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1700093 (1 of 13) © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Modulating Immunological Responses of Electrospun Fibers for Tissue Engineering

Nowsheen Goonoo

DOI: 10.1002/adbi.201700093

of the major issues remains the innate immune response elicited by the scaffold which often leads to fibrosis and eventu-ally failure of the scaffold.[2–4] For suc-cess of TE scaffolds, the latter need to be immune-tolerant and capable of stimu-lating stem cell recruitment, proliferation and differentiation.[5]

In addition to suitable mechanical properties and appropriate degradation behaviors, TE scaffolds need to be biocom-patible i.e., support the appropriate cellular activity, in order to optimize tissue regen-eration, without eliciting any undesirable local or systemic responses in the eventual host. However, any biomaterial implanted in vivo results in immune responses and the inflammation process is mainly driven by the scaffolds’ physicochemical properties.[6] Therefore, the primary goal is to minimize the risk of failure by regu-lating the response such that it promotes healing.[7] Scaffolds should be designed in such a way that they allow matrix deposi-

tion and tissue formation in the early stages without hampering tissue growth in the later stages. In particular, the rate of degra-dation of the scaffold should match that of new tissue formation. Material properties of the scaffolds impact on cell recruitment/differentiation, degradation rate and immune response.

Inflammation plays an important role in the early stages of wound healing.[8] Inflammation helps to remove necrotic and apoptotic cells, cleaved ECM molecules, and subsequently, to initiate angiogenesis and tissue repair.[9] In fact, inflamma-tory cells have been shown to play a role in regeneration.[10] However, excessive and chronic inflammation leads to the formation of a hostile environment for tissue regeneration and repair. Excessive inflammation and ECM remodeling lead to upregulation of matrix metalloproteinases (MMPs) and increased deposition of collagen type I and III.[11] It has been reported that controlled inflammatory response with a reduced expression of proinflammatory cytokines, transforming growth factor-beta (TGF-β) and overexpression of interleukin-10 (IL-10) led to better wound healing[12,13] and promoted mesenchymal stem cell (MSC)-mediated bone tissue regeneration.[14,15] It was rightly proposed that it is the quality and not the quantity of inflammation which influence the scaffolds’ success.[5] Hence, it is crucial that the beneficial aspects of the innate immune response be harnessed while limiting the potential detrimental aspects. The foreign body response and thus excessive inflam-mation following implantation of scaffolds may be modulated

The promise of tissue engineering is to improve or restore functions of impaired tissues or organs. However, one of the biggest challenges to its translation to clinical applications is the lack of tissue integration and func-tionality. The plethora of cellular and molecular events occurring following scaffold implantation is a major bottleneck. Recent studies confirmed that inflammation is a crucial component influencing tissue regeneration. Immuno-modulation or immune-engineering has been proposed as a poten-tial solution to overcome this key challenge in regenerative medicine. In this review, strategies to modify scaffold physicochemical properties through the use of the electrospinning technique to modulate host response and improve scaffold integration will be discussed. Electrospinning, being highly versatile allows the fabrication of ECM-mimicking scaffolds and also offers the possibility to control scaffold properties for instance, tailoring of fiber properties, chemical conjugation or physical adsorption of non-immunogenic materials on the scaffold surface, encapsulating cells or anti-inflammatory molecules within the scaffold. Such electrospun scaffold-based immune-engi-neering strategies can significantly improve the resulting outcomes of tissue engineering scaffolds.

Tissue Engineering

Dr. N. GoonooPhysical Chemistry IDepartment of Chemistry and Biology & Research Center of Micro and Nanochemistry and Engineering (Cµ)University of Siegen57076 Siegen, GermanyE-mail: [email protected]. N. GoonooBiomaterialsDrug Delivery & Nanotechnology UnitCentre for Biomedical and Biomaterials ResearchMSIRI BuildingUniversity of MauritiusRéduit, Mauritius

1. Introduction

Tissue engineering aims at improving or restoring compro-mised tissue functions and it involves the use of an extra-cellular matrix (ECM)-mimicking scaffold as a temporary structural template to drive tissue regeneration. The scaffold which may or may not be seeded with cells is then either incu-bated in a bioreactor or implanted in situ using the human body both as the bioreactor and cell source for remodeling of scaf-folds giving rise to neo-tissue.[1] Despite enormous advances in the field of tissue engineering (TE) over the last decade, one

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by careful control of physical and chemical properties of the scaffolds to promote tissue regeneration.

