-
strated 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 polariza-
tion [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 pro-
inflammatory 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
Table 3
Studies investigating monocytes, macrophages, osteoclasts or MNGCs on dental/orthopedic implant surfaces.
Author Year Main finding
Takebe et al. 2003 Surface roughness increased J774A.1 macrophage adhesion and release of BMP2.
Refai et al. 2004 Surface roughness increases RAW 264.7 macrophage secretion of pro-inflammatory cytokines. LPS further increases this finding.
Tan et al. 2006 Surface roughness increased pro-inflammatory cytokine production in J774A.1 macrophages
Makihira et al. 2007 Surface roughness increased osteoclast differentiation of RAW 264.7 macrophages cultured with RANKL.
Valles et al. 2008 Titanium particles influence macrophage cross-talk with osteoblasts and modulate IL6 and PGE2 expression.
Chehroudi et al. 2009 Bone formation on rough surfaces is preceded by macrophage accumulation on rough titanium surfaces in vivo
Hefti et al. 2010 Osteoclasts resorb bone in similar shapes and sizes to titanium implant surfaces
Milleret et al. 2011 Alkaline treatment and hyperhydrophilic surfaces improve nucleated cell adhesion and blood clot thickness
Hamlet 2011 Hydrophilic titanium surfaces reduces pro-inflammatory cytokine gene expression in RAW 264.7 macrophages
Scislowska-Czarnecka
et al.
2012 Ceramic modifications to porous titanium can modulate gene expression of RAW 264.7 macrophages.
Brinkmann et al. 2012 Surface roughness increases osteoclast differentiation and actin rings on titanium surfaces
Barth et al. 2012 Surface roughness promotes macrophage differentiation towards the M2 phenotype responsible for enhanced wound repair
Ghrebi et al. 2013 Surface topography modulates cell spreading and increases vinculin distribution and ERK1/2 signaling in macrophages.
Alfarsi et al. 2014 Hydrophilic SLA significantly downregulated 10 pro-inflammatory cytokines in macrophages.
Thalji et al. 2014 Clinical study demonstrating that early osseointegration of human dental implants involve an abundance of upregulated activated
macrophage genes
Ma et al. 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
Lu et al. 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. 1996 Osteoclasts capable of resorbing BCP bone grafts
Benahmed
et al.
1996 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.
Benahmed
et al.
1997 LPS increases biomaterial degradation by human monocytes/macrophages in vitro
Silva et al. 2003 Macrophages seeded on BCP release higher levels of calcium when compared to neighboring cells.
Rice et al. 2003 The percentage of TCP within the BCP granules was a governing factor in macrophage cellular response.
Curran et al. 2005 The percentage content of TCP was a more significant factor than granule size on macrophage pro-inflammatory release.
Xia et al. 2006 Established a model to investigate cell-mediated cement degradation in vitro
Fellah et al. 2007 The BCP micro particles <20 micro m initiated an inflammatory response which might play an important role in osteogenesis.
Fellah et al. 2010 The smallest microparticles decreased the viability of macrophages and enhanced the secretion of pro-inflammatory cytokines (IL-6 and TNF-alpha)
Egli et al. 2011 Thermal treatment of calcium phosphates affect osteoclast activity in vitro
Gamblin et al. 2014 BCP bone grafts with MSCs increased osteoblast gene expression and macrophages at 2 and 4 weeks, osteoclastogenesis at 4 and 8 weeks.
Davison et al. 2014a Liposomal clodronate inhibition of macrophage/osteoclastogenesis inhibits osteoinduction in beta-TCP scaffolds
Davison et al. 2014b Osteoclast activation occurs more favorably on submicron topographies
Davison et al. 2014c On submicrostructured TCPs, osteoclasts survived, fused, differentiated, and extensively resorbed TCP whereas FBGC had no ability to resorb TCP.
Kweon et al. 2014 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 eare 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
R.J. Miron, D.D. Bosshardt / Biomaterials 82 (2016) 1e1912

