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Biomaterials 32 (2011) 6692e6709

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

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
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
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 15 March 2011
Accepted 26 May 2011
Available online 28 June 2011

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.

Keywords:
Immune response to biomaterials
Immunomodulation
Extracellular matrix
Biomaterial integration

1. Introduction

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 transmembrane protein; ECM, extracellular matrix; EGF, epidermal growth factor; ENA78, epithelial cell-derived neutrophil attractant-78; ERK, extracellular signalregulated 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, interferon 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 metalloproteinases; NO, nitric oxide; NOS2, nitric oxide synthase 2; PAMPs, pathogenassociated molecular patterns; PDGF, platelet-derived growth factor; PEG, polyethylene glycol; PEO, poly(ethylene oxide); PGs, proteoglycans; PMN, polymorphonuclear 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;
TReg, regulatory T lymphocytes; VEGF, vascular endothelial growth factor.
* Corresponding author. Department of Dermatology, Venerology and Allergology, 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).
0142-9612/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2011.05.078

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, Williams 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 presented 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.

S. Franz et al. / 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 initiates 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, respectively. 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 catalyzed 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 concentrations of thrombin released by FXII on biomaterials are suggested
to activate platelets which in turn release mediators of the coagulation 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 biomaterial 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

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[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 anaphylatoxins C3a and C5a (reviewed in Ref. [22]). Activation of 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 demonstrated 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 can spontaneously adsorb to the
biomaterial surface while adopting a new conformation mimicking
that of surface bound C3b of the alternative pathway [24]. The bound
C3 then promotes the assembly of the initiating C3 convertase of the
alternative pathway. C3b arising from the proteolytic cleavage of C3
by coagulation factors including FXIIa and thrombin can also directly
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 convertases 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 implantation site [26]. Both anaphylatoxins can contribute to the onset of
inammatory responses at implantation sites through their multitude of effector functions including triggering of mast cell degranulation, 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 vitronectin 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

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S. Franz et al. / Biomaterials 32 (2011) 6692e6709

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

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 activation 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, phagocytosis, 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 immunocompetent 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 inammation 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 by cells 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 macrophages and dendritic cells (DCs) via pattern recognition receptors
(PRR) such as scavenger receptors, toll-like receptors (TLR) and Ctype 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, preferentially 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, inammatory cells e predominantely polymorphonuclear leukocytes
(PMNs, granulocytes) e migrate from the 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

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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 MIP1b [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 implantation 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 chemokines 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 woundhealing 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 MCP1 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.

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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 immunity 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 contributing to the production of ECM and by activation of broblasts
[67,68,70e72]. Although wound-healing macrophages exert antiinammatory 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, prostaglandins, 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 macrophages, 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 macrophages 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 adaptivity 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 macrophage chemotaxis toward each other [85].
Moreover, the properties of the biomaterial surface are important 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

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 inammation 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 macrophages 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 connective 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 pharmacological 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 dramatically change the cellular environment around the biomaterial and
to modify migration, differentiation and active state of macrophages and other immune cells [101]. Increased levels of MMP-9
have been suggested to be indicative of inammation and associated with poor wound healing [102]. An environment rich in MMP9 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 alternative activation state. The fusion-inducing cytokines IL-4 and IL13 are known to promote wound-healing macrophages during
inammation [67,71,72]. This transition is reected by alterations

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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 activated macrophages [64]. Over time the majority of these
inammatory cytokines is down-regulated in favor of IL-10, TGFb, 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. Moreover, 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 phenotype switch [64,105]. These ndings support the dogma that
implant surface properties dictate macrophage responses. More
importantly, they show that macrophage behavior is predominantly 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, downregulation 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 counterregulated 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 to biomaterials. DCs are key players 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 (TReg) (reviewed in
Refs. [113,114]). Whether DCs induce an immunogenic or a tolerogenic T cell response depend on many factors including the state of
DC maturation and the cytokine environment [114]. According to the
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]. Semimature DCs expressing MHC and co-stimulatory molecules but
unable to produce pro-inammatory cytokines such as IL-12, TNFa,

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IL-6 and IL-1b have been described to convert nave T cells into
CD4/CD25 TReg 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 regulators, various cytokines and growth factors including IL-6 and TNFa
at low concentrations as well as IL-16, granulocyte colony stimulating 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 component. 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 lectins. 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 serum stimulated 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 alternative 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 maturation 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 histocompatibility 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 (TNFa and IL-6) known to further amplify DC maturation by
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, respectively [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 macrophages 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 inammatory 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 inammatory 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]. Nevertheless, 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, T cell 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 lymphocytes 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 system described so far occur in vivo in the presence
of the ECM. Several ECM proteins are potent regulators of monocyte/
macrophage adhesion and activation and are involved in DC
migration and maturation [139,140]. Bidirectional interactions of
ECM, cells and growth factors/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 threedimensional scaffold for cellular organization. In addition it regulates 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 nonsulfated GAG. It is also not attached to a protein core whereas the
sulfated GAGs are linked to serine rich proteins to form proteoglycans (PGs) [144]. The glycan matrix of the ECM serves as lubricant and provides a reservoir for signaling molecules. Additionally,

S. Franz et al. / Biomaterials 32 (2011) 6692e6709

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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.

