Review

Immune responses to implants e A review of the implications for the design

of immunomodulatory biomaterials
Sandra Franz
a, d,
*
, Stefan Rammelt
b, d
, Dieter Scharnweber
c, d
, Jan C. Simon
a, d
a
Department of Dermatology, Venerology and Allergology, University Leipzig, 04103 Leipzig, Germany
b
Department of Trauma and Reconstructive Surgery, Dresden University Hospital Carl Gustav Carus, 01307 Dresden, Germany
c
Max Bergmann Center for Biomaterials, University of Technology Dresden, 01069 Dresden, Germany
d
Collaborative Research Center (SFB-TR67), Matrixengineering, Department of Dermatology, Venereology and Allergology, Philipp-Rosenthal-Str. 23, 04103 Leipzig, Germany
a r t i c l e i n f o
Article history:
Received 15 March 2011
Accepted 26 May 2011
Available online 28 June 2011
Keywords:
Immune response to biomaterials
Immunomodulation
Extracellular matrix
Biomaterial integration
a b s t r a c t
A key for long-term survival and function of biomaterials is that they do not elicit a detrimental immune
response. As biomaterials can have profound impacts on the host immune response the concept emerged
to design biomaterials that are able to trigger desired immunological outcomes and thus support the
healing process. However, engineering such biomaterials requires an in-depth understanding of the host
inammatory and wound healing response to implanted materials.
One focus of this review is to outline the up-to-date knowledge on immune responses to biomaterials.
Understanding the complex interactions of host response and material implants reveals the need for and
also the potential of immunomodulating biomaterials. Based on this knowledge, we discuss strategies
of triggering appropriate immune responses by functional biomaterials and highlight recent approaches
of biomaterials that mimic the physiological extracellular matrix and modify cellular immune responses.
2011 Elsevier Ltd. All rights reserved.
1. Introduction
The paradigm on the nature of biomaterials has been intensely
rened within the last decades. Rather than being an inert material
designed to minimize potentially deleterious host responses, Wil-
liams recently dened a biomaterial as .a substance that has been
engineered to take a form which, alone or as part of a complex system,
is used to direct, by control of interactions with components of living
systems, the course of any therapeutic or diagnostic procedure,. [1].
All materials when implanted into living tissue initiate a host
response that reects the rst steps of tissue repair. Modern
implant design is directed on making use of this immune response
to improve implant integration while avoiding its perpetuation
leading to chronic inammation and foreign body reactions, and
thus loss of the intended function [2]. Directing these processes
requires an in-depth understanding of the immunological
processes that take place at the interface between biomaterial and
host tissue.
This review outlines the current knowledge on immune
responses to biomaterials and debates on biomaterials designed to
elicit appropriate host immune responses. Evidence will be pre-
sented that biomaterials mimicking the physiological extracellular
matrix (ECM) may have the potential to modulate the immune
system by enhancing or suppressing normal immune cell
functions.
Abbreviations: aECM, articial ECM; BMP, bone morphogenetic protein; CCL, CC
chemokine ligand; COX-2, cyclooxygenase-2; CS, chondroitin sulfate; CXCL, CXC
chemokine ligand; DC, dendritic cells; DC-STAMP, dendritic cell-specic trans-
membrane protein; ECM, extracellular matrix; EGF, epidermal growth factor; ENA-
78, epithelial cell-derived neutrophil attractant-78; ERK, extracellular signal-
regulated kinases; FBGC, foreign body giant cells; FBR, foreign body response;
FGF, broblast growth factor; GAGs, glycosaminoglycans; GM-CSF, granulocyte
macrophage colony stimulating factor; HA, hyaluronic acid; HMW-HA, high
molecular weight HA; IL, interleukin; IL-1ra, IL-1 receptor antagonist; IFNg, inter-
feron gamma; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase;
MCP, monocyte chemotactic protein; MDC, macrophage-derived chemokine; MFI,
mean uorescence index; MHC-II, major histocompatibility complex class II
molecules; MIP, macrophage inammatory protein; MMPs, matrix metal-
loproteinases; NO, nitric oxide; NOS2, nitric oxide synthase 2; PAMPs, pathogen-
associated molecular patterns; PDGF, platelet-derived growth factor; PEG, poly-
ethylene glycol; PEO, poly(ethylene oxide); PGs, proteoglycans; PMN, poly-
morphonuclear leukocytes; PRR, pattern recognition receptors; RGD,
arginineeglycineeaspartic acid; ROS, reactive oxygen species; TF, tissue factor;
TGF-b, transforming growth factor beta; TH1, T helper 1 cells; TIMP, tissue inhibitor
of metalloproteinases; TLR, toll-like receptors; TNFa, tumor-nekrose-faktor alpha;
T
Reg
, regulatory T lymphocytes; VEGF, vascular endothelial growth factor.
* Corresponding author. Department of Dermatology, Venerology and Allergol-
ogy, Max Brger Research Centre, University Leipzig, Johannisallee 30, 04103
Leipzig, Germany. Tel.: 49 341 9725880; fax: 49 341 9725878.
E-mail address: sandra.franz@medizin.uni-leipzig.de (S. Franz).
Contents lists available at ScienceDirect
Biomaterials
j ournal homepage: www. el sevi er. com/ l ocat e/ bi omat eri al s
0142-9612/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2011.05.078
Biomaterials 32 (2011) 6692e6709
2. Host responses toward biomaterials
Biomaterial implantation is always accompanied by injury
through the surgical procedure. Injury to the tissue or organ initi-
ates an inammatory response to the biomaterial starting with the
formation of a provisional matrix. Implantation of engineered
cellematerial hybrids elicits an adaptive immune reaction toward
the cellular component that inuences the host response to the
material component. When degradable devices are applied the
immune response is additionally affected by degradation products
and surface changes of the biomaterial that occur due to the
degradation process.
2.1. Onset of the inammatory response: blood proteins and
alarmins induce granulocyte/monocyte activation
Nanoseconds after the rst contact with tissue, proteins from
blood and interstitial uids adsorb to the biomaterial surface. This
layer of proteins determines the activation of coagulation cascade,
complement system, platelets and immune cells and guides their
interplay which results in the formation of a transient provisional
matrix and the onset of the inammatory response [3,4] (Fig. 1A).
The following section only delineates the complex interactions of
coagulation, complement and platelets that occur on biomaterial
surfaces. For a more detailed depiction we refer to recent reviews
on biomaterial-associated protein adsorption, coagulation and
complement activation [3,4].
2.1.1. Coagulation, complement and adhesion proteins e signals for
integrins
Factor XII (FXII) and tissue factor (TF) are the initiators of the
intrinsic and extrinsic system of the coagulation cascade, respec-
tively. The intrinsic system is induced by contact activation of FXII
on negatively charged substrates followed by a downstream
cascade of protein reactions resulting in the release of thrombin
[4,5]. Indeed, auto-activation of FXII has been shown to be cata-
lyzed by surface contact with biomaterials [6]. It was suggested that
contact activation of FXII is specically promoted by biomaterials
with anionic surfaces by imposing specic orientation on the
surface adsorbed FXII facilitating its activation [7]. However, new
ndings demonstrate differences in the displacement of competing
proteins on hydrophilic and hydrophobic surfaces to be the cause
for enhanced FXII activation at negatively charged biomaterials
[8,9]. Although auto-activated FXII on biomaterials initiates the
generation of thrombin, the amount produced is not sufcient to
induce clot formation [10]. Blood coagulation on biomaterials has
been recently demonstrated to require the combination of both
contact activation and platelet adhesion and activation [10,11].
Thrombin is one of the main activators of platelets. Low concen-
trations of thrombin released by FXII on biomaterials are suggested
to activate platelets which in turn release mediators of the coagu-
lation process and expose negatively charged phospholipids, thus
providing the supposed catalytic surface for the coagulation
cascade [10,12]. Platelet-derived prothrombinases (factor Va and
factor Xa) of the extrinsic coagulation pathway assemble on the
activated platelet surface and become activated. Subsequent
thrombin formation occurs [12] that further activates platelets and
coagulation factors of the intrinsic and extrinsic system amplifying
the coagulation cascade on biomaterial surfaces [13]. Furthermore,
thrombin initiates the cleavage of brinogen to brin forming the
primary brous mesh around the biomaterial.
Fibrinogen is also known to spontaneously adsorb to biomate-
rial surfaces [14,15]. It was shown that an adhesion related
conformational change of brinogen resulted in the exposure of
two integrin binding domains capable to activate phagocytes
[14,15]. It is suggested that phagocytes sense brinogen adherent to
biomaterials as brin thus launching an inammatory response
occurring physiologically following clot formation [14]. Adsorbed
brinogen also serves as adhesion substrate for platelets [16,17].
Besides thrombin and the adsorbed brinogen, TF expressed on
damaged cells or on activated leukocytes at the implantation site as
well as activated complement can induce activation of biomaterial
attached platelets [18].
It is well established that complement is activated upon contact
with biomaterial [19e21]. In inammation complement activation
occurs via three distinct pathways, the alternative, the classical and
the lectin pathway which all converge on the level of C3 convertase
activation that mediates formation and release of the anaphylatox-
ins C3a andC5a (reviewedinRef. [22]). Activationof complement on
biomaterial surfaces has been shown to be predominantly triggered
by the classical and alternative pathway although there are also
reports of lectin mediated complement activation [19e21].
However, complement activation is always associated with the
biomaterial adsorbed protein layer. Attached IgG has been demon-
strated to bind C1q resulting in the assembly of C1, the rst enzyme
of the classical pathway that promotes the initiation of the classical
C3 convertase [23]. Additionally, C3 canspontaneouslyadsorb to the
biomaterial surface while adopting a newconformation mimicking
that of surface boundC3bof the alternative pathway[24]. The bound
C3 thenpromotes the assembly of the initiating C3 convertase of the
alternative pathway. C3b arising fromthe proteolytic cleavage of C3
bycoagulationfactors including FXIIa andthrombincanalsodirectly
attach to biomaterial surfaces and form an initiating C3 convertase
[4,25]. Action of the C3 convertase results in the generation of C3b
that binds to the biomaterial protein layer to form more C3 con-
vertases thus launching the amplication loop of the alternative
complement pathway [19,24]. Once the complement cascade is
started high amounts of C3a and C5a are generated at the implan-
tation site [26]. Both anaphylatoxins can contribute to the onset of
inammatory responses at implantation sites through their multi-
tude of effector functions including triggering of mast cell degran-
ulation, increasing vascular permeability, attracting and activating
of granulocytes and monocytes and inducing granulocyte ROS
(reactive oxygen species) release [22]. The induced complement
proteins have also been shown to support platelet adhesion and
activation on biomaterial surfaces [27] which in turn can propagate
the coagulation cascade [10,12] but also promote TF expression by
monocytes and granulocytes on biomaterial surfaces [28,29]. It
becomes apparent that the coagulation cascade and complement
system closely interact on the biomaterial surface and modulate
each others activity [18]. The cross-talk between both systems
operates synergistically in inammatory cell activation.
Adhesion proteins of the ECM including bronectin and vitro-
nectin have also been described to attach to biomaterial surfaces
[30]. Whereas brinogen and complement mainly contribute to the
activation of inammatory cells, bronectin and vitronectin are
critical in regulating the inammatory response to biomaterials.
Both proteins have been described to promote the fusion of
macrophages to foreign body giant cells on biomaterial surfaces
[31,32]. However, these proteins also support adhesion and
spreading of osteoblastic cells to biomaterials which represents
a crucial step for integration of orthopedic implants [33]. These
seemingly opposed functions of bronectin and vitronectin
exemplarily reect the high effector capacity of the adsorbed
protein layer. Depending on the environment at the implant site,
proteins adhering to biomaterials may either initiate and foster
inammation or assist healing.
Cell adhesion and activation on biomaterials primarily occurs via
interaction of adhesion receptors with the adsorbed proteins. In this
review we focus on the interaction of biomaterials with
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6693
Fig. 1. Immune response toward biomaterials. A) Adsorption of blood proteins and activation of the coagulation cascade, complement and platelets result in the priming and
activation of PMNs, monocytes and resident macrophages. B) Danger signals (alarmins) released from damaged tissue additionally prime the immune cells for enhanced function
via PRR engagement. C) The acute inammatory response is dominated by the action of PMNs. PMNs secrete proteolytic enzymes and ROS, corroding the biomaterial surface. IL-8
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6694
immunocompetent cells. Integrins represent the major adhesion
receptors of leukocytes [34]. Protein ligands of integrins include
brinogen, factor X, iC3b, bronectin, vitronectin [35] e all of which
have been shown to attach to biomaterial surfaces [36]. Indeed, cell
adhesion to protein-coated biomaterials and subsequent cell acti-
vation have been described to be mediated by integrins [37e40].
Initial adhesion and spreading of phagocytes are primarily achieved
through b2 integrins [14,37] which in turn leads to a change in the
receptor prole including the up-regulation and enabling of further
integrins [34]. Adherent monocytes differentiating to macrophages
initiate b1 integrins [41] which in conjunction with b2 integrins
mediate adhesion during biomaterial-associated macrophage fusion
[32,40,42]. Clustering of integrins may also induce motility, phago-
cytosis, degranulation, as well as the release of ROS and cytokines,
events which play an important role in the inammatory response
toward biomaterials.
2.1.2. Danger signals and pathogen recognition receptor
Besides recognition of biomaterials through adhesion receptors,
there are other receptoreligand interactions activating immuno-
competent cells. Danger signals (also referred to as alarmins) have
long been ignored as potential activators of leukocytes following
biomaterial implantation (Fig. 1B). The role of alarmins in inam-
mation has been reviewed in detail elsewhere [43]. Alarmins are
the endogenous equivalent of pathogen-associated molecular
patterns (PAMPs) and include heat shock proteins, HMGB1 (high
mobility group box 1), ATP and uric acid. They are rapidly released
following injury bycells dying in a non-programmed way (necrosis)
to signal associated tissue damage. Like PAMPs, alarmins are
recognized by cells of the innate immune system such as macro-
phages and dendritic cells (DCs) via pattern recognition receptors
(PRR) such as scavenger receptors, toll-like receptors (TLR) and C-
type lectins which promote inammation and immunity.
There is evidence that induced danger signals are capable of
immune cell activation at biomaterial surfaces [44,45]. Upon
biomaterial application alarmins may be released or induced by
cells at the implant site that had been damaged due to the surgical
procedure. Proteolytic enzymes leaking from the injured cells may
additionally trigger the generation of extracellular danger signals
[46]. These signals include brinogen or cleaved ECM components
such as collagen peptides, hyaluronic acid (HA), bronectin and
laminin that adsorb to biomaterials. Thus, a coated biomaterial
surface itself might act as a danger signal (Fig. 1A). Soluble or
biomaterial-associated alarmins can interact with PPRs, preferen-
tially TLRs on leukocytes and propagate the inammatory response.
Indeed, recent studies demonstrate a role of TLR4 in the host
response to biomaterials in vitro and in vivo [47,48].
2.1.3. PMN activation
Immediately following injury and protein deposition, inam-
matory cells e predominantely polymorphonuclear leukocytes
(PMNs, granulocytes) e migrate fromthe blood toward the implant
site. Circulating PMNs are rapidly recruited to infection sites by
host- and pathogen-derived mediators, acting as a rst line of
defense against invading pathogens. The role of granulocytes in the
innate immune response is extensively reviewed elsewhere [49].