Electrospinning has been recognized as a scaffold fabrication technique with enormous potential. The cost-effectiveness, sim-plicity as well as the possibility of controlling fiber morphology and fiber deposition pattern makes electrospinning a powerful method to fabricate tissue-engineered scaffolds with a defined micro/nanoarchitecture in terms of fiber size and fiber orien-tation.[16,17] Accordingly, electrospun substrates have been used for a wide range of tissue engineering applications including neural, cardiovascular, bone, and skin tissue engineering.[18–20] It is essential to understand the scaffold-host inflammatory reactions not only to predict the fate of these materials fol-lowing implantation but also to modulate the immune system to improve tissue regeneration and healing. A top priority is the development of innovative approaches to control the immune system via modification of scaffold design principles. In this review, the possibility to control the innate immune responses of electrospun scaffolds by (i) changing the scaffold chemistry (ii) modifying the physical properties of the scaffold, (iii) con-trolling the release of anti- or proinflammatory cytokines from scaffolds, and (iv) using cell-based therapy methods will be discussed.

2. Inflammation in Tissue Engineering: The Balance between Scaffold Integration and Isolation

The human immune system consists of innate and adap-tive immune systems which play a significant role in reacting against any foreign implanted material. Host reactions to the implanted scaffold determine the success of integration and its biological performance.[21]

Host reactions following scaffold implantation can be catego-rized into 8 different stages namely blood-material interactions, provisional matrix formation, inflammation, development of granulation tissue, foreign body reaction, and fibrous capsule development (fibrosis).[22–27] The timing and magnitude of these steps depend on the nature of the scaffold. In the early stages of scaffold implantation, disruption caused in the vasculature causes leaking of blood around the scaffold, initiating a blood-material interaction cascade. Plasma components such as pro-teins, sugars, lipids and ions adsorb to the surface of the scaffold within minutes.[28,29] Factors such as surface topography, rough-ness, chemistry, hydrophobicity and surface charge determine the types and amount of adsorbed molecules and also influence the recruitment and attachment of tissue derived inflammatory, vascular and stromal cells over the next few hours.[30–33] Due to presence of platelets in blood exudate, a provisional fibrin-rich clot is then formed over the injury site.[34,35] This clot acts as a depot for cytokines and growth factors which send signals to initiate wound repair. It also serves as a provisional matrix for cell attachment and migration.[36] Complement proteins from plasma together with specific structures such as danger associated molecular patterns (DAMPs) initiate the innate immune response, triggering the release of several proin-flammatory cytokines and chemokines which, in turn induce

directed chemotaxis of other innate inflammatory cells.[37] This process is subsequently taken over by the production of inflammatory cytokines or chemokines such as IL-8.[38–42] Poly-morphonuclear neutrophils (PMNs) which are essential to the innate immune response, help to remove cellular debris by phagocytosis and also allow elimination of pathogens via the release of reactive oxygen species (ROS) and inflammatory cytokines namely IL-1β, IFN-γ and TNF-α.[21,25,26,43,44] Simulta-neously, monocytes migrate to the active site and differentiate into macrophages and as a result of this, acute inflammation, and subsequently, chronic inflammation occurs.[45] In a normal inflammatory reaction, phagocytes engulf and digest the for-eign body. However, as the scaffolds are much larger than the phagocytes, “frustrated” phagocytosis may occur whereby enzymes are released to degrade the material.[46] Acute inflam-mation stimulates differentiation of monocytes towards the M1 macrophage phenotype which is considered as the proinflam-matory type. M1 macrophages amplify local inflammation but they are also important for clearing the injury site.[47,48] In the case of biocompatible implanted materials, the acute inflamma-tory response is usually resolved within one week and chronic inflammation generally lasts no longer than 2 weeks and is confined to the implantation site.[49]

Depending on the scaffold nature and properties there are 2 possibilities for the next stages of host response (Figure 1A). The formation of granulation tissue and extensive produc-tion of collagen around the scaffold leads to the formation of a fibrous capsule, isolating the scaffold from the surrounding tissues and leading to impaired tissue regeneration.[50] On the other hand, scaffold integration and hence neo tissue forma-tion is successful if an anti-inflammatory response occurs following chronic inflammation. During this process, Treg cells migrate to the inflamed site. Recent evidence indicated that CD4+CD25+Foxp3+ Treg cells could induce alternative activation of human macrophages/monocytes in vitro and upregulated expression of CD206 (macrophage mannose receptor) and CD163 (hemoglobin scavenger receptor), an increased production of CCL18, as well as an enhanced phago-cytic capacity. Further in depth mechanistic studies revealed

Nowsheen Goonoo is currently a Georg Forster Postdoctoral Fellow (awarded by the Alexander Von Humboldt Foundation) at the University of Siegen, Germany. She received her PhD in Chemistry from the University of Mauritius, Mauritius in 2015. Her Ph.D. thesis focused on the elabora-tion of polydioxanone-based

scaffolds for tissue engineering applications. The main aim of her Postdoctoral research is the development of nanoen-gineered polymeric blend scaffolds for bone regeneration and for wound healing; with particular focus on harnessing the potential of seaweed derived biopolymers.