the osteopromotive potential of bone substitute materials towards
their immunomodulatory behavior thus emphasizing the impor-
tance 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 andthat
they are indispensable for osteogenesis and that their heteroge-
neity and plasticity render macrophages a primer target for im-
mune 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 cal-
cium 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 phagocy-
tosis 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 character-
izing 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 macro-
phage function in vitro [194]. They described an in vitro phenom-
enon 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 biocom-
patibility 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 mac-
rophages 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
m
m 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
m
m than for 40e80 and 80e200
m
m. The number of
macrophages was relatively higher for the smallest than for the
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, investiga-
tion 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) demon-
strated 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 sur-
vived, fused, differentiated, and extensively resorbed the substrate;
however, on microstructured TCP, osteoclast survival, TRAP acti-
vation, 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 di-
mensions of 1
m
m may be responsible for triggering osteoinduc-
tion 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, macrostruc-
ture, and surface chemistry emicrostructural dimensions are
critical in promoting osteoclastogenesis and triggering ectopic bone
formation [204]. Shiwaku et al. showed that osteoclast differenti-
ation is partially impaired by increased HA content, but not by the
presence of micropores within BCP scaffolds, thus favoring osteo-
blast crosstalk [205]. The combination of the above mentioned
studies have provided rational that 1) surface micro and nano-
topography 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 osteo-
clasts 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].
R.J. Miron, D.D. Bosshardt / Biomaterials 82 (2016) 1e19 13

Inhibition of foreign body giant cell formation could be achieved by
4-hexylresorcinol (4HR) through suppression of diacylglycerol ki-
nase 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 re-
ceptor (CaSR) pathway. It was also found that these macrophages
significantly upregulated BMP2 following stimulation with beta-
TCP, indicating that macrophages may participate in osteogenesis
[190]. Thereafter, this same group performed 2 studies demon-
strating that the osteoimmunomodulatory properties of magne-
sium 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 incor-
poration 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 IFN-
gamma, a T-helper1 cytokine that is a known inducer of the clas-
sically activated M1 macrophage (1e3). Interestingly, tissue mac-
rophages 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 sug-
gested 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 LY6C
hi
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 contrib-
utors 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. Howev-
er, it was recently shown that these ‘foam-cells’are also able to self-
renew 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 sup-
pression 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 ‘foam-
cells’) 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 ther-
apeutic 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 sys-
tems around bone grafting materials completely abolished their
osteoinductive activity [3]. Taken together, these findings strongly
confirm the essential roles of OsteoMacs in normal bone develop-
ment, 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 differ-
ences 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 tis-
sues. How different are MNGCs observed around biomaterial im-
plants 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 macro-
phages 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.
R.J. Miron, D.D. Bosshardt / Biomaterials 82 (2016) 1e1914

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 ho-
meostasis 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 mem-
branes, 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 mate-
rials been compared directly in well-conducted studies. Instead
most studies to date have focused on individual classes of bio-
materials 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 Osteo-
Mac 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 ma-
terial 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 ho-
meostasis 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 ho-
meostasis 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 consid-
eration that other studies demonstrated that revisited joint re-
placements extracted following failure consistently found FBGCs
predominantly expressing inflammatory M1 factors
[146,225e227]. Therefore, it remains a valuable therapeutic option
to better understand the patho-physiological regulation of these
cell types for therapeutic benefit.
It also remains interesting to note that strategies are now
implemented in atherosclerosis research to reduce M2 macrophage
MNGCs (described as ‘foam-cells’) in order to reduce ectopic bone
formation. In light of these findings, it remains of great interest to
collaborate with researchers in these fields to better understand
how pathology in certain fields is of potential benefit in others. In
conclusion, it is clear that our incomplete understanding of tissue
macrophages and fusion to MNGCs has thus far hindered thera-
peutic advances and possibilities of new technologies but should
with time improve clinical settings. It thus becomes vital that more
research be performed on this relatively understudied cell-type
called OsteoMac to further improve the development of osteo-
compatible and osteo-promotive bone biomaterials.
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