PMN recruitment to implantation sites is triggered by host derived
chemoattractants released from activated platelets and endothelial
cells as well as injured cells (Fig. 1A,B). Mast cell degranulation and
associated histamine release have been shown to play a role in
directing PMNs and monocytes to implanted biomaterials in mice
and humans [50,51]. Reaching the implantation site, PMNs
encounter the protein-coated biomaterial surface and subsequent
engagement of integrins and PRRs on the PMN surface triggers
a phagocytic response and degranulation [52,53] (Fig. 1C). PMNs
usually secrete proteolytic enzymes and ROS to promote and foster
pathogen killing [49]. At the implant site, the destructive agents
may corrode material surfaces as described for polyurethane [54].
Furthermore, the cytotoxic components damage surrounding
tissue, prolonging the inammatory response. Another adverse
effect of biomaterial-induced PMN activation is the metabolic
exhaustion and depletion of the granulocytes oxidative resources.
Due to the continuous release of ROS the microbial killing capacity
of PMNs is dramatically reduced, which has been related to severe
biomaterial-centered infections [55].
PMNs also represent a signicant source of immunoregulatory
signals which they synthesize upon activation [56], interleukin-8
(IL-8) being among the most prominent chemokines. The primary
targets of IL-8 are PMNs themselves. Various studies have reported
granulocyte migration and prolonged presence of granulocytes
within chitosan materials [57,58] due to persistent autocrine PMN
attraction by IL-8 [59]. Activated PMNs also secrete MCP-1 and MIP-
1b [49]. Both chemokines are known as potent chemoattractants
and activation factors for monocytes, macrophages, immature DCs
and lymphocytes [60]. Increased release of these chemokines by
PMNs suppresses further PMN inltration in favor of mononuclear
cell inux [61]. Due to a lack of further activation signals PMN
undergo apoptosis after having done their job as phagocytes and
are engulfed by macrophages [61]. Within the rst two days after
biomaterial implantation PMN typically disappear from implanta-
tion sites [62].
2.2. Chronic inammation: dual role of macrophages as
inammatory mediators and wound healing regulators in the
foreign body reaction
Chronic inammation develops as inammatory stimuli persist
at the implant site with macrophages representing the driving force
in perpetuating immune responses [63]. Monocytes arriving at the
implantation site undergo a phenotypic change differentiating to
macrophages. Their activation leads to further dissemination of
chemoattractants. Macrophages attached to the biomaterial can
foster invasion of additional inammatory cells by secreting che-
mokines like IL-8, MCP-1, MIP-1b [64] (Fig. 1D).
Macrophages also play a critical role in wound healing and
tissue regeneration. Phagocytosis of wound debris, release of
enzymes important for tissue reorganization and of cytokines and
growth factors inducing migration and proliferation of broblasts
are mediated by macrophages and constitute the initial steps
toward effective tissue regeneration [65]. These different functions
are typically promoted by different macrophage subsets, originally
referred to as M1 (classically activated) and M2 (alternatively
activated) macrophages [66]. Based on fundamental macrophage
functions involved in maintaining homeostasis these subsets have
been reclassied into classically activated, regulatory, and wound-
healing macrophages [67] (Fig. 2). The latter two arise from
released from PMNs enhances PMN inux and priming. In the transition from acute to chronic inammation, PMNs stop secreting IL-8 in favor of cytokines promoting immigration
and activation of monocytes and macrophages. D) Macrophages are the driving force of chronic inammation. Constant release of inammatory mediators like TNFa, IL-6, and MCP-
1 results in permanent activation of macrophages. Fusion-inducing stimuli like IL-4 and IL-13 promote the fusion of macrophages to FBGC, which form a highly degradative
environment on the biomaterial surface. Furthermore, FBGC promote ECM remodeling and broblast activation resulting in excessive brosis and biomaterial encapsulation. E)
Macrophage-derived cytokines and PRR engagement activate DCs on the biomaterial surface. Depending on the nature of the stimulus, DCs mature to either immunogenic or
tolerogenic subtypes, amplifying or suppressing the inammatory response.
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6695
a subdivision of the alternatively activated macrophage subset.
Activation and function of these macrophage phenotypes is
excellently reviewed by Mosser and Edwards [67]. The different
macrophage populations are generated in response to either
endogenous stimuli released by damaged cells or innate immune
cells following injury or infection or to adaptive immune signals
produced by antigen-specic immune cells [68,69]. Classically
activated macrophages are typically triggered by interferon-g
(IFNg) released by T helper 1 (TH1) cells during adaptive immu-
nity or by natural killer (NK) cells during innate immunity and by
TNFa produced by antigen presenting cells [67,68]. Classical
stimulation prime macrophages to secrete inammatory cytokines
and to perform microbicidal activity mediated by increased
synthesis of ROS and nitrogen radicals making them to a crucial
part of host defense [67,68]. Wound-healing macrophages are
generated in response to IL-4 produced by basophils, mast cells
and granulocytes in early innate immune responses or by TH2
cells during adaptive immune responses [67,70]. Interleukin-4
programs macrophages to down-regulate pro-inammatory
mediators and to promote wound healing processes by contrib-
uting to the production of ECM and by activation of broblasts
[67,68,70e72]. Although wound-healing macrophages exert anti-
inammatory activities they are not capable of down-regulating
immune responses. Regulatory macrophages also arise during
innate and adaptive immune responses. They are triggered
in response to a variety of signals including apoptotic cells, pros-
taglandins, IL-10, immune complexes, glucocorticoids [67,73].
However, to become fully activated the macrophages need
a second signal such as a PRR-ligand [67,73,74]. The main task of
regulatory macrophages is to limit inammation and to dampen
immune responses which they achieve by release of high levels of
IL-10, a very potent immunosuppressive cytokine [67,73,74].
An immune response involves the action of all types of macro-
phages, classical activated macrophages in the early phase and
regulatory and wound-healing macrophages in the resolution
stage. It is still debated whether inammatory macrophages
emigrate from the site of inammation to give rise for regulatory
and wound-healing macrophages [75] or whether the macro-
phages alter their functional phenotype in response to progressive
changes of signals during the course of inammation [76]. There are
reports showing that macrophages retain their functional adap-
tivity and adjust their phenotype to changing environmental
stimuli [76,77]. The remarkable plasticity of macrophages is making
them to an interesting target in the context of immunomodulation.
2.2.1. Foreign body giant cell formation
Macrophages that attach and recognize a foreign material show
typically a classically activated phenotype secreting inammatory
cytokines, ROS, and degradative enzymes and displaying high
phagocytic capacity. Single macrophages are able to phagocytose
particles up to a size of 5 mm [78]. If the particle size is larger,
macrophages attempt to coalesce to FBGCs. The cytokines IL-4 and
IL-13 have been identied to induce macrophage fusion on
biomaterial surfaces in vivo and in vitro [79e81]. Activated
T lymphocytes (CD4 cells) at the implant site are assumed to be
the source of both IL-4 and IL-13 and have been shown to enhance
macrophage fusion on biomaterials [82]. However, a recent study
investigating synthetic biomaterials in nude mice revealed CD4
cells not to be essential for induction of a foreign body response
(FBR) [83]. Although IL-4 was not present in the T cell-decient
setting, macrophage fusion was not impaired due to unaffected
levels of IL-13 at the implant site. It was suggested that mast cells
most likely serve as a source of IL-13 at the onset of inammation
and also sustain IL-13 production during the chronic inammatory
response to the biomaterial. Chemoattractant CCL2 was also
reported to be involved in FBGC formation [84] though not by
recruiting cells to the implant site but rather by guiding macro-
phage chemotaxis toward each other [85].
Moreover, the properties of the biomaterial surface are impor-
tant for FBGC formation. Since biomaterials are immediately
covered, it is the adsorbed protein layer that renders the surface
fusogenic. A variety of proteins including collagen, bronectin,
Fig. 2. Classication of macrophages. Macrophages represent a heterogeneous population of cells with different phenotypic proles performing distinct functions in host defense,
wound healing, and immune regulation. This plasticity enables macrophages to adapt their functions to environmental cues. Adapted from Refs. [67,68].
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6696
laminin, brinogen, and vitronectin have been tested on their
capability to promote FBGC formation [32]. Although all proteins
mediate initial monocyte adhesion, only vitronectin supports
macrophage adhesion and fusion [32]. It has been shown that b1
and b2 integrins play a role in macrophage adhesion during
biomaterial-associated fusion [42]. Nevertheless, both integrins
seem not to mediate the crucial adhesion step required for
macrophage fusion [86]. It is proposed that fusion-inducing stimuli
and initial adhesion to the biomaterial induces the expression of
multiple fusogenic molecules including mannose receptor (CD206)
[87], CD44 [88], CD47 [89], DC-STAMP [90], and E-cadherin [91]
rendering macrophages capable for fusion [85].
2.2.2. Release of ROS, degradative enzymes and MMPs
If the FBGCs do not succeed in phagocytosing the foreign
material, they remain at the biomaterialetissue interface and shape
podosomal structures forming a closed compartment between
their surface and the underlying substrate [92]. Various studies
have shown that following fusion to FBGC, macrophages display
a reduced phagocytic activity in coincidence with enhanced
degradative capacity [93], a phagocyte-specic phenomenon
referred to as frustrated phagocytosis. In an attempt to resorb the
non-phagocytosable biomaterial, FBGCs secrete protons, enzymes,
and ROS into the compartment between them [94,95]. This will
lead to resorption of materials that are susceptible to degradation
[96]. If the biomaterial is completely resorbed, associated inam-
mation may resolve as the causative agent is no longer present.
Matrix metalloproteinases (MMPs) are macrophage-derived
proteolytic enzymes involved in the foreign body reaction to
biomaterials [97e100]. The collagenases MMP-8 and MMP-13 and
the gelatinases MMP-2 and MMP-9 could be detected in macro-
phages adhering to collagen disks post explantation [97]. Combined
action of both gelatinases and collagenases has been suggested to
promote degradation of collagen implants with MMP-9 as key
enzyme. MMP-9 was also found to be induced in macrophages and
broblasts during tissue remodeling in response to natural
hydroxyapatite implanted in rats [100]. In this study MMP-9 alone
was unable to breakdown the xenograft implant, but was involved
in angiogenesis and ECM remodeling of the peri-implant connec-
tive tissue. Macrophages and FBGC cultured in vitro on various
biomaterials have been shown to express MMP-9 [98]. The study
further demonstrated reduced macrophage fusion by pharmaco-
logical inhibition of MMP-1, -8, -13, and -18 implicating a role of
these MMPs in the fusion process. Although the MMP blocking
assays of this study did not show an involvement of MMP-9 in the
FBGC formation [98] comprehensive investigations analyzing the
foreign body reaction in MMP-9 null mice clearly demonstrated an
involvement of MMP-9 in macrophage fusion [99]. The study also
revealed that MMP-9 may play a pivotal role in biomaterial
encapsulation and angiogenesis [99].
The action of MMPs at the implant site is assumed to dramati-
cally change the cellular environment around the biomaterial and
to modify migration, differentiation and active state of macro-
phages and other immune cells [101]. Increased levels of MMP-9
have been suggested to be indicative of inammation and associ-
ated with poor wound healing [102]. An environment rich in MMP-
9 at the biomaterial site may thus perpetuate inammation and act
counter-regulatory on the wound healing process.
2.2.3. Fusion-induced macrophage phenotype switch
Fusion to FBGCs is typically associated with a phenotype
switch of the macrophages from a classical to a more alter-
native activation state. The fusion-inducing cytokines IL-4 and IL-
13 are known to promote wound-healing macrophages during
inammation [67,71,72]. This transition is reected by alterations
of the cytokine prole at the biomaterial site [64,103e105]. Early
upon biomaterial recognition macrophages release IL-6, IL-1b,
TNFa, IL-8, MCP-1, RANTES, ENA-78 similar to classically acti-
vated macrophages [64]. Over time the majority of these
inammatory cytokines is down-regulated in favor of IL-10, TGF-
b, MDC, Eotaxin-2 as well as IL1 receptor antagonist (IL1ra) that
resembles the anti-inammatory cytokine/chemokine prole of
alternatively activated macrophages. However, the activation
state of fusing macrophages is not completely identical to the
alternative phenotype since they still produce pro-inammatory
RANTES and MCP-1 [64], ROS and degradative enzymes. More-
over, biomaterial-adherent macrophages sustain or even increase
their IL-6 and TNFa production on materials that do not promote
macrophage fusion without mandatorily undergoing a pheno-
type switch [64,105]. These ndings support the dogma that
implant surface properties dictate macrophage responses. More
importantly, they show that macrophage behavior is predomi-
nantly governed by the fusion event rather than adhesion.
2.2.4. Impaired wound healing and excessive brosis
Little knowledge exists on the involvement of macrophages/
FBGCs in the healing response to biomaterials. Successful tissue
repair requires resolution of the inammation through the release
of anti-inammatory mediators, cleavage of chemokines, down-
regulation of inammatory mediators and receptors, and
apoptosis of immune cells [106]. At implantation sites, FBGCs
produce anti-inammatory cytokines (IL-10, IL-1ra) [64,103e105].
However, their immunosuppressive activity may be counter-
regulated by the proteolytic and pro-oxidant microenvironment
due to continuous release of ROS and degradative enzymes around
the biomaterial [106,107]. FBGCs that have formed in response to
IL-4 may release pro-brotic factors such as transforming growth
factor beta (TGF-b) and platelet-derived growth factor (PDGF) that
trigger the action of broblasts and endothelial cells as shown for
IL-4 induced alternatively activated macrophages in vitro [71,72].
Activated broblasts start to synthesize and to deposit collagen that
often results in material encapsulation [108,109]. However, the
release of pro-brotic factors by FBGCs has never been clearly
described. Although the mechanism is not well understood, it is
assumed that continuous action of FBGCs result in prolonged
broblast activation and excessive biomaterial-associated matrix
deposition [110,111].
2.3. Dendritic cell responses to biomaterials
Synthetic biomaterials including ceramics, polymers or metallic
materials are typically not immunogenic and are thought not to
initiate an adaptive immune response, except for cases of metal
hypersensitivity. However, lymphocytes have been found at sites of
synthetic implants [44,112] suggesting their involvement in
immune responses tobiomaterials. DCs are keyplayers of innate and
adaptive immunity. They elicit adaptive immune responses by their
ability of antigen presentation and T cell priming. Additionally, DCs
possess immunoregulatory capacities as they play a role in the
induction of antigen-specic T cell tolerance, T cell anergy and the
activation and expansion of regulatory T cells (T
Reg
) (reviewed in
Refs. [113,114]). Whether DCs induce an immunogenic or a tolero-
genic T cell response depend on many factors including the state of
DCmaturationandthe cytokineenvironment [114]. Accordingtothe
current concept of DC functions immature and semi-mature DCs are
promoters of tolerance whereas fully mature DCs induce immunity
[113]. Antigen presentation in the absence of co-stimulation by
immature DCs typically promotes T cell anergy [115,116]. Semi-
mature DCs expressing MHC and co-stimulatory molecules but
unable to produce pro-inammatory cytokines such as IL-12, TNFa,
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6697
IL-6 and IL-1b have been described to convert nave T cells into
CD4/CD25 T
Reg
or IL-10 secreting CD4 T cells (Tr1) [117e119].