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, syndecans 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 intracellular 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].

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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, membranetype (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 osteoclasts [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 fragments 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 transcription by cytokines and integrin clustering and after secretion by
endogenous inhibitors including a2-macroglobulin and TIMPs
[161e163]. Whereas cytokine and integrin signaling up- or downregulate MMP expression, the protease inhibitors and here specifically 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 dysregulation 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 threedimensional scaffold for cells, it also plays a highly functionalized
role in directing cell behavior by spatially and temporarily
concerted interaction with other cells, ECM components, degrading
enzymes and signaling molecules all of which being relevant to
tissue repair, host responses to biomaterial and ultimately biomaterial 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 biomaterials, 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 inammation and macrophage activation are crucial for tissue repair [171,172].
With growing knowledge on the processes of wound healing and host
responses to biomaterials the eld of biomaterial engineering has
begun to address the development of materials with immunomodulating 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
modulation of functions of innate and adaptive 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 molecules 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 and conformation of serum proteins 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 chemistrydependent 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 low integrin
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 biomaterials 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, proliferation, 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 nm to 2 mm were 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

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Fig. 4. Current strategies in the design of immunomodulating biomaterials. A) Alterations of physicochemical features of biomaterials like chemistry or topography. B) Functionalization 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.

4.2. Immunomodulation by incorporation of bioactive molecules


Current methods of biomaterial functionalization include
specic surface coatings and the incorporation of bioactive molecules 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-arginineasparagine) 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

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S. Franz et al. / Biomaterials 32 (2011) 6692e6709

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). Glucocorticoids are potent suppressors of immune responses (reviewed
in Ref. [199]). They inhibit inammatory cell activation by abrogating the synthesis of inammatory mediators including several
cytokines and chemokines, prostaglandins, leukotrienes, proteolytic enzymes, free oxygen radicals and nitric oxide (NO). Simultaneously, they promote resolution of inammation and of adaptive
immune response by enhancing anti-inammatory cytokine
release and suppressing cellular [T helper (Th)1-directed] immunity 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 antiangiogenic effect could be overcome [202]. Though, dexamethasone 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 themselves 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), TGFb, 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 immunomodulatory (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 monocytes [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 physiologic 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 susceptibility 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 polysaccharides (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 immunogenicity of the materials restrain their use. A comparative study
evaluating the morphologic host tissue response to ve commercially 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 ECMmimetic biomaterials has also been exploited to improve the biological acceptance and integration of synthetic implants. The rst
coatings that were developed made either use of adhesive domains
of ECM proteins or ECM-derived proteins 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

and higher expression of bone-specic matrix proteins associated


with increased early bone remodeling around the modied biomaterials were observed [227] indicating a modulatory capacity of the
collagen matrices on both immune and tissue cells. Collagen coatings seem to promote specic cell implant interactions via integrin
b1 as presented for 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 from these 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 osteoblast 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 modications of GAGs to inuence their interaction with specic growth
factors. Within the natural ECM the sulfate groups in GAGs represent
one important growth factor binding site. Thus, incorporation of

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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 interacting with bone cell stimulating factors.
In vitro experiments revealed aECM containing 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 coatings 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 boneforming 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
evaluating osseointegration of implants coated with aECM composed
of collagen, GAG and BMP-4 [240]. Interestingly, collageneGAG
coatings alone proved to be as effective as those preloaded with BMP4. The similar effects of both coatings might be due to the fact that
only a very low amount of growth factor was used. Another explanation is the interaction of the collageneGAG matrix with endogenous 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 whole proteins (osteocalcin [241]), peptides (phosphoserine as
an active part of osteopontin [242]), or functionalities (sodium citrate
[242]) had positive effects on bone remodeling around hydroxyapatiteecollagen 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).

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S. Franz et al. / Biomaterials 32 (2011) 6692e6709

the inammatory response which in turn has an important inuence on bone regeneration and wound healing (Fig. 4B). Natural
occurring GAGs have been identied to bind and modify inammatory 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 resulted 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

found that the immunosuppressive activity of CS is based on reduced


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 antiinammatory activity of CS was not only found at the level of
chondrocytes and synovial cells but also on circulating peripheral
blood mononuclear cells [248] suggesting an ubiquitous action of CS.
Thus, CS may have the capacity to act as an immunosuppressive
agent in a variety of inammatory conditions associated with NF-KB
activation including the foreign body reaction. Furthermore, both CS
and HA were identied as antioxidants capable of reducing free
radicals, protecting both cells and materials from ROS damage [249].
CS may therefore provide biomaterials with both immunosuppressive 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 immunostimulatory 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

further tissue injury. However, high molecular weight (HMW)-HA


might continue acting anti-inammatory even at sites of inammation and promote and healing. In a model of non-infectious lung
injury, up-regulation of endogenous HMW-HA production protected 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 TReg [255]. In
uninjured or healing tissue, HMW-HA provides an important signal
for TReg 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 important 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 costimulatory molecules CD80 and CD86 (Fig. 6B) as well as release
of IL-12p40 (Fig. 6B), signals through which DCs direct differentiation 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 stimulation 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 investigating 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

6705

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 implantation 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.
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