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that CD4+CD25+CD127lowFoxp3+ Treg cells produced IL-10, IL-4, and IL-13 which are involved in the suppression of the proinflammatory cytokine responses.[51] Moreover, in vivo

studies showed that CD4+CD25+ Treg cells efficiently promoted the differentiation of M2 macrophages at least in part through arginase, IL-10 and TGF-b pathways as well as inhibited M1

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Figure 1. A) Summary of macrophage subtypes and their functions in TE[53–58] and B) overview of immune response to scaffold following implantation.[59]



macrophage induction by CD4+CD25−T cells.[52] The M2 macro-phage phenotype is considered to be anti-inflammatory and allow stem cell differentiation. M2 macrophages are subdivided into M2a, M2b and M2c and they all have different cytokine and surface marker expressions (Figure 1A). In general, M1 are inflammatory and microbicidal, whereas M2 are immunomod-ulatory, reparative, and poorly microbicidal (Figure 1A).[53–58]

Scaffolds designed for TE are generally immune-stimulatory due to their chemical nature and the presence of other antigens.[5] In fact, the scaffold, immune system and stem cells are linked by a 3-way interaction and much effort is being put in the design and functionalization of scaffolds in order to drive the inflammation process towards regeneration and vascularization. For successful tissue repair, the initial pro-inflammatory response caused by the scaffold must be accompanied by a pro-healing response and for this to occur, the shift of macrophage phenotype from M1 to M2 is crucial. A multitude of cells and factors are involved in the pro-inflammatory and pro-healing processes, which eventually lead to tissue engraftment and regeneration (Figure 1B).

In summary, a balanced cooperation between pro-inflam-matory and pro-resolution players is essential. Modulating the innate immune response, instead of completely suppressing it is the best way for successful tissue regeneration.

3. Scaffold Success Dependent on the Interaction between Immune Cells and other Cell Populations

It is becoming increasingly clear that since immune cells are influenced by scaffold features, their resulting phenotypes and behavior will, in turn, affect stem cell differentiation and survival.[60] The success of TE scaffolds depends on the recruitment of stem cells and pro-angiogenic cells to the site of the implant as well as the complex interactions between these cells. Immune cells influence the recruitment, activa-tion, differentiation, proliferation and survival of both stem cells and pro-angiogenic cells either directly (via cell to cell contact or by paracrine factors) or indirectly (via modification of the surrounding environment i.e., digestion of scaffold).

3.1. Immune Cell/Stem Cell Cross Talk

Mesenchymal stem cells (MSCs) are found in most tissues and help in tissue regeneration.[61,62] They are often used in TE applications mainly due to their ability to differentiate towards mesodermal lineages and to modulate the immune microenvironment.[63–65] In particular, immune-modulatory factors released by MSCs for e.g., TGS-6, PGE2, IL-6 and CXCL1 have been found to reduce excessive production of pro-inflammatory cytokines by PMNs and macrophages, thereby improving wound healing.[66] More importantly, MSCs polarize macrophages towards an M2 phenotype via a bidirectional cross-talk.[67,68] MSCs induced the expression of M2 markers (IL-10, IL-4, CD206, Arg1) and decreased M1 marker expressions (IL-6, IL-1β, MCP-1 and iNOS) in a co-culture model of macrophages and mouse-derived MSCs.[68] Macrophages, in turn stimulated the production of M2-inducing cytokines.[68]

3.2. Immune Cell/Proangiogenic Cell Cross Talk

Tissue survival and function are largely determined by func-tional vasculature. Due to their ability to recruit pro-angio-genic cells and increase their differentiation and blood vessel formation, immune cells such as macrophages are the main regulators of vascularization. Based on traditional paradigms, M2 macrophages were considered to be pro-angiogenic while the M1 phenotype was considered to be anti-angiogenic.[69,70] However, a recent study indicated that the roles of mac-rophages in angiogenesis were much more complex and in fact, M1, M2a and M2c phenotypes support angiogenesis in different ways.[71] In particular, M1 macrophages secrete highest amounts of vascular endothelial growth factor (VEGF- a well-established angiogenic molecule); M2a produce highest levels of PDGF-BB (chemoattractant for stabilizing pericytes and help to promote anastomosis of sprouting endothelial cells) and M2c macrophages secrete the highest levels of MMP-9 (protease involved in vascular remodeling). In sum-mary, this study suggests that coordinated efforts both by M1 and M2 macrophages are required for angiogenesis and vascularization.[71]