The tolerogenic activity of semi-mature DCs can be additionally
enhanced by their release of IL-10 [113,114]. Cytokines have been
shown to play the major part in the induction of tolerogenicity.
Besides IL-10 and TGF-b, two important immunosuppressive regu-
lators, various cytokines and growth factors including IL-6 and TNFa
at low concentrations as well as IL-16, granulocyte colony stimu-
lating factor and hepatocyte growth factor have been reported to
generate DCs with tolerogenic activity [114,120e125]. With respect
to biomaterial application induction of tolerogenic DCs at the
implant site would provide a powerful means to limit the immune
response and to promote wound healing and biomaterial
integration.
The role of DCs in biomaterial application has mostly been
addressed in the presence of an immunogenic biological compo-
nent. Biomaterials were found to exert adjuvant effects since they
potentiated immune responses toward co-delivered antigens [126].
However, acting as an adjuvant requires the capability to prime DCs
that has been shown to necessitate direct cell-material contact
[127]. It is assumed that biomaterials activate DCs by triggering
receptors and signaling cascades of the pathogen recognition
system [44] (Fig. 1B and E). As described above, DCs may sense
materials using PRRs including toll-like receptors and C-type lec-
tins. The receptor-activating ligands are danger signals constituted
by proteins and carbohydrate moieties in the adsorbed protein
layer. A recent study suggests a substrate-dependent DC activation.
Albumin or whole serumstimulated DCs to the release of IL-10 or to
prime IL-4 producing T cells as typical for Th2 type responses [128].
In contrast, DCs cultured on collagen and vitronectin generated
high levels of IL-12p40 that correlated with the release of IFNg by T
cells indicating a Th1 type response [128]. Because collagen and
vitronectin function both as danger signals and adhesion substrates
for integrins, integrin signaling cannot be ruled out as an alterna-
tive mechanism of DC activation to PRR engagement. Indeed, DC
integrin binding to ECM proteins and subsequent DC maturation
has been shown, and for bronectin a dependency of DC matura-
tion on b1 integrin binding was demonstrated [129,130].
Various biomaterial polymers (alginate, agarose, chitosan, HA,
poly(lactic-co-glycolic acid)) have been shown to exert differential
effects on DC maturation and activation [44,131,132]. DCs activated
upon biomaterial contact develop an immunogenic phenotype
similar to LPS-activated DCs which is characterized by increased
expression of co-stimulatory molecules (CD80/CD86), major histo-
compatibility complex class II (MHC-II) molecules and the DC
maturation marker CD83 [44,127]. Biomaterial-matured DCs are
capable to promote T cell proliferation and secrete inammatory
cytokines (TNFaandIL-6) knowntofurther amplifyDCmaturationby
autocrine stimulation [44,127]. Nevertheless, some of the synthetic
materials tested (e.g. alginate or HA) restrained DC maturation [131].
These cells develop a tolerogenic phenotype [125] that, upon
encountering and presenting antigens, induce T cell tolerance.
Depending on which PRR is engaged, DC maturation can be
promoted or inhibited leading to immunity or tolerance, respec-
tively [133]. Whereas immunogenic DCs may prolong the immune
response to biomaterials and delay wound healing, tolerogenic DCs
are capable to down-regulate the immune cells and resolve
inammation. Thus, induction of tolerogenic DC by designing the
surface chemistry appears to be a promising strategy of modulating
immune responses to biomaterials to improve biocompatibility.
2.4. T lymphocyte responses to biomaterials
T lymphocytes have been shown to adhere to synthetic
biomaterials in vitro [62]. In co-culture with macrophages, they
were found attached predominantly to macrophages and not to the
biomaterial surface [82]. T lymphocytes have been demonstrated to
promote macrophage adhesion and fusion via paracrine effects [82]
(Fig. 1D). However, close association of lymphocytes and macro-
phages also suggests direct signaling which has been shown to
dominate at later time points of their interaction [134]. During the
initial response to biomaterials, lymphocytes and macrophages
predominantly release inammatory mediators [134e136]. These
include the cytokines IL-1b, IL-6, TNFa and the chemokines IL-8,
MCP, MIP-1b, ENA-78 all of which attract and activate inamma-
tory effector cells such as neutrophils, monocytes, T lymphocytes
and natural killer cells. Interestingly, release of IL-1b and TNFa
declined over time in favor of IL-10 and MMP-9, tissue inhibitor of
MMPs (TIMP)-1 and TIMP-2 e important mediators for ECM
remodeling in wound healing [135]. These data nicely demonstrate
the capability of T lymphocyteemacrophage interactions in guiding
the inammatory phases of the foreign body reaction. The absence
of T cell activation in in vitro studies, however, questions the effect
of lymphocytes in these processes. Neither IL-2 nor IFNg were
detected as a response to lymphocytes alone or in co-culture with
macrophages to different synthetic biomaterials including silicone
rubber, elasthane 80A or polyethylene terephthalate [135e137]. On
the other hand, activated T cells were identied during inamma-
tory biomaterial responses in vivo, suggesting the presence of the
complete inammatory environment as requirement for T cell
activation in response to synthetic materials [137,138]. Neverthe-
less, the question remains how T cell activation is mediated during
foreign body reaction. Synthetic biomaterials do not serve as an
antigen. Given that the biomaterial is not degradable and that no
bacteria transiently attach to its surface, Tcell activation via antigen
presentation does not occur. It has been suggested, that synthetic
biomaterials may present functional groups on their surfaces acting
as mitogens [137]. Mitogens are lectins that can trigger lympho-
cytes by cross-linking of glycoproteins on the lymphocyte surface.
To date, mitogenic capabilities of biomaterials have not been
demonstrated.
3. Extracellular matrix e a native modulator of cell activity in
immune responses and tissue repair
The direct interactions of biomaterials and components of the
hosts immune systemdescribed so far occur in vivo in the presence
of the ECM. Several ECMproteins are potent regulators of monocyte/
macrophage adhesion and activation and are involved in DC
migration and maturation [139,140]. Bidirectional interactions of
ECM, cells andgrowthfactors/cytokines determine cellular behavior
during all phases of biomaterial integration and wound healing
including inammation, cell proliferation and tissue remodeling.
Moreover the ECM provides mechanical support and a three-
dimensional scaffold for cellular organization. In addition it regu-
lates cell behavior, storage and mobilization of signaling molecules
as well as proteolytic degradation [141] (Fig. 3A). Collagen, elastin
and brin form a brous structure that provides tensile strength or
elasticity [142]. Non-brous proteins (predominantly bronectin
and laminin) linked to this scaffold supply domains for cellematrix
interaction [143]. The protein scaffold is embedded in a gelatinous,
negatively charged matrix composed of glycosaminoglycans
(GAGs). GAGs are long, unbranched carbohydrate chains consisting
of repeating disaccharide units [144]. The sugar chains can be
modied by sulfate groups as in chondroitin sulfate (CS), heparan
sulfate, dermatan sulfate and keratin sulfate. HA is the only non-
sulfated GAG. It is also not attached to a protein core whereas the
sulfated GAGs are linked to serine rich proteins to form proteo-
glycans (PGs) [144]. The glycan matrix of the ECM serves as lubri-
cant and provides a reservoir for signaling molecules. Additionally,
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6698
it participates in a variety of biological processes including
cellematrix interactions and activation of enzymes and mediators
like growth factors and cytokines [145].
Cells including those involved in inammation attach to the ECM
via various surface receptors including integrins, selectins, synde-
cans and CD44 (Fig. 3B). They either recognize specic adhesive
domains in the protein scaffold or bind to components of the glycan
matrix e.g. HA, heparan sulfate and CS. This usually triggers intra-
cellular signals that direct adhesion, migration, proliferation,
differentiation, protein synthesis, and secretion [139,141,146]. These
cellular functions are additionally regulated by signaling molecules
(growth factors, cytokines and chemokines) that are trapped in the
ECM. However, the ECM not only serves as a reservoir for signaling
molecules, it rather regulates their distribution and mode of action.
Components of the ECM (mostly PGs) retain the soluble mediators
for example via electrostatic interactions between the negatively
charged sulfate groups of the PGs and the positively charged surface
of the signaling molecule [147]. This interaction has different
biological consequences since it affects the local concentration,
biological activity, and stabilization of growth factors [148,149]
(Fig. 3C). Secreted growth factors usually have a very short half-life
due to their high susceptibility to proteolytic degradation. Linkage
to the ECM protects them from enzymatic cleavage. Moreover,
binding to the ECM prevents growth factor diffusion within the
compartment, providing a local store of functional molecules that
persists long after their release has stopped [148]. This may result in
a local concentration of growth factors needed for effective receptor
signaling. Additionally, growth factor activity may be enhanced by
localization within the ECM, allowing interaction with its specic
ligands [149]. On the other hand, some growth factors may become
inactive when bound to the ECM and can only act on their target
when released by matrix proteolysis [141]. This requires action of
ECM-degrading enzymes expressed by cells regulated by growth
factors and ECM adhesion domains. Thus, growth factors and ECM
proteins collaborate in creating a distinct cellular environment or
niche that regulates tissue regeneration [150].
Fig. 3. Structure and function of the ECM. A) ECM composition. B) Cellematrix interaction: engagement of cell surface receptors with proteins and proteoglycans of the ECM triggers
intracellular signaling events. C) Cytokineematrix interaction: proteoglycans bind growth factors and cytokines. Interaction with the ECM protects the signaling molecules from
enzymatic cleavage, enriches their local concentration and enhances or reduces their action on cells.
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6699
Proteolytic degradation of the ECM is also an essential feature of
tissue repair and remodeling, and enables cell migration and
invasion [148]. Numerous cell-secreted and cell-activated enzymes
with ECM-degrading capacities have been identied including
MMPs, elastase, and plasmin. MMPs play a predominant role in
ECM degradation since they process and degrade virtually all
structural ECM proteins. There are over 25 known MMPs grouped
into collagenases, gelatinases, stromelysins, matrilysins, mem-
branetype (MT)-MMPs and others performing multiple, sometimes
overlapping, functions in ECM proteolysis (reviewed in Ref. [151]).
However, MMPs are not only involved in ECM breakdown they also
target non-ECM proteins, cell surface molecules and ECM-bound
growth factors and cytokines and thus inuencing cellular
behavior [152]. For example, repressed T cell activity due to
a down-regulation of the IL-2 receptor is mediated by MMP-9 that
cleaves the cytokine receptor from the T cell surface [152,153].
Membranetype-MMP-1-mediated shedding of syndecan-1,
a transmembrane protein involved in cellecell and cellematrix
adhesion has been shown to stimulate cell migration [154].
Vascular endothelial growth factor (VEGF) restrained in the ECM
becomes mobilized by MMP-induced proteolysis to exert its
angiogenic activity and to serve as chemotactic signal for osteo-
clasts [155,156]. Several MMPs (MMPs-2, -9, -13 and -14) are
involved in the release of the ECM-bound latent TGF-b complex and
its processing into an active ligand that controls proliferation,
differentiation and other functions [151,157,158]. MMP-mediated
proteolysis of bronectin generates fragments inducing cell
migration but blocking cell proliferation [151]. Angiogenic frag-
ments are released from collagen by MMP-2 and -9 [151,159].
Laminin-5 when cleaved by MMP-2, -3, -12, -13 and -14 exposes
neo-epitopes promoting cell migration whereas MMP-8-derived
laminin-5 fragments do not [151,160].
The action of MMPs is controlled at two levels, during tran-
scription by cytokines and integrin clustering and after secretion by
endogenous inhibitors including a
2
-macroglobulin and TIMPs
[161e163]. Whereas cytokine and integrin signaling up- or down-
regulate MMP expression, the protease inhibitors and here specif-
ically TIMPs bind to the MMPs thereby determining their activity
[163]. Precise control of MMP activity is crucial to ensure ECM
remodeling under normal physiological conditions as dysregula-
tion has been implicated in many diseases such as brosis, cancer,
arthritis and vascular disease [162,164e166].
Taken together, the ECM not only provides support and a three-
dimensional scaffold for cells, it also plays a highly functionalized
role in directing cell behavior by spatially and temporarily
concerted interaction with other cells, ECMcomponents, degrading
enzymes and signaling molecules all of which being relevant to
tissue repair, host responses to biomaterial and ultimately bioma-
terial integration [167].
4. Modulating immune responses to biomaterials
For a long time biomaterial engineering focused on inert
biomaterials. The concept of biologically inert materials is to
minimize the host response by avoiding cellematerial interactions.
Inert biomaterials are recognized as foreign by the host but remain
essentially unchanged and tolerated due to their encapsulation in
brous tissue [168]. However, it has been realized that permitting
specic cell responses may in fact be benecial for biomaterial
integration and improve implant performance [169]. For example
osseointegration of titanium implants, classically inert biomate-
rials, could be improved by modication of the material surface
allowing migration and adhesion of bone-forming cells from the
surrounding tissues onto the implant [170].
Controlled tissue responses at the implant site are assumed to
encourage wound healing. Increasing comprehension of the healing
process point out that immune responses associated with inamma-
tion and macrophage activation are crucial for tissue repair [171,172].
Withgrowing knowledge onthe processes of woundhealing andhost
responses to biomaterials the eld of biomaterial engineering has
begun to address the development of materials with immunomo-
dulating capacities. Ideally, the materials should affect normal
immune cell function such that they promote healing and implant
integration while sustaining specic implant function [2]. There are
several publications reporting of modulated immune responses
induced by silicons and blends of polydioxanon (PDO) and elastin or
collagen [173e175]. The very comprehensive studies addressing
modulationof functions of innate andadaptive immune cells revealed
suppressed NK cell activity in response to all biomaterials whereas
macrophage functions remained unaffected[173e175]. Blends of PDO
and elastin or collagen were also shown to exert immunosuppressive
effects on T and B cell-mediated immunity [174,175].
On the one hand the studies clearly point out the need for testing
of biomaterials on their effects on both acquired and innate immune
responses as a component of biocompatibility assessment [174,175].
On the other hand the studies nicely demonstrate the potential of
biomaterials to modulate immune cell function encouraging the
design of biomaterials capable of eliciting appropriate immune
responses at implantation sites. Current strategies in the design of
such biomaterials include alteration of material surface properties
either passively via physicochemical features or actively with mole-
cules or matrices designed to systematically target cell behavior.
4.1. Immunomodulation by surface modications of biomaterials
Passive modulation of biomaterial surface properties aims at
limiting macrophage adhesion, activation and fusion to FBGCs
(Fig. 4A). Type, level andconformationof serumproteins that adsorb
to biomaterial surfaces depend on the terminal chemistry of the
biomaterial [36,38,39,176]. The adsorbed protein layer usually
provides binding sites for protein-specic receptors (integrins,
PRRs) on PMNs, monocytes and macrophages. Surface chemistry-
dependent modulation of the protein layer may thus enable
different receptor binding and signaling in the immune cells leading
to altered cellular responses. Indeed, comprehensive proteomics
studies revealed macrophages to change their protein expression
proles and cytokine/chemokine responses when cultured on
surface-modied polymers displaying hydrophobic, hydrophilic,
and/or ionic chemistries [64,177]. Macrophages attaching to
hydrophilic and anionic biomaterial surfaces providing lowintegrin
binding sites were shown to experience low integrin-mediated cell
spreading, leading to macrophage apoptosis [178]. These ndings
provide a clue for surface modication of biomaterials to elicit
desired PMN and macrophage activities.
