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. 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