Macrophage infiltration within the scaffold is the initial crucial step during tissue regeneration. Indeed, as summa-rized in Figure 2, macrophage phenotype changes and cell migration are critical during a neo-artery formation. Briefly, CD68+ macrophages initially observed within the scaffold top surface increase continuously. CD206+ cells then migrate progressively from the surrounding tissue into the scaffold and increase significantly in number. This is accompanied by the formation of capillaries. Endothelial cell coverage extends significantly until complete endothelialization is achieved, forming an endothelium similar to that in native artery. MYH+ cells organize circumferentially resulting in the forma-tion of arterial media. Loosely distributed α-SMA+ cells within the scaffold move to the luminal side of the latter forming a dense layer. This α-SMA+ cell layer then transforms into MYH+ cells. α-SMA+ and MYH+ cells synthesize ECM and eventually organize into the artery wall.[72]

4. Immune-engineering Strategies of Electrospun Scaffolds

Immunomodulation of electrospun TE scaffolds may be achieved by various strategies such as (i) changing the scaffold chemistry (ii) modifying the physical properties of the scaf-fold, (iii) controlling the release of anti- or pro-inflammatory cytokines from scaffolds, and (iv) using cell-based therapy methods.

4.1. Immuno-modulation via Scaffold Chemistry

The surface chemistry of scaffolds influences protein adsorp-tion and subsequently cell behavior. Therefore, tuning surface properties and changing the surface chemistry of TE scaffolds directly influences the biological behavior of immune factors.

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4.1.1. Use of Biomimetic ECM Components

Scaffolds that are composed of or mimic ECM components can create a microenvironment which is conducive to tissue regen-eration. A common way to use ECM components to create an immune-compatible scaffold (native scaffold) is via decellulari-zation of tissues or organs. Decellularization allows removal of cells and immunogenic components such as genetic material, membrane antigens and MHC/HLA molecules from tissues/organs leaving behind only ECM components with a specific combination of structural and functional characteristics and a 3D architecture.[73] These biomimetic scaffolds provide excellent microenvironment for cell attachment but offer poor control over structural properties. The processing parameters used to generate these ECM-based materials significantly determine their final fate upon implantation. Indeed, good decellularization protocols i.e., detergent choice and treatment with specific enzymes are critical to prevent fibrous encapsulation of the native scaffolds. In particular, special care must be taken to avoid the presence of non-self-antigens such as α-galactosidase (α-Gal) epitope which stimulates the humoral responses and produce anti-α-Gal anti-bodies which in turn lead to excessive scaffold degradation and block ECM sites from stem cell recognition.[74,75]

4.1.2. Choice of Polymeric Materials

In contrast to native scaffolds, those fabricated from natural and synthetic polymers can be specifically designed to resemble a given structure, and their physico-chemical properties tailored depending on their end applications. Inflammatory reactions produced from electrospun scaffolds are strongly dependent on the chemical nature of the material. Indeed, different cytokines were expressed following culture of peripheral blood mono-nuclear cells (PBMCs) on electrospun polyhydroxybutyrate (PHB), polycaprolactone (PCL), poly(l-lactic acid) (PLA) and col-lagen.[76] Implantation of electrospun collagen, PHB and PLA fibers led to the infiltration of M2 macrophages in both healthy and infarcted myocardium. This observation was consistent with the fact that collagen and PHB induced expression of the M2 cytokines namely IL-13 and IL-10, respectively, in PBMCs.

On the other hand, electrospun PCL scaffolds provoked a chronic M1 response.

In addition, several naturally occurring polymers have intrinsic anti-inflammatory signals for example high molecular weight hyaluronic acid (HA)[77–79] and chitosan.[80] Hyaluronic acid, a major glycosaminoglycan of the ECM, has differential signaling based on its molecular weight.[79] In particular, mac-rophages undergo phenotypic changes corresponding to either (1) pro-inflammatory response for low molecular weight HA or (2) pro-resolving response for high molecular weight HA. Regardless of the initial polarization state of macrophages, low molecular weight HA induced a classically activated-like state, confirmed by upregulation of pro-inflammatory genes, such as nos2, tnf, il12b, and cd80, and enhanced secretion of nitric oxide and TNF-α. On the other hand, high molecular weight HA promoted an alternatively activated-like state, confirmed by up regulation of pro-resolving gene transcription, including arg1, il10, and mrc1, and enhanced arginase activity.[79] More-over, thicker granulomas and a significant foreign body reac-tion were noted in animals treated with polyethylene tereph-thalate (PET)/chitosan fibers compared to those treated with PET fibers only.[80] In addition, the inflammatory response to implanted chitosan scaffolds was shown to be more intense with increasing degree of acetylation (DA).[81] This study indi-cates that acetyl and amine functional groups play important roles in tissue repair and regeneration.[81]