Another strategy for guiding macrophage responses to bioma-
terials is the variation of roughness and surface topography
[179,180]. In their natural environment cells respond to ECM
components in the nanometer scale in terms of adhesion, prolif-
eration, migration, and gene expression. Imprinting of patterns at
micron and nanometer scales on material surfaces may mimic the
natural topography of the ECM [179]. Topographic patterns are
known to affect function of broblasts [181], epithelial cells [182],
and endothelial cells [183]. A differing response of macrophages to
micron-structured biomaterials has been demonstrated recently in
the context of the foreign body reaction [184]. Parallel gratings
imprinted on polymeric surfaces with line width ranging from
250 nmto 2 mmwere shown to affect macrophage morphology and
cytokine secretion in vitro, and macrophage adhesion in vivo
independent of the biomaterial surface chemistry [184].
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6700
4.2. Immunomodulation by incorporation of bioactive molecules
Current methods of biomaterial functionalization include
specic surface coatings and the incorporation of bioactive mole-
cules such as adhesion sites, growth factors, anti-inammatory
mediators or drugs either alone or combined [185].
4.2.1. Providing of integrin adhesion sites
Functionalization of biomaterial surfaces with specic integrin
binding sites represents a powerful strategy in directing responses
of inammatory cells (Fig. 4A). Attachment of short oligopeptide
sequences that make up receptor binding domains within adhesive
proteins have been shown to promote cell-specic adhesion and
function on biomaterials with arginineeglycineeaspartic acid
(RGD) being the most prominent domain (reviewed in Refs.
[186,187]). The RGD and PHSRN (proline-histidine-serine-arginine-
asparagine) domains of bronectin were identied to be crucial in
regulating macrophage function via a5b1 and anb3 integrin
signaling in vitro and in vivo [188,189]. Both domains, when
imprinted at a specic orientation on the biomaterial, were found
Fig. 4. Current strategies in the design of immunomodulating biomaterials. A) Alterations of physicochemical features of biomaterials like chemistry or topography. B) Func-
tionalization of biomaterials by incorporation of bioactive molecules like integrin adhesion sites as well as growth factors and anti-inammatory mediators. C) Coating of
biomaterials with articial ECM e mimicking the biofunctions of the natural ECM as a tool for modulating immune cell behavior. Collagen provides natural binding sites for cell
adhesion receptors (e.g. integrins). Proteoglycans are capable to interact with endogenous cytokines and growth factors allowing for their specic presentation to target cells.
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6701
to initiate distinct intracellular outside-in signal pathways
mediating macrophage adhesion and function upon ligation with
integrins [190]. As one consequence, RGD and PHSRN domains
mediated the formation of FBGC on biomaterial surfaces [189,190].
The incorporation of integrin ligands to biomaterials is often
combined with polyethylene glycol (PEG) coatings (PEGylation),
which renders biomaterial surfaces non-interactive (also referred
to as non-fouling) [191,192]. Surfaces modied with PEG resist
protein adsorption and are thus protected against passive cell
attachment and subsequent cell activation [193]. The advantage of
these combined approaches relies on the prevention of nonspecic
cellematerial interaction in favor of specic cell activation elicited
by recognition of integrin adhesion sites on the biomaterial surface
[194,195]. Further non-fouling coatings include dextran-based gels,
poly(ethylene oxide) (PEO), and alginate [196e198].
4.2.2. Coupling of anti-inammatory drugs to biomaterials
Another method of rendering biomaterials immunomodulatory
is the incorporation of anti-inammatory factors (Fig. 4A). Gluco-
corticoids are potent suppressors of immune responses (reviewed
in Ref. [199]). They inhibit inammatory cell activation by abro-
gating the synthesis of inammatory mediators including several
cytokines and chemokines, prostaglandins, leukotrienes, proteo-
lytic enzymes, free oxygen radicals and nitric oxide (NO). Simul-
taneously, they promote resolution of inammation and of adaptive
immune response by enhancing anti-inammatory cytokine
release and suppressing cellular [T helper (Th)1-directed] immu-
nity in favor of humoral (Th2-directed) immunity and tolerance.
Indeed, delivery at the implantation site of dexamethasone via
coupling to biomaterials results in reduced implant-associated
inammation as shown by decreased numbers of PMNs in the
initial inammatory phase and the absence of macrophages,
lymphocytes, and brous capsule formation in the later phase
[200,201]. However, an unwanted side effect of dexamethasone
treatment is the reduction of VEGF in the surrounding tissue
impeding angiogenesis and delaying wound healing [200,202]. By
combined administration of dexamethasone and VEGF this anti-
angiogenic effect could be overcome [202]. Though, dexametha-
sone needs to be supplied by the biomaterial throughout its
lifetime. As soon as delivery is subsided, the anti-inammatory
effects fade and PMNs and macrophages start an inammatory
response [203].
Loading of biomaterial surfaces with NO-releasing coatings has
evolved as an attractive strategy for durable control of immune
responses [204]. Continuous and slowly liberation of NO results in
reduced inammatory cell recruitment and performance at the
implant surface that sustains even after exhaustion of the NO
reservoir [205]. Down-regulation of inammatory cytokines such
as IL-6 and MCP-1 as well as induction of nitrosated proteins are
discussed to cause NO-mediated immunosuppression [206].
Furthermore, NO may induce macrophages to produce NO them-
selves explaining its long-lasting anti-inammatory activity [207].
4.2.3. Delivery of growth factors
A complex signaling network of growth factors, including
epidermal growth factor (EGF), broblast growth factor (FGF),
granulocyte macrophage colony stimulating factor (GM-CSF), TGF-
b, VEGF, and PDGF control adhesion, migration, proliferation, and
differentiation of broblasts, keratinocytes, and endothelial cells in
wound healing (reviewed in Ref. [208]). Although tissue cells are
the primary targets of the growth factors, biomaterials decorated
with these bioactive molecules can still be considered as immu-
nomodulatory (Fig. 4A). Wound healing in adult tissue is always
associated with an inammatory response [209] and there is a tight
cross-talk between immune cells and tissue cells regulating the
healing process [210e215]. Fibroblasts, for example, have been
shown to suppress MIP-1a release by activated macrophages [211]
and to differently modulate IL-10 and IL-12 production by mono-
cytes [212]. Monocytes develop broblast modulating activity as
seen by enhanced MMP-2 expression of broblasts only in response
to monocyte-derived GM-CSF. Vice versa, the activated broblasts
amplify GM-CSF production in monocytes suggesting synergistic
interactions during matrix remodeling [210]. Thus, modulating
broblast, endothelial cell and keratinocyte function by loading
biomaterials with growth factors also feeds back on the activity of
monocytes and macrophages [202,216e219]. Moreover, growth
factors also act directly on the immune cells, as shown for TGF-b1
and PDGF on macrophage chemotaxis and activation during wound
repair [220].
4.3. Designing immunomodulating biomaterials based on
articial ECM e concepts and recent ndings
The majority of functionalized biomaterials provide a single
bioactive signal. Presentation of several bioactive factors is closer to
the natural environment of the cells and may help tuning the
desired responses. Interaction with the ECM regulates cellular
behavior including migration, differentiation and proliferation
making the ECM an attractive tool for endowing biomaterials with
physiologic biofunctions (Fig. 4B).
4.3.1. Hydrogels
Considerable effort has been directed at mimicking the physi-
ologic ECM microenvironment in the design of tissue engineering
matrices. Natural and synthetic hydrogels are attractive matrices
due to their high water content and three-dimensional structure
resembling soft tissue [221]. Synthetic hydrogels are composed of
polymers like PEG that are bio-inert. In different approaches the
synthetic scaffolds have been rendered ECM-mimetic by grafting
them with typical biofunctions including the presentation of
receptor binding ligands for specic cell adhesion, the suscepti-
bility to proteolytic degradation and remodeling by cell-derived
proteases and the capability of binding and presentation of
growth factors (reviewed in Ref. [222]).
Hydrogels that are made of materials from natural sources
including ECM proteins (collagen, brin, gelatin) and poly-
saccharides (GAGs, dextran, alginate, chitosan) are already
provided with ECM-derived biofunctions [223]. Although they
usually have a low toxicity and rarely induce chronic inammatory
responses, the potential of pathogen transmission and immuno-
genicity of the materials restrain their use. A comparative study
evaluating the morphologic host tissue response to ve commer-
cially available ECM-derived biologic scaffolds revealed that the
scaffolds elicited distinct host responses depending on species of
origin, tissue of origin, processing methods, and/or method of
terminal sterilization [224]. However, natural ECM scaffolds are
widely used for tissue engineering in regenerative medicine due to
their simple design and economic fabrication in contrast to
synthetic hydrogels.
4.3.2. Articial ECM coatings for synthetic implants
The concept of modulating cellular responses through ECM-
mimetic biomaterials has also been exploited to improve the bio-
logical acceptance and integration of synthetic implants. The rst
coatings that were developed made either use of adhesive domains
of ECMproteins or ECM-derivedproteins as a whole. Various studies
reported of collagen coatings that supported cell attachment and
activity on implants in vitro and signicantly improved bone
maturation and mineralization at implantetissue interfaces in vivo
[225,226]. Early appearance of mononuclear phagocytozing cells
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6702
and higher expression of bone-specic matrix proteins associated
with increased early bone remodeling around the modied bioma-
terials were observed [227] indicating a modulatory capacity of the
collagen matrices on both immune and tissue cells. Collagen coat-
ings seem to promote specic cell implant interactions via integrin
b1as presentedfor rat calvarial osteoblasts [228]. Although, collagen
was shown to mediate adhesion and spreading of osteoblasts to the
implants, it had no effect on later processes such as proliferation,
differentiation, and mineralization of the osteoblasts [229].
Two conclusions can be drawn fromthese ndings. First, cellular
functions are not controllable only by promoting adhesion and
direct interaction with integrins and other cell surface receptors. It
also requires indirect modulation through inducing specic growth
factor responses. Second, to effectively modulate cell activity
biomaterial coatings should comprise the action of signaling
molecules. Biodegradable coatings locally releasing incorporated
growth factors like bone morphogenetic protein (BMP), insulin-like
growth factor (IGF), and TGF-b have been successfully tested to
stimulate fracture healing and to improve biomaterial performance
[230]. However, to yield proper biological effects, the growth
factors have to be supplied in non physiologically high amounts at
enormous costs. A more sophisticated approach therefore is to
utilize the growth factor regulating property of the ECM. Articial
ECM (aECM) coatings were developed by modifying collagen
matrices with GAGs and PGs [231] in order to create a biomaterial
environment mimicking the situation in vivo (Fig. 5A).
Proteoglycans are suggested to be important mediators of osteo-
blast attachment and adhesion. Osteoblasts cultured on titanium
were shown to synthesize sulfated GAGs that permeate the
cellebiomaterial interface and promote adhesion [232]. Additionally,
PGs should act as mediator between matrix and endogenous growth
factors, thus improving cell activity around implants and inuencing
biomaterial integration [233]. Recent investigations focus on modi-
cations of GAGs to inuence their interaction with specic growth
factors. Within the natural ECMthe sulfate groups in GAGs represent
one important growth factor binding site. Thus, incorporation of
additional sulfate groups to GAGs should improve the biological
properties of aECM coatings. For HA it was demonstrated that
modication with sulfate groups increased the binding afnity for
human BMP-4 [234]. Implant coatings containing sulfated GAGs may
thus allow for enhanced osteoinductive activity by specically inter-
acting with bone cell stimulating factors.
Invitro experiments revealed aECMcontaining sulfated GAGs like
CS promote cell adhesion as well as cell proliferation [235,236].
Several in vivo studies reported that addition of CS to collagen coat-
ings improved the osteoconductive properties of both titanium
implants [231,237] and hydroxyapatite bone cements [238,239].
When compared to uncoated implants and collagen matrices, bone
remodeling and new bone formation around the implants were
increased when CS was incorporated into the coatings (Fig. 5BeD). It
was suggested that CS mediates attachment of growth factors to the
ECM or cell surface thus stimulating both bone resorbing and bone-
forming cells [231,237e239]. However, whether a specic growth
factor response was modulated by the aECM coatings remains
unclear. An interesting hint in this respect was provided by a study
evaluatingosseointegrationof implants coatedwithaECMcomposed
of collagen, GAG and BMP-4 [240]. Interestingly, collageneGAG
coatings alone provedtobe as effective as those preloadedwithBMP-
4. The similar effects of both coatings might be due to the fact that
only a very low amount of growth factor was used. Another expla-
nation is the interaction of the collageneGAG matrix with endoge-
nous growth factors. The aECM coating might have stored growth
factors at the implant site and presented them to cell receptors
beneting bone formation. Besides GAGs, other ECM components,
either wholeproteins (osteocalcin[241]), peptides (phosphoserineas
anactive part of osteopontin[242]), or functionalities (sodiumcitrate
[242]) had positive effects on bone remodeling around hydrox-
yapatiteecollagen composite bone cements.
4.3.3. Immunomodulating effects of aECM coatings
ECM proteins are potent regulators of immune cell activity
[139,140]. Thus, aECM coatings may have the capability to control
Fig. 5. Use of aECM coatings for synthetic implants. A) General assembly of aECM coatings: collagen brils serve as protein substrate in which distinct glycosaminoglycans (GAGs)
are incorporated. B) Signicantly greater bone contact and increased new bone formation around titanium implants coated with type I collagen and chondroitin sulfate (Ti Coll CS)
as compared with uncoated titanium rods (Ti) in a rat tibia model [231] 7 days after implantation (Goldner Trichrome stain, original magnication 25). C) Signicantly increased
number of TRAP-positive osteoclasts (red) around the newly formed bone (B) at the surface of titanium implants coated with type I collagen and chondroitin sulfate (Ti Coll CS) as
a sign of increased bone remodeling 7 days after implantation [231]. Around the uncoated pins there is less bone contact and a brous interface (F) at the same time point
(enzymehistochemical stain against TRAP, original magnication 100). D) Increased new bone formation around Schanz screws coated with type I collagen and chondroitin sulfate
(Ti Coll CS) as compared to uncoated screws (Ti) in the medullary canal of sheep tibiae [237] 6 weeks after implantation (Goldner Trichrome stain, original
magnication 16).(For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article).
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6703
the inammatory response which in turn has an important inu-
ence on bone regeneration and wound healing (Fig. 4B). Natural
occurring GAGs have been identied to bind and modify inam-
matory factors such as interleukins and chemokines [243e245]. An
important immunoregulatory cytokine is IL-10, exhibiting both
suppressive and stimulatory effects on the immune system. IL-10
acts anti-inammatory on macrophages and DCs by reducing the
production of pro-inammatory cytokines and presenting antigens
to T cells. Sulfated GAGs have been reported to bind human IL-10
and to modulate its activity [244]. Interestingly, GAG-promoted
linkage of IL-10 to the surface of monocytes/macrophages resul-
ted in enhanced activity of IL-10 on the immune cells [244]. The
binding capability of the sulfated GAG was determined by grade of
sulfation, position of the sulfate groups (C4 or C6) and linkage of the
sulfate group to the sugar chain (N- or O-sulfation) indicating that
subtle differences in the GAG structure and/or sequence might be
sensed by signaling molecules and thus guides their interaction
with the ECM. Differential binding to subsets of various sulfated
GAGs was also observed for chemokines such as IL-8 [245]. Given
that GAG structures vary among tissues and that chemokine
interaction with GAG mediates their presentation to leukocytes, it
is conceivable that GAGs participate in determining the specicity
of leukocyte recruitment in vivo. This might be an important aspect
for the design of aECM coatings to modulate the invasion of
immune cells to the implantation site.
However, GAGs not only modulate the activity of inammatory
mediators they are also capable to directly interact with immune
cells. In this respect, CS and HA are of special interest because they
are known to act immunosuppressive on various cells. CS, for
example, is used as a therapeutic agent in osteoarthritis since it
exerts strong anti-inammatory effects in the joint [246]. It was
foundthat theimmunosuppressiveactivityof CSis basedonreduced
nuclear translocation of NF-KB, a key transcription factor of various
pro-inammatory mediators, due to an inhibition of the ERK1/2 and
p38MAPK pathway by CS [247]. Subsequent down-regulation of
expression of MMPs, IL-1b, TNFa, COX-2 and NOS2 resulted in
a reduced inammatory reaction. Of special note is that the anti-
inammatory activity of CS was not only found at the level of
chondrocytes and synovial cells but also on circulating peripheral
bloodmononuclear cells [248] suggestinganubiquitous actionof CS.
Thus, CS may have the capacity to act as an immunosuppressive
agent in a variety of inammatory conditions associated with NF-KB
activationincluding the foreignbody reaction. Furthermore, bothCS
and HA were identied as antioxidants capable of reducing free
radicals, protecting bothcells andmaterials fromROS damage [249].
CS may therefore provide biomaterials with both immunosuppres-
sive and antioxidant properties likely to modulate the inammatory
response toward resolution and healing.
The use of HA for aECM biomaterial coatings, however, is more
complex. HA features different immunomodulatory activities
depending on its molecular size [250]. Large HA polymers, as seen
under physiologic conditions, have anti-inammatory properties
and promote tissue integrity and quiescence. However, during
tissue injury and inammation, HA becomes cleaved into smaller
fragments that function as pro-inammatory and immunostimu-
latory mediators potentially serving as endogenous danger signal
triggering PRR of immunocompetent cells. Several studies have
reported of low molecular weight HA activating inammatory
cytokine and chemokine production in PMNs and monocytes/
macrophages as well as maturation of DCs via CD44 and/or TLR4
signaling, respectively [251e254]. Implant coating with HA might
therefore seem counterproductive, since cleavage may cause
Fig. 6. Immunomodulating effects of aECM coatings on dendritic cells. A) Composition of the tested aECM coatings. B, C) Articial ECM coatings attenuate DC maturation induced by
collagen and LPS-stimulation: immature DCs (iDCs) were generated by culture of human CD14 monocytes for 4 days in the presence of GM-CSF and IL-4. The iDCs were cultured
for 24 h on either aECM or collagen alone with and without co-stimulation with LPS at 10 ng/ml. Expression of DC maturation markers and release of IL-12, the main cytokine for
activation of T cell immunity were assessed by ow cytometry and ELISA, respectively. DC viability was assessed by staining with annexin V and propidium iodide. Viability of DC
after 24 h culture on aECM was the same as in controls (DC cultured on plastic) and in all experiments over 85% (data not shown). At least three independent experiments were
performed. ManneWhitney U test was used for statistical analysis (*p <0.05; **p <0.005). Error bars represent standard deviation.
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6704
further tissue injury. However, high molecular weight (HMW)-HA
might continue acting anti-inammatory even at sites of inam-
mation and promote and healing. In a model of non-infectious lung
injury, up-regulation of endogenous HMW-HA production pro-
tected against acute inammation due to reduced epithelial cell
apoptosis and accelerated tissue repair [252]. HMW-HA has also
been shown to promote the function of CD4CD25 T
Reg
[255]. In
uninjured or healing tissue, HMW-HA provides an important signal
for T
Reg
populations to persist, to resolve inammatory processes
and maintain homeostasis. There are reports of HA cross-linking to
cables at sites of inammation as part of a protective mechanism in
inammatory processes (reviewed in Ref. [256]). These HA cables
are pro-adhesive to leukocytes, but suppress their activation by
impeding the interaction with inammation-promoting receptors
on the underlying tissues. Moreover, the cross-linked HA structures
are suggested to sequester pro-inammatory mediators and to be
more resistant to degradation in injured tissue, both being impor-
tant events for limiting inammation.
Although all these ndings clearly emphasize the potential of
HMW-HA and CS to gear inammatory into regulatory processes
suggesting their potential use for immunomodulating biomaterial
coatings, data on controlling function of immunocompetent cells
by aECM composed of collagen and GAGs are lacking. Our group
recently addressed the immunomodulatory effects of aECM that
was generated utilizing the natural self-assembly potential of type I
collagen in combination with either HA or CS. HA was additionally
modied by attaching of sulfate groups at low (S1) or high levels
(S3) providing binding sites for endogenous growth factors and
inammatory mediators (Fig. 6A). Induction of tolerogenic DC
function by aECM would provide a powerful tool to promote
resolution of inammation and to modulate T cell activity at the
implantation site. In accordance with other studies [128] we found
that collagen alone provokes DC maturation (Fig. 6B). Culture of
immature DCs on collagen induces up-regulation of the co-
stimulatory molecules CD80 and CD86 (Fig. 6B) as well as release
of IL-12p40 (Fig. 6B), signals through which DCs direct differenti-
ation of T cells toward an immune response. Of note, incorporation
of the GAG derivates into the collagen matrix attenuated the
collagen driven DC maturation. Release of IL-12p40 and expression
of maturation marker (MHC, CD86, CD80) were down-regulated
following DC interaction with aECM (Fig. 6C). Moreover, DC
maturation induced by LPS, a potent activator of DCs, is also
diminished in the presence of aECM as seen by reduced expression
level of MHC and CD86 as well as decreased secretion of IL-12p40
(Fig. 6C). Dendritic cells that have been prevented to mature are
prone to develop a tolerogenic phenotype [113,114]. T cell stimu-
lation in an environment lacking of co-stimulation (as typical for
tolerogenic DC) can lead to T cell clonal anergy and adaptive
tolerance [257]. As one consequence the inammatory activity of T
cells at the implantation site could become compromised either by
T cell growth arrest or by down-regulating their effector functions.
Additionally T cells with a regulatory phenotype could be induced
capable to suppress nave T cell responses but also the activity of
innate immune cells like DC and macrophages through their release
of immunosuppressive IL-10 and TGF-b. We are currently investi-
gating T cell responses triggered by aECM-activated DC. Since our
data suggest immunomodulatory capacities of aECM for DC we will
further expand our investigations on innate immune cells including
PMNs and macrophages.
5. Conclusions
For a long time, biomaterial science focused on inert materials
which are recognized as foreign by the host but remain essentially
unchanged and tolerated due to their encapsulation in brous
tissue. However, it has been realized that permitting specic cell
responses may be benecial for biomaterial integration and
improve implant performance. Cellular responses at the implan-
tation site will also include immune responses to biomaterials
resulting in their rejection or degradation. With expanding
knowledge on these processes, biomaterial engineers have begun
to develop materials capable of avoiding such detrimental immune
responses. Current strategies for the design of these biomaterials
focus on alteration of material surfaces either by modication of
their physicochemical features or by rendering them biologically
active to systematically target cell behavior. Among the latter,
functionalization of biomaterials with articial ECM coatings
appears to be particularly promising.
Acknowledgements
This work was funded by the German Research Council (DFG
SFB-TR67, projects A3, B3, B5). We thank Markus Karsten for his
outstanding help in designing the graphics.
References
[1] Williams DF. On the nature of biomaterials. Biomaterials 2009;30:5897e909.
[2] Williams DF. On the mechanisms of biocompatibility. Biomaterials 2008;29:
2941e53.
[3] Wilson CJ, Clegg RE, Leavesley DI, Pearcy MJ. Mediation of biomaterialecell
interactions by adsorbed proteins: a review. Tissue Eng 2005;11:1e18.
[4] Gorbet MB, Sefton MV. Biomaterial-associated thrombosis: roles of coagu-
lation factors, complement, platelets and leukocytes. Biomaterials 2004;25:
5681e703.
[5] Schmaier AH. Contact activation: a revision. Thromb Haemost 1997;78:
101e7.
[6] Zhuo R, Siedlecki CA, Vogler EA. Autoactivation of blood factor XII at
hydrophilic and hydrophobic surfaces. Biomaterials 2006;27:4325e32.
[7] Chen X, Wang J, Paszti Z, Wang F, Schrauben JN, Tarabara VV, et al. Ordered
adsorption of coagulation factor XII on negatively charged polymer surfaces
probed by sum frequency generation vibrational spectroscopy. Anal Bioanal
Chem 2007;388:65e72.
[8] Vogler EA, Siedlecki CA. Contact activation of blood-plasma coagulation.
Biomaterials 2009;30:1857e69.
[9] Zhuo R, Siedlecki CA, Vogler EA. Competitive-protein adsorption in contact
activation of blood factor XII. Biomaterials 2007;28:4355e69.
[10] Sperling C, Fischer M, Maitz MF, Werner C. Blood coagulation on biomaterials
requires the combination of distinct activation processes. Biomaterials 2009;
30:4447e56.
[11] Fischer M, Sperling C, Werner C. Synergistic effect of hydrophobic and
anionic surface groups triggers blood coagulation in vitro. J Mater Sci Mater
Med 2010;21:931e7.
[12] Heemskerk JW, Bevers EM, Lindhout T. Platelet activation and blood coag-
ulation. Thromb Haemost 2002;88:186e93.
[13] Johne J, Blume C, Benz PM, Pozgajova M, Ullrich M, Schuh K, et al. Platelets
promote coagulation factor XII-mediated proteolytic cascade systems in
plasma. Biol Chem 2006;387:173e8.
[14] Hu WJ, Eaton JW, Ugarova TP, Tang L. Molecular basis of biomaterial-
mediated foreign body reactions. Blood 2001;98:1231e8.
[15] Tang L. Mechanisms of brinogen domains: biomaterial interactions.
J Biomater Sci Polym Ed 1998;9:1257e66.
[16] Savage B, Bottini E, Ruggeri ZM. Interaction of integrin alpha IIb beta 3 with
multiple brinogen domains during platelet adhesion. J Biol Chem 1995;270:
28812e7.
[17] Rodrigues SN, Goncalves IC, Martins MC, Barbosa MA, Ratner BD. Fibrinogen
adsorption, platelet adhesion and activation on mixed hydroxyl-/methyl-
terminated self-assembled monolayers. Biomaterials 2006;27:5357e67.
[18] Fischer M, Sperling C, Tengvall P, Werner C. The ability of surface charac-
teristics of materials to trigger leukocyte tissue factor expression. Biomate-
rials 2010;31:2498e507.
[19] Nilsson B, Ekdahl KN, Mollnes TE, Lambris JD. The role of complement in
biomaterial-induced inammation. Mol Immunol 2007;44:82e94.
[20] Lhotta K, Wurzner R, Kronenberg F, Oppermann M, Konig P. Rapid activation
of the complement system by cuprophane depends on complement
component C4. Kidney Int 1998;53:1044e51.
[21] Hed J, Johansson M, Lindroth M. Complement activation according to the
alternate pathway by glass and plastic surfaces and its role in neutrophil
adhesion. Immunol Lett 1984;8:295e9.
[22] Sarma JV, Ward PA. The complement system. Cell Tissue Res 2011;343:
227e35.
[23] Tengvall P, Askendal A, Lundstrom II. Ellipsometric in vitro studies on the
activation of complement by human immunoglobulins M and G after
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6705
adsorption to methylated silicon. Colloids Surf B Biointerfaces 2001;20:
51e62.
[24] Andersson J, Ekdahl KN, Larsson R, Nilsson UR, Nilsson B. C3 adsorbed to
a polymer surface can form an initiating alternative pathway convertase.
J Immunol 2002;168:5786e91.
[25] Johnson RJ. Complement activation during extracorporeal therapy:
biochemistry, cell biology and clinical relevance. Nephrol Dial Transplant
1994;9(Suppl. 2):36e45.
[26] Andersson J, Ekdahl KN, Lambris JD, Nilsson B. Binding of C3 fragments on
top of adsorbed plasma proteins during complement activation on a model
biomaterial surface. Biomaterials 2005;26:1477e85.
[27] KarpmanD, ManeaM, Vaziri-Sani F, Stahl AL, KristofferssonAC. Platelet activation
in hemolytic uremic syndrome. Semin Thromb Hemost 2006;32:128e45.
[28] Gorbet MB, Sefton MV. Material-induced tissue factor expression but not
CD11b upregulation depends on the presence of platelets. J Biomed Mater
Res A 2003;67:792e800.
[29] Morley DJ, Feuerstein IA. Adhesion of polymorphonuclear leukocytes to
protein-coated and platelet adherent surfaces. Thromb Haemost 1989;62:
1023e8.
[30] McFarland CD, Thomas CH, DeFilippis C, Steele JG, Healy KE. Protein
adsorption and cell attachment to patterned surfaces. J Biomed Mater Res
2000;49:200e10.
[31] Keselowsky BG, Bridges AW, Burns KL, Tate CC, Babensee JE, LaPlaca MC,
et al. Role of plasma bronectin in the foreign body response to biomaterials.
Biomaterials 2007;28:3626e31.
[32] McNally AK, Jones JA, Macewan SR, Colton E, Anderson JM. Vitronectin is
a critical protein adhesion substrate for IL-4-induced foreign body giant cell
formation. J Biomed Mater Res A 2008;86:535e43.
[33] Kilpadi KL, Chang PL, Bellis SL. Hydroxylapatite binds more serum proteins,
puried integrins, and osteoblast precursor cells than titanium or steel.
J Biomed Mater Res 2001;57:258e67.
[34] Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002;
110:673e87.
[35] Lowell CA, Berton G. Integrin signal transduction in myeloid leukocytes.
J Leukoc Biol 1999;65:313e20.
[36] Jenney CR, Anderson JM. Adsorbed serum proteins responsible for surface
dependent human macrophage behavior. J Biomed Mater Res 2000;49:
435e47.
[37] McNally AK, Anderson JM. Complement C3 participation in monocyte
adhesion to different surfaces. Proc Natl Acad Sci U S A 1994;91:10119e23.
[38] Keselowsky BG, Collard DM, Garcia AJ. Integrin binding specicity regulates
biomaterial surface chemistry effects on cell differentiation. Proc Natl Acad
Sci U S A 2005;102:5953e7.
[39] Keselowsky BG, Collard DM, Garcia AJ. Surface chemistry modulates focal
adhesion composition and signaling through changes in integrin binding.
Biomaterials 2004;25:5947e54.
[40] McNally AK, Macewan SR, Anderson JM. Alpha subunit partners to beta1 and
beta2 integrins during IL-4-induced foreign body giant cell formation.
J Biomed Mater Res A 2007;82:568e74.
[41] Werr J, Eriksson EE, Hedqvist P, Lindbom L. Engagement of beta2 integrins
induces surface expression of beta1 integrin receptors in human neutrophils.
J Leukoc Biol 2000;68:553e60.
[42] McNally AK, Anderson JM. Beta1 and beta2 integrins mediate adhesion
during macrophage fusion and multinucleated foreign body giant cell
formation. Am J Pathol 2002;160:621e30.
[43] Bianchi ME. DAMPs, PAMPs and alarmins: all we need to know about danger.