Clearly, the induction of macrophage polarization (M1 or M2) is dependent on the source and polymer nature (Table 1). Both native and synthetic scaffolds have pros and cons. The immune response triggered by scaffolds is further complicated by the fact that a given polymeric scaffold does not influence all cells of the immune system in the same manner. For instance, evaluations of innate immune responses of electrospun PDX/elastin fibers showed that significant immunosuppression was observed in nat-ural killer cell activity but not in macrophage functional assays.[82]

4.1.3. Use of Passive Non-Biofouling Strategies

Limiting nonspecific interaction of cells and proteins with the scaffold surfaces is critical, since these interactions can prove

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Figure 2. Proposed changes and migration of cells during the tissue remodelling process. Reproduced with permission.[72] Copyright 2014, Elsevier.



highly problematic for scaffold integration and success.[101] As a result, versatile and cost-effective strategies for rendering scaffold surfaces resistant to fouling by proteins and cells (non-biofouling strategies) are being developed to control protein adsorption, leucocyte recruitment and activation on electrospun fibers and hence prevent fibrous encapsulation of electrospun scaffolds. In this context, several polymers have been explored including polyacrylates,[102–104] oligosaccharides,[105,106] polymer mimics of phospholipids,[107,108] and poly(ethylene glycol) (PEG).[109] Indeed, coating or modification of electrospun fibers with non-fouling polymers resulted in improved immune responses. For example, subsequent coating of electrospun poly(L-lactide-co-D/L-lactide) fibers with an ultrathin plasma-polymerized allylamine (PPAAm) layer resulted in a signifi-cant increase in the implant-infiltrating tissue for the uncoated mats but not for the PPAAm-coated ones, thereby indicating enhanced inflammatory response for the uncoated fibers.[110] In addition, increasing the cationic surface charge of electrospun PCL/PEI fibers via methylation of primary amines significantly reduced inflammatory responses in murine macrophages com-pared to the untreated PCL/PEI fibers as confirmed by lower levels of the inflammatory cytokines TNF-α and IFN-γ.[111]

4.2. Immunomodulation using Bioactive Strategies

Bioactive strategies involve the delivery of anti-inflammatory and/or pro-wound healing molecules. The delivery of soluble pharmacological anti-inflammatory agents such as dexa-methasone (dex),[112,113] metronidazole[114] or neuregulin-1[115] from electrospun fibers was shown to lead to reduced inflam-mation and foreign body response. Dex loaded PLLA fibers induced the formation of a much thinner inflammatory capsule compared to PLLA fibers after 2 and 4 weeks post implantation.[102] In line with this, the release of dex from electrospun polyethylene oxide (PEO)/ poly(ε-caprolactone) (PCL) fibers led to reduced acute inflammatory response, in correlation with the expression of inflammation related genes

(Figure 3).[113] Metronidazole-loaded PCL nanofibers evoked a less severe inflammatory response than pure PCL nanofibers, as indicated by thinner fibrous capsule formation around the scaffold 7 weeks post implantation.[114] In addition, release of the growth factor, neuregulin-1 (Nrg) from electrospun poly(lactide-co-glycolide) (PLGA)-based fibers led to an increase

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Figure 3. Expression of inflammation related genes after culture of LPS-induced Raw 264.7 macrophage cells on PCL, 11.4%PEO-88.6%PCLand 23.1%PEO-76.9%PCL with or without containing DEX (10−4 M) for 24 h. Cells seeded on TCP with or without LPS treatment, serve as negative and posi-tive reference groups of inflammation. Data represent relative mRNA levels of target genes normalized with reference genes, expressed as a percentage of negative control of inflammation (TCP) cells, which was set to 100%. Differences between groups were assessed by Mann–Whitney test p < 0.05: a) versus positive control of inflammation (TCP + LPS); b) versus negative control of inflammation (TCP); c) versus each control fiber. Reproduced with permission.[113] Copyright 2015, Royal Society of Chemistry.

Table 1. Summary of a selection of polymers and their predominant reaction.