J Leukoc Biol 2007;81:1e5.
[44] Babensee JE. Interaction of dendritic cells with biomaterials. Semin Immunol
2008;20:101e8.
[45] Bennewitz NL, Babensee JE. The effect of the physical form of poly(lactic-co-
glycolic acid) carriers on the humoral immune response to co-delivered
antigen. Biomaterials 2005;26:2991e9.
[46] Kono H, Rock KL. How dying cells alert the immune system to danger. Nat
Rev Immunol 2008;8:279e89.
[47] Grandjean-Laquerriere A, Tabary O, Jacquot J, Richard D, Frayssinet P,
Guenounou M, et al. Involvement of toll-like receptor 4 in the inammatory
reaction induced by hydroxyapatite particles. Biomaterials 2007;28:400e4.
[48] Rogers TH, Babensee JE. Altered adherent leukocyte prole on biomaterials
in toll-like receptor 4 decient mice. Biomaterials 2010;31:594e601.
[49] Kobayashi SD, Voyich JM, Burlak C, DeLeo FR. Neutrophils in the innate
immune response. Arch Immunol Ther Exp (Warsz) 2005;53:505e17.
[50] Tang L, Jennings TA, Eaton JW. Mast cells mediate acute inammatory
responses to implanted biomaterials. Proc Natl Acad Sci U S A 1998;95:
8841e6.
[51] Zdolsek J, Eaton JW, Tang L. Histamine release and brinogen adsorption
mediate acute inammatory responses to biomaterial implants in humans.
J Transl Med 2007;5:31.
[52] Nimeri G, Ohman L, Elwing H, Wettero J, Bengtsson T. The inuence of
plasma proteins and platelets on oxygen radical production and F-actin
distribution in neutrophils adhering to polymer surfaces. Biomaterials 2002;
23:1785e95.
[53] Nimeri G, Majeed M, Elwing H, Ohman L, Wettero J, Bengtsson T. Oxygen
radical production in neutrophils interacting with platelets and surface-
immobilized plasma proteins: role of tyrosine phosphorylation. J Biomed
Mater Res A 2003;67:439e47.
[54] Labow RS, Meek E, Santerre JP. Neutrophil-mediated biodegradation of
medical implant materials. J Cell Physiol 2001;186:95e103.
[55] Patel JD, Krupka T, Anderson JM. iNOS-mediated generation of reactive
oxygen and nitrogen species by biomaterial-adherent neutrophils. J Biomed
Mater Res A 2007;80:381e90.
[56] Scapini P, Lapinet-Vera JA, Gasperini S, Calzetti F, Bazzoni F, Cassatella MA. The
neutrophil as acellular sourceof chemokines. Immunol Rev2000;177:195e203.
[57] Hidaka Y, Ito M, Mori K, Yagasaki H, Kafrawy AH. Histopathological and
immunohistochemical studies of membranes of deacetylated chitin deriva-
tives implanted over rat calvaria. J Biomed Mater Res 1999;46:418e23.
[58] VandeVord PJ, Matthew HW, DeSilva SP, Mayton L, Wu B, Wooley PH.
Evaluation of the biocompatibility of a chitosan scaffold in mice. J Biomed
Mater Res 2002;59:585e90.
[59] Park CJ, Gabrielson NP, Pack DW, Jamison RD, Wagoner Johnson AJ. The effect
of chitosan on the migration of neutrophil-like HL60 cells, mediated by IL-8.
Biomaterials 2009;30:436e44.
[60] Yamashiro S, Kamohara H, Wang JM, Yang D, Gong WH, Yoshimura T.
Phenotypic and functional change of cytokine-activated neutrophils:
inammatory neutrophils are heterogeneous and enhance adaptive immune
responses. J Leukoc Biol 2001;69:698e704.
[61] Gilroy DW. The endogenous control of acute inammation e from onset to
resolution. Drug Discov Today Ther Strat 2004;1:313e9.
[62] Anderson JM, Rodriguez A, Chang DT. Foreign body reaction to biomaterials.
Semin Immunol 2008;20:86e100.
[63] Hamilton JA. Nondisposable materials, chronic inammation, and adjuvant
action. J Leukoc Biol 2003;73:702e12.
[64] Jones JA, Chang DT, Meyerson H, Colton E, Kwon IK, Matsuda T, et al. Pro-
teomic analysis and quantication of cytokines and chemokines from
biomaterial surface-adherent macrophages and foreign body giant cells.
J Biomed Mater Res A 2007;83:585e96.
[65] Broughton G, Janis JE, Attinger CE. The basic science of wound healing. Plast
Reconstr Surg 2006;117:12Se34S.
[66] Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemo-
kine system in diverse forms of macrophage activation and polarization.
Trends Immunol 2004;25:677e86.
[67] Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activa-
tion. Nat Rev Immunol 2008;8:958e69.
[68] Martinez FO, Sica A, Mantovani A, Locati M. Macrophage activation and
polarization. Front Biosci 2008;13:453e61.
[69] Zhang X, Mosser DM. Macrophage activation by endogenous danger signals.
J Pathol 2008;214:161e78.
[70] Stein M, Keshav S, Harris N, Gordon S. Interleukin 4 potently enhances
murine macrophage mannose receptor activity: a marker of alternative
immunologic macrophage activation. J Exp Med 1992;176:287e92.
[71] Gratchev A, Guillot P, Hakiy N, Politz O, Orfanos CE, Schledzewski K, et al.
Alternatively activated macrophages differentially express bronectin and
its splice variants and the extracellular matrix protein betaIG-H3. Scand J
Immunol 2001;53:386e92.
[72] Gratchev A, Kzhyshkowska J, Utikal J, Goerdt S. Interleukin-4 and dexa-
methasone counterregulate extracellular matrix remodelling and phagocy-
tosis in type-2 macrophages. Scand J Immunol 2005;61:10e7.
[73] Mosser DM. The many faces of macrophage activation. J Leukoc Biol 2003;73:
209e12.
[74] Gerber JS, Mosser DM. Reversing lipopolysaccharide toxicity by ligating the
macrophage Fc gamma receptors. J Immunol 2001;166:6861e8.
[75] Bellingan GJ, Xu P, Cooksley H, Cauldwell H, Shock A, Bottoms S, et al.
Adhesion molecule-dependent mechanisms regulate the rate of macrophage
clearance during the resolution of peritoneal inammation. J Exp Med 2002;
196:1515e21.
[76] Stout RD, Jiang C, Matta B, Tietzel I, Watkins SK, Suttles J. Macrophages
sequentially change their functional phenotype in response to changes in
microenvironmental inuences. J Immunol 2005;175:342e9.
[77] Franz S, Hoeve MA, Wickert S, Janko C, Dranseld I. Clearance of apo Nph
induces an immunosuppressive response in pro-inammatory type-1 and
anti-inammatory type-2 MPhi. Autoimmunity 2009;42:275e7.
[78] Xia Z, Triftt JT. A review on macrophage responses to biomaterials. Biomed
Mater 2006;1:R1e9.
[79] DeFife KM, Jenney CR, McNally AK, Colton E, Anderson JM. Interleukin-13
induces human monocyte/macrophage fusion and macrophage mannose
receptor expression. J Immunol 1997;158:3385e90.
[80] Kao WJ, McNally AK, Hiltner A, Anderson JM. Role for interleukin-4 in
foreign-body giant cell formation on a poly(etherurethane urea) in vivo.
J Biomed Mater Res 1995;29:1267e75.
[81] McNally AK, Anderson JM. Interleukin-4 induces foreign body giant cells
from human monocytes/macrophages. Differential lymphokine regulation of
macrophage fusion leads to morphological variants of multinucleated giant
cells. Am J Pathol 1995;147:1487e99.
[82] Brodbeck WG, Macewan M, Colton E, Meyerson H, Anderson JM. Lympho-
cytes and the foreign body response: lymphocyte enhancement of macro-
phage adhesion and fusion. J Biomed Mater Res A 2005;74:222e9.
[83] Rodriguez A, Macewan SR, Meyerson H, Kirk JT, Anderson JM. The foreign
body reaction in T-cell-decient mice. J Biomed Mater Res A 2009;90:106e13.
[84] Kyriakides TR, Foster MJ, Keeney GE, Tsai A, Giachelli CM, Clark-Lewis I, et al.
The CC chemokine ligand, CCL2/MCP1, participates in macrophage fusion
and foreign body giant cell formation. Am J Pathol 2004;165:2157e66.
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6706
[85] Helming L, Gordon S. Molecular mediators of macrophage fusion. Trends Cell
Biol 2009;19:514e22.
[86] Helming L, Gordon S. Macrophage fusion induced by IL-4 alternative acti-
vation is a multistage process involving multiple target molecules. Eur J
Immunol 2007;37:33e42.
[87] McNally AK, DeFife KM, Anderson JM. Interleukin-4-induced macrophage
fusion is prevented by inhibitors of mannose receptor activity. Am J Pathol
1996;149:975e85.
[88] Cui W, Ke JZ, Zhang Q, Ke HZ, Chalouni C, Vignery A. The intracellular domain
of CD44 promotes the fusion of macrophages. Blood 2006;107:796e805.
[89] Han X, Sterling H, Chen Y, Saginario C, Brown EJ, Frazier WA, et al. CD47,
a ligand for the macrophage fusion receptor, participates in macrophage
multinucleation. J Biol Chem 2000;275:37984e92.
[90] Yagi M, Miyamoto T, Sawatani Y, Iwamoto K, Hosogane N, Fujita N, et al. DC-
STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant
cells. J Exp Med 2005;202:345e51.
[91] Moreno JL, Mikhailenko I, Tondravi MM, Keegan AD. IL-4 promotes the
formation of multinucleated giant cells from macrophage precursors by
a STAT6-dependent, homotypic mechanism: contribution of E-cadherin.
J Leukoc Biol 2007;82:1542e53.
[92] Anderson JM. Multinucleated giant cells. Curr Opin Hematol 2000;7:40e7.
[93] Xia ZD, Zhu TB, Du JY, Zheng QX, Wang L, Li SP, et al. Macrophages in degra-
dation of collagen/hydroxylapatite(CHA), beta-tricalciumphosphate ceramics
(TCP) articial bone graft. Aninvivo study. ChinMedJ (Engl) 1994;107:845e9.
[94] Christenson EM, Anderson JM, Hiltner A. Oxidative mechanisms of poly(-
carbonate urethane) and poly(ether urethane) biodegradation: in vivo and
in vitro correlations. J Biomed Mater Res A 2004;70:245e55.
[95] Santerre JP, Woodhouse K, Laroche G, Labow RS. Understanding the
biodegradation of polyurethanes: from classical implants to tissue engi-
neering materials. Biomaterials 2005;26:7457e70.
[96] Kalbacova M, Roessler S, Hempel U, Tsaryk R, Peters K, Scharnweber D, et al.
The effect of electrochemically simulated titanium cathodic corrosion
products on ROS production and metabolic activity of osteoblasts and
monocytes/macrophages. Biomaterials 2007;28:3263e72.
[97] Luttikhuizen DT, van Amerongen MJ, de Feijter PC, Petersen AH,
Harmsen MC, van Luyn MJ. The correlation between difference in foreign
body reaction between implant locations and cytokine and MMP expression.
Biomaterials 2006;27:5763e70.
[98] Jones JA, McNally AK, Chang DT, Qin LA, Meyerson H, Colton E, et al. Matrix
metalloproteinases and their inhibitors in the foreign body reaction on
biomaterials. J Biomed Mater Res A 2008;84:158e66.
[99] MacLauchlan S, Skokos EA, Meznarich N, Zhu DH, Raoof S, Shipley JM, et al.
Macrophage fusion, giant cell formation, and the foreign body response
require matrix metalloproteinase 9. J Leukoc Biol 2009;85:617e26.
[100] Zambuzzi WF, Paiva KB, Menezes R, Oliveira RC, Taga R, Granjeiro JM. MMP-
9 and CD68() cells are required for tissue remodeling in response to natural
hydroxyapatite. J Mol Histol 2009;40:301e9.
[101] Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell
behavior. Annu Rev Cell Dev Biol 2001;17:463e516.
[102] Liu Y, Min D, Bolton T, Nube V, Twigg SM, Yue DK, et al. Increased matrix
metalloproteinase-9 predicts poor wound healing in diabetic foot ulcers.
Diabetes Care 2009;32:117e9.
[103] Gretzer C, Emanuelsson L, Liljensten E, Thomsen P. The inammatory cell
inux and cytokines changes during transition from acute inammation to
brous repair around implanted materials. J Biomater Sci Polym Ed 2006;17:
669e87.
[104] Anderson JM, Jones JA. Phenotypic dichotomies in the foreign body reaction.
Biomaterials 2007;28:5114e20.
[105] Rodriguez A, Meyerson H, Anderson JM. Quantitative in vivo cytokine analysis
at synthetic biomaterial implant sites. J Biomed Mater Res A 2009;89:152e9.
[106] Eming SA, Krieg T, Davidson JM. Inammation in wound repair: molecular
and cellular mechanisms. J Invest Dermatol 2007;127:514e25.
[107] Moseley R, Hilton JR, Waddington RJ, Harding KG, Stephens P, Thomas DW.
Comparison of oxidative stress biomarker proles between acute and
chronic wound environments. Wound Repair Regen 2004;12:419e29.
[108] Ratner BD. Reducing capsular thickness and enhancing angiogenesis around
implant drug release systems. J Control Release 2002;78:211e8.
[109] Ratner BD, Bryant SJ. Biomaterials: where we have been and where we are
going. Annu Rev Biomed Eng 2004;6:41e75.
[110] Diegelmann RF, Evans MC. Wound healing: an overview of acute, brotic and
delayed healing. Front Biosci 2004;9:283e9.
[111] Stramer BM, Mori R, MartinP. The inammation-brosis link? AJekyll andHyde
role for blood cells during wound repair. J Invest Dermatol 2007;127:1009e17.
[112] Prantl L, Fichtner-Feigl S, Hofstaedter F, Lenich A, Eisenmann-Klein M,
Schreml S. Flow cytometric analysis of peripheral blood lymphocyte subsets
in patients with silicone breast implants. Plast Reconstr Surg 2008;121:
25e30.
[113] Lutz MB, Schuler G. Immature, semi-mature and fully mature dendritic cells:
which signals induce tolerance or immunity? Trends Immunol 2002;23:
445e9.
[114] Rutella S, Danese S, Leone G. Tolerogenic dendritic cells: cytokine modula-
tion comes of age. Blood 2006;108:1435e40.
[115] Hawiger D, Inaba K, Dorsett Y, Guo M, Mahnke K, Rivera M, et al. Dendritic
cells induce peripheral T cell unresponsiveness under steady state conditions
in vivo. J Exp Med 2001;194:769e79.
[116] Steinman RM, Hawiger D, Liu K, Bonifaz L, Bonnyay D, Mahnke K, et al.
Dendritic cell function in vivo during the steady state: a role in peripheral
tolerance. Ann N Y Acad Sci 2003;987:15e25.
[117] Jonuleit H, Schmitt E, Schuler G, Knop J, Enk AH. Induction of interleukin 10-
producing, nonproliferating CD4() T cells with regulatory properties by
repetitive stimulation with allogeneic immature human dendritic cells. J Exp
Med 2000;192:1213e22.
[118] Menges M, Rossner S, Voigtlander C, Schindler H, Kukutsch NA, Bogdan C,
et al. Repetitive injections of dendritic cells matured with tumor necrosis
factor alpha induce antigen-specic protection of mice from autoimmunity.
J Exp Med 2002;195:15e21.
[119] Vigouroux S, Yvon E, Biagi E, Brenner MK. Antigen-induced regulatory T
cells. Blood 2004;104:26e33.
[120] Geissmann F, Revy P, Regnault A, Lepelletier Y, Dy M, Brousse N, et al. TGF-
beta 1 prevents the noncognate maturation of human dendritic Langerhans
cells. J Immunol 1999;162:4567e75.
[121] Sato K, Yamashita N, Matsuyama T. Human peripheral blood monocyte-
derived interleukin-10-induced semi-mature dendritic cells induce anergic
CD4() and CD8() T cells via presentation of the internalized soluble
antigen and cross-presentation of the phagocytosed necrotic cellular frag-
ments. Cell Immunol 2002;215:186e94.
[122] Rutella S, Bonanno G, Procoli A, Mariotti A, de Ritis DG, Curti A, et al.
Hepatocyte growth factor favors monocyte differentiation into regulatory
interleukin (IL)-10IL-12low/neg accessory cells with dendritic-cell
features. Blood 2006;108:218e27.
[123] Rutella S, Bonanno G, Pierelli L, Mariotti A, Capoluongo E, Contemi AM, et al.
Granulocyte colony-stimulating factor promotes the generation of regula-
tory DC through induction of IL-10 and IFN-alpha. Eur J Immunol 2004;34:
1291e302.
[124] Della BS, Nicola S, Timofeeva I, Villa ML, Santoro A, Berardi AC. Are
interleukin-16 and thrombopoietin new tools for the in vitro generation of
dendritic cells? Blood 2004;104:4020e8.
[125] Frick JS, Grunebach F, Autenrieth IB. Immunomodulation by semi-mature
dendritic cells: a novel role of toll-like receptors and interleukin-6. Int J
Med Microbiol 2010;300:19e24.
[126] Babensee JE, Stein MM, Moore LK. Interconnections between inammatory and
immune responses in tissue engineering. Ann N Y Acad Sci 2002;961:360e3.
[127] Yoshida M, Babensee JE. Poly(lactic-co-glycolic acid) enhances maturation of
human monocyte-derived dendritic cells. J Biomed Mater Res A 2004;71:
45e54.
[128] Acharya AP, Dolgova NV, Clare-Salzler MJ, Keselowsky BG. Adhesive
substrate-modulation of adaptive immune responses. Biomaterials 2008;29:
4736e50.
[129] Kohl K, Schnautz S, Pesch M, Klein E, Aumailley M, Bieber T, et al. Subpop-
ulations of human dendritic cells display a distinct phenotype and bind
differentially to proteins of the extracellular matrix. Eur J Cell Biol 2007;86:
719e30.
[130] Brand U, Bellinghausen I, Enk AH, Jonuleit H, Becker D, Knop J, et al. Inuence
of extracellular matrix proteins on the development of cultured human
dendritic cells. Eur J Immunol 1998;28:1673e80.
[131] Babensee JE, Paranjpe A. Differential levels of dendritic cell maturation on
different biomaterials used in combination products. J Biomed Mater Res A
2005;74:503e10.
[132] Yoshida M, Babensee JE. Differential effects of agarose and poly(lactic-co-
glycolic acid) on dendritic cell maturation. J Biomed Mater Res A 2006;79:
393e408.
[133] Padovan E, Landmann RM, De LG. How pattern recognition receptor trig-
gering inuences T cell responses: a new look into the system. Trends
Immunol 2007;28:308e14.
[134] Chang DT, Colton E, Anderson JM. Paracrine and juxtacrine lymphocyte
enhancement of adherent macrophage and foreign body giant cell activation.
J Biomed Mater Res A 2009;89:490e8.
[135] Chang DT, Jones JA, Meyerson H, Colton E, Kwon IK, Matsuda T, et al.
Lymphocyte/macrophage interactions: biomaterial surface-dependent
cytokine, chemokine, and matrix protein production. J Biomed Mater Res A
2008;87:676e87.
[136] Marques AP, Reis RL, Hunt JA. Cytokine secretion from mononuclear cells
cultured in vitro with starch-based polymers and poly-L-lactide. J Biomed
Mater Res A 2004;71:419e29.
[137] Rodriguez A, Anderson JM. Evaluation of clinical biomaterial surface effects
on T lymphocyte activation. J Biomed Mater Res A 2010;92:214e20.
[138] Katzin WE, Feng LJ, Abbuhl M, Klein MA. Phenotype of lymphocytes asso-
ciated with the inammatory reaction to silicone gel breast implants. Clin
Diagn Lab Immunol 1996;3:156e61.
[139] de Fougerolles AR, Koteliansky VE. Regulation of monocyte gene expression
by the extracellular matrix and its functional implications. Immunol Rev
2002;186:208e20.
[140] Forster R, valos-Misslitz AC, Rot A. CCR7 and its ligands: balancing immunity
and tolerance. Nat Rev Immunol 2008;8:362e71.
[141] Hynes RO. The extracellular matrix: not just pretty brils. Science 2009;326:
1216e9.
[142] Daamen WF, van Moerkerk HT, Hafmans T, Buttafoco L, Poot AA,
Veerkamp JH, et al. Preparation and evaluation of molecularly-dened col-
lageneelastineglycosaminoglycan scaffolds for tissue engineering. Bioma-
terials 2003;24:4001e9.
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6707
[143] Badylak SF. Regenerative medicine and developmental biology: the role of
the extracellular matrix. Anat Rec B New Anat 2005;287:36e41.
[144] Dudhia J. Aggrecan, aging and assembly in articular cartilage. Cell Mol Life Sci
2005;62:2241e56.
[145] Taylor KR, Gallo RL. Glycosaminoglycans and their proteoglycans: host-
associated molecular patterns for initiation and modulation of inamma-
tion. FASEB J 2006;20:9e22.
[146] Korpos E, Wu C, Song J, Hallmann R, Sorokin L. Role of the extracellular
matrix in lymphocyte migration. Cell Tissue Res 2010;339:47e57.
[147] Kreuger J, Spillmann D, Li JP, Lindahl U. Interactions between heparan sulfate
and proteins: the concept of specicity. J Cell Biol 2006;174:323e7.
[148] Schultz GS, Wysocki A. Interactions between extracellular matrix and
growth factors in wound healing. Wound Repair Regen 2009;17:153e62.
[149] Rosso F, Giordano A, Barbarisi M, Barbarisi A. From cell-ECM interactions to
tissue engineering. J Cell Physiol 2004;199:174e80.
[150] Chen Q, Sivakumar P, Barley C, Peters DM, Gomes RR, Farach-Carson MC,
et al. Potential role for heparan sulfate proteoglycans in regulation of
transforming growth factor-beta (TGF-beta) by modulating assembly of
latent TGF-beta-binding protein-1. J Biol Chem 2007;282:26418e30.
[151] Mott JD, Werb Z. Regulation of matrix biology by matrix metalloproteinases.
Curr Opin Cell Biol 2004;16:558e64.
[152] McCawley LJ, Matrisian LM. Matrix metalloproteinases: theyre not just for
matrix anymore! Curr Opin Cell Biol 2001;13:534e40.
[153] Sheu BC, Hsu SM, Ho HN, Lien HC, Huang SC, Lin RH. A novel role of met-
alloproteinase in cancer-mediated immunosuppression. Cancer Res 2001;
61:237e42.
[154] Endo K, Takino T, Miyamori H, Kinsen H, Yoshizaki T, Furukawa M, et al.
Cleavage of syndecan-1 by membrane type matrix metalloproteinase-1
stimulates cell migration. J Biol Chem 2003;278:40764e70.
[155] Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, et al. Matrix
metalloproteinase-9 triggers the angiogenic switch during carcinogenesis.
Nat Cell Biol 2000;2:737e44.
[156] Engsig MT, Chen QJ, Vu TH, Pedersen AC, Therkidsen B, Lund LR, et al. Matrix
metalloproteinase 9 and vascular endothelial growth factor are essential for
osteoclast recruitment into developing long bones. J Cell Biol 2000;151:
879e89.
[157] Dallas SL, Rosser JL, Mundy GR, Bonewald LF. Proteolysis of latent trans-
forming growth factor-beta (TGF-beta)-binding protein-1 by osteoclasts. A
cellular mechanism for release of TGF-beta from bone matrix. J Biol Chem
2002;277:21352e60.
[158] Yu Q, Stamenkovic I. Cell surface-localized matrix metalloproteinase-9
proteolytically activates TGF-beta and promotes tumor invasion and angio-
genesis. Genes Dev 2000;14:163e76.
[159] Hangai M, Kitaya N, Xu J, Chan CK, Kim JJ, Werb Z, et al. Matrix metal-
loproteinase-9-dependent exposure of a cryptic migratory control site in
collagen is required before retinal angiogenesis. Am J Pathol 2002;161:
1429e37.
[160] Pirila E, Sharabi A, Salo T, Quaranta V, Tu H, Heljasvaara R, et al. Matrix
metalloproteinases process the laminin-5 gamma 2-chain and regulate
epithelial cell migration. Biochem Biophys Res Commun 2003;303:1012e7.
[161] Baker AH, Edwards DR, Murphy G. Metalloproteinase inhibitors: biological
actions and therapeutic opportunities. J Cell Sci 2002;115:3719e27.
[162] Murphy G, Nagase H. Progress in matrix metalloproteinase research. Mol
Aspects Med 2008;29:290e308.
[163] Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of met-
alloproteinases: structure, function, and biochemistry. Circ Res 2003;92:
827e39.
[164] Egeblad M, Werb Z. New functions for the matrix metalloproteinases in
cancer progression. Nat Rev Cancer 2002;2:161e74.
[165] Galis ZS, Khatri JJ. Matrix metalloproteinases in vascular remodeling and
atherogenesis: the good, the bad, and the ugly. Circ Res 2002;90:251e62.
[166] Smeets TJ, Kraan MC, Galjaard S, Youssef PP, Smith MD, Tak PP. Analysis of
the cell inltrate and expression of matrix metalloproteinases and granzyme
B in paired synovial biopsy specimens from the cartilageepannus junction in
patients with RA. Ann Rheum Dis 2001;60:561e5.
[167] Streuli C. Extracellular matrix remodelling and cellular differentiation. Curr
Opin Cell Biol 1999;11:634e40.
[168] Ratner BD, Allan AS, Schoen FJ, Lemons JE. Biomaterials science: an intro-
duction to materials in medicine. 2nd ed.; 2004.
[169] Ratner BD. The engineering of biomaterials exhibiting recognition and
specicity. J Mol Recognit 1996;9:617e25.
[170] Rungsiyakull C, Li Q, Sun G, Li W, Swain MV. Surface morphology optimi-
zation for osseointegration of coated implants. Biomaterials 2010;31:
7196e204.
[171] Lucas T, WaismanA, RanjanR, Roes J, KriegT, Muller W, et al. Differential roles of
macrophages in diverse phases of skin repair. J Immunol 2010;184:3964e77.
[172] Mirza R, DiPietro LA, Koh TJ. Selective and specic macrophage ablation is
detrimental to wound healing in mice. Am J Pathol 2009;175:2454e62.
[173] Bradley SG, White Jr KL, McCay JA, Brown RD, Musgrove DL, Wilson S, et al.
Immunotoxicity of 180 day exposure to polydimethylsiloxane (silicone)
uid, gel and elastomer and polyurethane disks in female B6C3F1 mice. Drug
Chem Toxicol 1994;17:221e69.
[174] Smith MJ, White Jr KL, Smith DC, Bowlin GL. In vitro evaluations of innate
and acquired immune responses to electrospun polydioxanone-elastin
blends. Biomaterials 2009;30:149e59.
[175] Smith MJ, Smith DC, Bowlin GL, White KL. Modulation of murine innate and
acquired immune responses following in vitro exposure to electrospun
blends of collagen and polydioxanone. J Biomed Mater Res A 2010;93:
793e806.
[176] Scotchford CA, Gilmore CP, Cooper E, Leggett GJ, Downes S. Protein
adsorption and human osteoblast-like cell attachment and growth on
alkylthiol on gold self-assembled monolayers. J Biomed Mater Res 2002;59:
84e99.
[177] Dinnes DL, Marcal H, Mahler SM, Santerre JP, Labow RS. Material surfaces
affect the protein expression patterns of human macrophages: a proteomics
approach. J Biomed Mater Res A 2007;80:895e908.
[178] Brodbeck WG, Patel J, Voskerician G, Christenson E, Shive MS, Nakayama Y,
et al. Biomaterial adherent macrophage apoptosis is increased by hydrophilic
and anionic substrates in vivo. Proc Natl Acad Sci U S A 2002;99:10287e92.
[179] Yim EK, Leong KW. Signicance of synthetic nanostructures in dictating
cellular response. Nanomedicine 2005;1:10e21.
[180] Fink J, Fuhrmann R, Scharnweber T, Franke RP. Stimulation of monocytes and
macrophages: possible inuence of surface roughness. Clin Hemorheol
Microcirc 2008;39:205e12.
[181] Schulte VA, Diez M, Moller M, Lensen MC. Surface topography induces
broblast adhesion on intrinsically nonadhesive poly(ethylene glycol)
substrates. Biomacromolecules 2009;10:2795e801.
[182] Andersson AS, Backhed F, von EA, Richter-Dahlfors A, Sutherland D,
Kasemo B. Nanoscale features inuence epithelial cell morphology and
cytokine production. Biomaterials 2003;24:3427e36.
[183] Dalby MJ, Riehle MO, Johnstone H, Affrossman S, Curtis AS. In vitro reaction
of endothelial cells to polymer demixed nanotopography. Biomaterials 2002;
23:2945e54.
[184] Chen S, Jones JA, Xu Y, Low HY, Anderson JM, Leong KW. Characterization of
topographical effects on macrophage behavior in a foreign body response
model. Biomaterials 2010;31:3479e91.
[185] Boontheekul T, Mooney DJ. Protein-based signaling systems in tissue engi-
neering. Curr Opin Biotechnol 2003;14:559e65.
[186] Hersel U, Dahmen C, Kessler H. RGD modied polymers: biomaterials for
stimulated cell adhesion and beyond. Biomaterials 2003;24:4385e415.
[187] Shin H, Jo S, Mikos AG. Biomimetic materials for tissue engineering.
Biomaterials 2003;24:4353e64.
[188] Kao WJ, Lee D, Schense JC, Hubbell JA. Fibronectin modulates macrophage
adhesion and FBGC formation: the role of RGD, PHSRN, and PRRARV
domains. J Biomed Mater Res 2001;55:79e88.
[189] Kao WJ, Liu Y. Utilizing biomimetic oligopeptides to probe bronectin-
integrin binding and signaling in regulating macrophage function in vitro
and in vivo. Front Biosci 2001;6:D992e9.
[190] Kao WJ, Lee D. In vivo modulation of host response and macrophage
behavior by polymer networks grafted with bronectin-derived biomimetic
oligopeptides: the role of RGD and PHSRN domains. Biomaterials 2001;22:
2901e9.
[191] Schmidt DR, Kao WJ. Monocyte activation in response to polyethylene glycol
hydrogels grafted with RGD and PHSRN separated by interpositional spacers
of various lengths. J Biomed Mater Res A 2007;83:617e25.