Polymer Type of macrophages Reaction of macrophages when in contact with material

Cytokine production

Technique used to detect response

Refs

Polyurethane (PU) Human monocytes (THP-1) Pro-inflammatory and

anti-inflammatory

High/high Immunolabeling [83]

Poly(lactid acid) Human monocyte derived Pro-inflammatory and

anti-inflammatory

High/high ELISA [84–86]

Poly(ethylene oxide) Human peripheral blood derived macrophage

and murine macrophages

Mainly pro-inflammatory High ELISA and RT-PCR [87,88]

Polycaprolactone Human peripheral blood mononuclear cells, peripheral

blood mononuclear cells (PBMCs)

Mainly anti-inflammatory High Immunolabeling [89,90]

Polydioxanone (PDX) Mouse bone marrow-derived macrophages Mainly anti-inflammatory High ELISA [91]

Collagen bone marrow-derived macrophages, U937 macrophage Mainly pro-inflammatory High Immunolabeling, ELISA [92–96]

Silk Human monocytes, RAW 264.7 murine macrophage cells Mainly pro-inflammatory High RT-qPCR, ELISA [97,98]

Keratin Human monocytes (THP-1) Pro-inflammatory and

anti-inflammatory

Low/high ELISA [99]

Chitosan Human monocytes Pro-inflammatory Low ELISA [84,100]



in the M2:M1 macrophage following 1 month implantation in vivo, suggesting that the initial immune reaction to the scaffold evolved into a more constructive response over time.[115]

Furthermore, controlled release of the anti-inflammatory molecules is required to reduce the foreign body response effectively. Indeed, no change in severe inflammatory response between PCL fibers and dex loaded PCL fibers were noted due to a severe burst release of dex in vitro.[102]

In addition, natural anti-inflammatory compounds such as curcumin[116] and genistein[117] have also been incorporated within electrospun polymeric fibers. The production of excess nitric oxide (NO) within the implant site leads to inflammatory response and tissue damage. The incorporation of curcumin within electrospun PCL fibers was shown to lead to a signifi-cant decrease in NO production from 26 µM to 10 µM in LPS stimulated RAW264.7 mouse macrophages, indicative of the suppression of inflammatory responses.[116] Genistein-loaded electrospun PEO/PDLLA fibers suppressed the in vitro secre-tion of TNF-α, relative to those of pure PEO and PDLLA.[117] In addition, the amount of genistein released depended on the PEO content in the blend fiber. The higher the PEO content, the higher the amount of genistein released, leading to enhanced anti-inflammatory effects.

Chemokines and their receptors regulate the local inflamma-tory response by directly modulating cellular infiltration around the scaffold.[118] Moreover, cytokines and growth factors influ-ence the phenotype of immune cells directly. The delivery of cytokines may be achieved via direct inclusion or through the use of nucleic-acid based strategies.[119] For direct inclusion, electro-spun PCL scaffolds were conjugated with IL-10. This resulted in the promotion of macrophages towards an anti-inflammatory/wound healing state (M2 phenotype) by inducing arginase-1 (Arg1).[120] IL-10 conjugated PCL nanofibers (PCL-IL10) success-fully induced macrophage polarization towards the M2 activated state not only within the scaffold but also in the adjacent tissue surrounding the sciatic nerve, suggesting that cells do not nec-essarily have to be in constant contact with the scaffold to alter its phenotype (Figure 4). IL-10 desorbed from the PCL-IL10 scaf-fold promoted macrophage polarization towards the M2 state beyond the scaffold surface. The macrophages in contact with IL-10 were then “switched on” to produce endogenous IL-10. In contrast, macrophages around the PCL scaffold expressed high levels of ED1 and low levels of Arg1 and CD206, indicative of the M1 phenotype.

Modulation of scaffold properties as a function of time i.e., dynamic modulation is an important aspect to consider given

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Figure 4. Examples of macrophage M2 polarization at 3 days post scaffold implantation. Transmitted light and confocal images (Z-stack projection) superimposed showing ED1+ and Arg1+ (A–B) and ED1+ and CD206+ (C–D) cells in the scaffold (above coarse dotted lines) and surrounding perineural/connective tissue (below coarse dotted lines) from nerves that received IL10-biofunctionalised (PCL-IL10, A and C) or control (PCL, B and D) scaffold implants. Double positive cells are shown as yellow. Magnified regions are indicated by the inserts. The sciatic nerve is visible in panel A (below fine dotted line). Scale bars = 100 µm. Scaffolds were wrapped around the sciatic nerve in rats. Reproduced with permission.[120] Copyright 2015, Elsevier.