[192] Zhu J, Tang C, Kottke-Marchant K, Marchant RE. Design and synthesis of
biomimetic hydrogel scaffolds with controlled organization of cyclic RGD
peptides. Bioconjug Chem 2009;20:333e9.
[193] VandeVondele S, Voros J, Hubbell JA. RGD-grafted poly-L-lysine-graft-
(polyethylene glycol) copolymers block non-specic protein adsorption
while promoting cell adhesion. Biotechnol Bioeng 2003;82:784e90.
[194] Dalsin JL, Hu BH, Lee BP, Messersmith PB. Mussel adhesive protein mimetic
polymers for the preparation of nonfouling surfaces. J Am Chem Soc 2003;
125:4253e8.
[195] Shen M, Pan YV, Wagner MS, Hauch KD, Castner DG, Ratner BD, et al. Inhi-
bition of monocyte adhesion and brinogen adsorption on glow discharge
plasma deposited tetraethylene glycol dimethyl ether. J Biomater Sci Polym
Ed 2001;12:961e78.
[196] Monchaux E, Vermette P. Bioactive microarrays immobilized on low-fouling
surfaces to study specic endothelial cell adhesion. Biomacromolecules
2007;8:3668e73.
[197] Wang DA, Ji J, Sun YH, Shen JC, Feng LX, Elisseeff JH. In situ immobilization of
proteins and RGD peptide on polyurethane surfaces via poly(ethylene oxide)
coupling polymers for human endothelial cell growth. Biomacromolecules
2002;3:1286e95.
[198] Duggal S, Fronsdal KB, Szoke K, Shahdadfar A, Melvik JE, Brinchmann JE.
Phenotype and gene expression of human mesenchymal stem cells in algi-
nate scaffolds. Tissue Eng Part A 2009;15:1763e73.
[199] Franchimont D, Kino T, Galon J, Meduri GU, Chrousos G. Glucocorticoids and
inammation revisited: the state of the art. NIH clinical staff conference.
Neuroimmunomodulation 2002;10:247e60.
[200] Morais JM, Papadimitrakopoulos F, Burgess DJ. Biomaterials/tissue interac-
tions: possible solutions to overcome foreign body response. AAPS J 2010;
12:188e96.
[201] Patil SD, Papadimitrakopoulos F, Burgess DJ. Dexamethasone-loadedpoly(lactic-
co-glycolic) acid microspheres/poly(vinyl alcohol) hydrogel composite coatings
for inammation control. Diabetes Technol Ther 2004;6:887e97.
[202] Patil SD, Papadmitrakopoulos F, Burgess DJ. Concurrent delivery of dexa-
methasone and VEGF for localized inammation control and angiogenesis.
J Control Release 2007;117:68e79.
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6708
[203] Norton LW, Koschwanez HE, Wisniewski NA, Klitzman B, Reichert WM.
Vascular endothelial growth factor and dexamethasone release from non-
fouling sensor coatings affect the foreign body response. J Biomed Mater Res
A 2007;81:858e69.
[204] Hetrick EM, Prichard HL, Klitzman B, Schoensch MH. Reduced foreign body
response at nitric oxide-releasing subcutaneous implants. Biomaterials
2007;28:4571e80.
[205] Gifford R, Batchelor MM, Lee Y, Gokulrangan G, Meyerhoff ME, Wilson GS.
Mediation of in vivo glucose sensor inammatory response via nitric oxide
release. J Biomed Mater Res A 2005;75:755e66.
[206] Schwentker A, Vodovotz Y, Weller R, Billiar TR. Nitric oxide and wound
repair: role of cytokines? Nitric Oxide 2002;7:1e10.
[207] Bogdan C. Regulation of lymphocytes by nitric oxide. Methods Mol Biol
2011;677:375e93.
[208] Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth
factors and cytokines in wound healing. Wound Repair Regen 2008;16:
585e601.
[209] Martin P, Leibovich SJ. Inammatory cells during wound repair: the good,
the bad and the ugly. Trends Cell Biol 2005;15:599e607.
[210] Chung AS, Kao WJ. Fibroblasts regulate monocyte response to ECM-derived
matrix: the effects on monocyte adhesion and the production of inam-
matory, matrix remodeling, and growth factor proteins. J Biomed Mater Res
A 2009;89:841e53.
[211] Oshikawa K, Yamasawa H, Sugiyama Y. Human lung broblasts inhibit
macrophage inammatory protein-1alpha production by
lipopolysaccharide-stimulated macrophages. Biochem Biophys Res Commun
2003;312:650e5.
[212] Vancheri C, Mastruzzo C, Tomaselli V, Sortino MA, DAmico L, Bellistri G, et al.
Normal human lung broblasts differently modulate interleukin-10 and
interleukin-12 production by monocytes: implications for an altered
immune response in pulmonary chronic inammation. Am J Respir Cell Mol
Biol 2001;25:592e9.
[213] Schirmer C, Klein C, von BM, Simon JC, Saalbach A. Human broblasts
support the expansion of IL-17 producing T-cells via up-regulation of IL-23
production by dendritic cells. Blood; 2010.
[214] Saalbach A, Klein C, Schirmer C, Briest W, Anderegg U, Simon JC. Dermal
broblasts promote the migration of dendritic cells. J Invest Dermatol 2010;
130:444e54.
[215] Sundararaj KP, Samuvel DJ, Li Y, Sanders JJ, Lopes-Virella MF, Huang Y.
Interleukin-6 released from broblasts is essential for up-regulation of
matrix metalloproteinase-1 expression by U937 macrophages in coculture:
cross-talking between broblasts and U937 macrophages exposed to high
glucose. J Biol Chem 2009;284:13714e24.
[216] Li B, Davidson JM, Guelcher SA. The effect of the local delivery of platelet-
derived growth factor from reactive two-component polyurethane scaf-
folds on the healing in rat skin excisional wounds. Biomaterials 2009;30:
3486e94.
[217] De la Riva B, Sanchez E, Hernandez A, Reyes R, Tamimi F, Lopez-Cabarcos E,
et al. Local controlled release of VEGF and PDGF from a combined brushite-
chitosan system enhances bone regeneration. J Control Release 2010;143:
45e52.
[218] Pieper JS, Hafmans T, van Wachem PB, van Luyn MJ, Brouwer LA,
Veerkamp JH, et al. Loading of collagen-heparan sulfate matrices with bFGF
promotes angiogenesis and tissue generation in rats. J Biomed Mater Res
2002;62:185e94.
[219] Stefonek TJ, Masters KS. Immobilized gradients of epidermal growth factor
promote accelerated and directed keratinocyte migration. Wound Repair
Regen 2007;15:847e55.
[220] Hosgood G. Wound healing. The role of platelet-derived growth factor and
transforming growth factor beta. Vet Surg 1993;22:490e5.
[221] Hoffman AS. Hydrogels for biomedical applications. Adv Drug Deliv Rev
2002;54:3e12.
[222] Zhu J. Bioactive modication of poly(ethylene glycol) hydrogels for tissue
engineering. Biomaterials 2010;31:4639e56.
[223] Geutjes PJ, Daamen WF, Buma P, Feitz WF, Faraj KA, van Kuppevelt TH. From
molecules to matrix: construction and evaluation of molecularly dened
bioscaffolds. Adv Exp Med Biol 2006;585:279e95.
[224] Valentin JE, Badylak JS, McCabe GP, Badylak SF. Extracellular matrix bio-
scaffolds for orthopaedic applications. A comparative histologic study. J Bone
Joint Surg Am 2006;88:2673e86.
[225] Nagai M, Hayakawa T, Fukatsu A, Yamamoto M, Fukumoto M, Nagahama F,
et al. In vitro study of collagen coating of titanium implants for initial cell
attachment. Dent Mater J 2002;21:250e60.
[226] Morra M, Cassinelli C, Meda L, Fini M, Giavaresi G, Giardino R. Surface analysis
and effects on interfacial bone microhardness of collagen-coated titanium
implants: a rabbit model. Int J Oral Maxillofac Implants 2005;20:23e30.
[227] Rammelt S, Schulze E, Bernhardt R, Hanisch U, Scharnweber D, Worch H,
et al. Coating of titanium implants with type-I collagen. J Orthop Res 2004;
22:1025e34.
[228] Geissler U, Hempel U, Wolf C, Scharnweber D, Worch H, Wenzel K. Collagen
type I-coating of Ti6Al4V promotes adhesion of osteoblasts. J Biomed Mater
Res 2000;51:752e60.
[229] Becker D, Geissler U, Hempel U, Bierbaum S, Scharnweber D, Worch H, et al.
Proliferation and differentiation of rat calvarial osteoblasts on type I
collagen-coated titanium alloy. J Biomed Mater Res 2002;59:516e27.
[230] Schmidmaier G, Lucke M, Schwabe P, Raschke M, Haas NP, Wildemann B.
Collective review: bioactive implants coated with poly(D,L-lactide) and
growth factors IGF-I, TGF-beta1, or BMP-2 for stimulation of fracture healing.
J Long Term Eff Med Implants 2006;16:61e9.
[231] Rammelt S, Illert T, Bierbaum S, Scharnweber D, Zwipp H, Schneiders W.
Coating of titanium implants with collagen, RGD peptide and chondroitin
sulfate. Biomaterials 2006;27:5561e71.
[232] Nakamura HK, Butz F, Saruwatari L, Ogawa T. A role for proteoglycans in
mineralized tissue-titanium adhesion. J Dent Res 2007;86:147e52.
[233] Stadlinger B, Pilling E, Huhle M, Mai R, Bierbaum S, Scharnweber D, et al.
Evaluation of osseointegration of dental implants coated with collagen,
chondroitin sulphate and BMP-4: an animal study. Int J Oral Maxillofac Surg
2008;37:54e9.
[234] Hintze V, Moeller S, Schnabelrauch M, Bierbaum S, Viola M, Worch H, et al.
Modications of hyaluronan inuence the interaction with human bone
morphogenetic protein-4 (hBMP-4). Biomacromolecules 2009;10:3290e7.
[235] Wollenweber M, Domaschke H, Hanke T, Boxberger S, Schmack G, Gliesche K,
et al. Mimicked bioarticial matrix containing chondroitin sulphate on
a textile scaffold of poly(3-hydroxybutyrate) alters the differentiation of adult
human mesenchymal stem cells. Tissue Eng 2006;12:345e59.
[236] Bierbaum S, Douglas T, Hanke T, Scharnweber D, Tippelt S, Monsees TK, et al.
Collageneous matrix coatings on titanium implants modied with decorin
and chondroitin sulfate: characterization and inuence on osteoblastic cells.
J Biomed Mater Res A 2006;77:551e62.
[237] Rammelt S, Heck C, Bernhardt R, Bierbaum S, Scharnweber D, Goebbels J,
et al. In vivo effects of coating loaded and unloaded Ti implants with
collagen, chondroitin sulfate, and hydroxyapatite in the sheep tibia. J Orthop
Res 2007;25:1052e61.
[238] Schneiders W, Reinstorf A, Ruhnow M, Rehberg S, Heineck J, Hinterseher I,
et al. Effect of chondroitin sulphate on material properties and bone
remodelling around hydroxyapatite/collagen composites. J Biomed Mater
Res A 2008;85:638e45.
[239] Schneiders W, Reinstorf A, Biewener A, Serra A, Grass R, Kinscher M, et al. In
vivo effects of modication of hydroxyapatite/collagen composites with and
without chondroitin sulphate on bone remodeling in the sheep tibia.
J Orthop Res 2009;27:15e21.
[240] Stadlinger B, Pilling E, Mai R, Bierbaum S, Berhardt R, Scharnweber D, et al.
Effect of biological implant surface coatings on bone formation, applying
collagen, proteoglycans, glycosaminoglycans and growth factors. J Mater Sci
Mater Med 2008;19:1043e9.
[241] Rammelt S, Neumann M, Hanisch U, Reinstorf A, Pompe W, Zwipp H, et al.
Osteocalcin enhances bone remodeling around hydroxyapatite/collagen
composites. J Biomed Mater Res A 2005;73:284e94.
[242] Schneiders W, Reinstorf A, Pompe W, Grass R, Biewener A, Holch M, et al.
Effect of modication of hydroxyapatite/collagen composites with sodium
citrate, phosphoserine, phosphoserine/RGD-peptide and calcium carbonate
on bone remodelling. Bone 2007;40:1048e59.
[243] Mummery RS, Rider CC. Characterization of the heparin-binding properties
of IL-6. J Immunol 2000;165:5671e9.
[244] Salek-Ardakani S, Arrand JR, Shaw D, Mackett M. Heparin and heparan sulfate
bind interleukin-10 and modulate its activity. Blood 2000;96:1879e88.
[245] Witt DP, Lander AD. Differential binding of chemokines to glycosamino-
glycan subpopulations. Curr Biol 1994;4:394e400.
[246] du Souich P, Garcia AG, Verges J, Montell E. Immunomodulatory and anti-
inammatory effects of chondroitin sulphate. J Cell Mol Med 2009;13:1451e63.
[247] Vallieres M, du SP. Modulation of inammation by chondroitin sulfate.
Osteoarthritis Cartilage 2010;18(Suppl. 1):S1e6.
[248] Herrero-Beaumont G, Marcos ME, Sanchez-Pernaute O, Granados R,
Ortega L, Montell E, et al. Effect of chondroitin sulphate in a rabbit model
of atherosclerosis aggravated by chronic arthritis. Br J Pharmacol 2008;
154:843e51.
[249] Campo GM, Avenoso A, Campo S, Ferlazzo A, Altavilla D, Micali C, et al.
Aromatic trap analysis of free radicals production in experimental collagen-
induced arthritis in the rat: protective effect of glycosaminoglycans treat-
ment. Free Radic Res 2003;37:257e68.
[250] Stern R, Asari AA, Sugahara KN. Hyaluronan fragments: an information-rich
system. Eur J Cell Biol 2006;85:699e715.
[251] Sconocchia G, Campagnano L, Adorno D, Iacona A, Cococcetta NY, Boffo V,
et al. CD44 ligation on peripheral blood polymorphonuclear cells induces
interleukin-6 production. Blood 2001;97:3621e7.
[252] Jiang D, Liang J, Fan J, Yu S, Chen S, Luo Y, et al. Regulation of lung injury and
repair by Toll-like receptors and hyaluronan. Nat Med 2005;11:1173e9.
[253] Yamawaki H, Hirohata S, Miyoshi T, Takahashi K, Ogawa H, Shinohata R, et al.
Hyaluronan receptors involved in cytokine induction in monocytes. Glycobiology
2009;19:83e92.
[254] Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, Ahrens T, et al. Oligo-
saccharides of hyaluronan activate dendritic cells via toll-like receptor 4.
J Exp Med 2002;195:99e111.
[255] Bollyky PL, Falk BA, Wu RP, Buckner JH, Wight TN, Nepom GT. Intact
extracellular matrix and the maintenance of immune tolerance: high
molecular weight hyaluronan promotes persistence of induced CD4CD25
regulatory T cells. J Leukoc Biol 2009;86:567e72.
[256] Day AJ, de la Motte CA. Hyaluronan cross-linking: a protective mechanism in
inammation? Trends Immunol 2005;26:637e43.
[257] Schwartz RH. T cell anergy. Annu Rev Immunol 2003;21:305e34.
S. Franz et al. / Biomaterials 32 (2011) 6692e6709 6709