that the TE process is time dependent. For instance, the scaffold must slowly degrade away so as to create space for the newly growing tissue. The mechanical properties should not drop dras-tically but instead should decrease slowly with time, ensuring that the cells have adequate support during the initial growth stages. Indeed, much attention is being given to the design of active scaffolds instead of passive ones.[121–123] Electromechani-cally active polypyrole (PPy) coated PLGA scaffold provided an optimum microenvironment including electromechanical stimulation that supported and directly stimulated induced pluripotent stem cells, with excellent cell viability after stimula-tion.[121] Stimulation of rat pheochromocytoma 12 (PC12) cells on aligned PPy-PLGA fibers resulted in longer neurites and more neurite-bearing cells than stimulation on random PPy-PLGA fibers, suggesting a combined effect of electrical stimula-tion and topographical guidance.[123] In addition, it was recently shown that the thrombogenic and inflammatory responses of electrospun heparin-doped polypyrole coated PCL fibers may be modulated under AC electrical stimulation.[124]

4.3. Immunomodulation using Physical Scaffold Properties

4.3.1. Topography, Fiber Diameter and Pore Size

Substrate topography not only plays an important role in influencing cellular behaviour but also influences the inflam-matory response activated by macrophages, especially in the early inflammation stage.[125] Indeed, macrophages adherent to flat PLLA film induced a stronger inflammation reaction compared to electrospun scaffolds.[125] Moreover, the thick-ness of fibrous capsules formed on electrospun PCL mats were significantly thinner than on the corresponding film (4.13 ± 0.31 v/s 37.7 ± 0.25 µm).[126] Several studies have shown that fiber diameter and pore size play key roles in modula-tion of the innate immune response. A strong correlation was noted between increasing fiber diameter and increased expression of the M2 markers Arg1, TGF-β1, VEGF & bFGF, along with decreased expression of the M1 marker inducible nitric oxide synthase (iNOS).[90] In particular, unstimulated bone marrow derived macrophages (BMMΦ (M0)) not only acquire a functional M2-like phenotype when in contact with bigger fiber/pore size scaffold but possess similar angiogenic

properties as a pre-polarized M2. Furthermore, compared to fiber diameter, pore size was found to be a more crucial regu-lator of Arg 1 expression and BMMΦ phenotype modulation towards an M2 phenotype.[90] This may be explained by the fact that macrophage infiltration is easier in large diameter/pore size scaffolds and once inside the electrospun mat, they orient themselves in 3 dimensional space, with a natural spread mor-phology. In contrast, being unable to penetrate within small fiber diameter/pore size scaffolds, macrophages remain at the surface of the electrospun mats. They then undergo ‘frustrated phagocytosis’ in an attempt to invade the scaffold. Such macro-phages acquire tissue destructive roles typically associated with the M1 phenotype.[90] The signalling mechanism is still unclear but the myeloid differentiation factor 88 (MyD88) is believed to be a key component involved.

In line with the work of Bowlin and co-workers,[83] macro-phages cultured on thicker fiber scaffolds were shown to polarize preferentially into the M2 phenotype, while those cul-tured on thinner fiber scaffolds expressed M1 phenotype.[66] This consequently mediated the regeneration of cell-free PCL grafts into neo-arteries in vivo.

Similarly, in another study, it was demonstrated that com-pared to micron-sized electrospun polyurethane fibers, nano-sized ones resulted in minimal macrophage responses in vitro and in vivo and induced only mild foreign body reactions.[127]

4.3.2. Fiber Alignment

The influence of fiber alignment on the induced inflammatory response is still unclear due to contradictory findings. Com-pared to random fibers, the implantation of aligned fibers in vivo elicited thinner fibrous capsule formation (4.13 ± 0.31 v s−1 7.55 ± 0.54 µm), thereby resulting in minimized host response, and enhanced tissue-scaffold integration.[126] This observation was rationalized by the fact that cells could penetrate within the aligned fibers leading to reduced severity of foreign body reaction. Indeed, they showed that monocyte adhered poorly on aligned fibers as suggested by the rounded morphology (Figure 5A). On the other hand, the well spread monocyte morphology observed on random fibers indicated good cell adhesion (Figure 5B).[126] In another study, the authors con-cluded that fiber alignment had no influence on macrophage

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Figure 5. SEM images of monocytes at day 0 (2 h) on (A) PCL aligned fibers and (B) PCL random fibers. The scale bar is 10 µm. Adapted with permission.[126] Copyright 2010, Wiley.



activation and secretion of pro-inflammatory cytokines and chemokines.[125]

4.3.3. Fiber Roughness

Several studies have indicated that the guidance (cell shape) and activation (phenotype) of macrophages are influenced by nanoscale topographical features.[128–132] Macrophages can be sensitive to nanotopography as small as 30 nm.[133] On patterned surfaces, the cells were better spread and extended microspikes usually perpendicular to the direc-tion of the patterns.[128] Moreover, the degree of orientation of the macrophages increased with increasing depth and with decreasing width of grooves.[128] Secretion of cytokines IL-6 and IL-1β from macrophages decreased (60 ng/ml and 4 ng/ml to 20 ng ml−1 and 2.5 ng ml−1 respectively) with increasing size of the nanoscale features (16 nm to 68 nm).[129] Importantly, the elongation of bone marrow derived macrophages and expres-sion of phenotypic markers associated with a pro-healing M2 phenotype was highest on intermediate groove sizes ranging from 400 nm to 5 µm in width.[131]

In accordance with these studies, RAW 264.7 cells were found to be less elongated on fibers with nanoscale depressions compared to those cultured on smooth fibers (Figure 6).[134] However, no significant differences in the number of adherent cells or cell metabolism were noted. The nanotopographical fea-tures on the fibers were formed as a result of phase separation during the electrospinning process.[134]

In summary, physical features of electrospun scaffolds such as fiber diameter, pore size, fiber alignment, and fiber

topography influence macrophage morphology, spreading and polarization (Table 2).

4.4. Immuno-modulation using Cell-Based Strategies

Macrophages not only play vital roles in the first line of host defenses but also regulate the recruitment, proliferation and differentiation of other types of cells including fibroblasts, endothelial cells, keratinocytes etc.[135,136] Furthermore, recent studies have led to the realization that immune cells can be used for the indirect induction of desired biological events such as angiogenesis. Under appropriate conditions, macrophages can produce several pro-angiogenic factors and this aspect can be harnessed to promote tissue regeneration. Indeed, it has been demonstrated that co-culturing macrophages with other cell lines is a viable method to improve tissue regeneration.[137] For instance, the treatment of a co-culture system consisting of human outgrowth endothelial cells (OECs) and primary osteo-blasts with macrophages (induced from THP-1) resulted in a higher number of microvessel-like structures formed by OECs in contrast to the co-culture.[137] This observation correlated with a significantly higher concentration of the pro-angiogenic VEGF in cell culture supernatants of triple-cultures and was accompanied by an increase in the expression of different pro-inflammatory cytokines, such as IL-6, IL-8 and TNF-α.[137] In addition to macrophages, fibroblasts are also involved in for-eign body formation. They can modulate adaptive immune responses by suppressing allogenic proliferation of peripheral blood mononuclear cells (PBMC) and allogeneic T-cell activa-tion.[138,139] Co-culturing fibroblasts with macrophages reduced

Adv. Biosys. 2017, 1700093

Figure 6. SEM images of (A) smooth fibers and (B) fibers with nanoscale depressions, Fluorescence microscopy images of RAW 264.7 macrophages labeled with actin (green-cytoskeleton), vinculin (red-cell adhesions) and DAPI (blue- nuclei) on (C) smooth fibers and (D) fibers with nanoscale depres-sions. Adapted with permission.[134] Copyright 2013, American Chemical Society.



inflammation by releasing TSG6 and Cox-2 and favored macro-phage polarization to M2, thereby resolving inflammation and promoting tissue-regeneration.[140] Hence, the synergistic inter-actions and cross-communication between macrophages and fibroblasts can be a means to immune-modulate inflammation and foreign responses.

Prior in vitro and in vivo investigations have suggested that the use of mesenchymal stromal cells (MSCs), by virtue of their ability to modulate the inflammatory environment, may improve tissue regeneration.[141–144] However, the mechanisms by which MSCs regulate inflammation for improved wound healing still remain unclear. MSCs allow modulation of the innate immune response by producing factors that directly pro-tect fibroblasts from harmful cytokines, such as IL-1β.[143,145] In addition, as reported by Manning et al.[146] co-culture of MSCs with M1 macrophages successfully suppressed the effects of M1 macrophages on fibroblasts by inducing a phenotypic switch from a pro-inflammatory macrophage phenotype to an anti-inflammatory macrophage phenotype. Results obtained by Cho et al.[147] further confirmed the immuno-modulatory char-acteristics of MSCs as indicated by the preferential shift of the macrophage phenotype from M1 to M2 during co-culture of MSCs with macrophages.

A 3-way interaction was found to exist between macrophages, MSCs and the scaffold whose combined influences dictate the macrophage immunophenotype and MSC multipotency.[148] Indeed, a material-dependent effect was noted when MSCs were cultured in combination with macrophages on either col-lagen or gelatin/PEG scaffolds. In fact, MSCs encapsulated within collagen scaffolds promoted chondrogenic differen-tiation while the gelatin/PEG scaffold enhanced adipogenic differentiation as confirmed by IL-6, IL-10, TNF-α and IL-12 expressions.[148]

Overall, these studies indicated that the co-culture of mac-rophages in combination with other cells on electrospun scaffolds can reverse inflammation and lead to positive effects on tissue regeneration. Macrophages can be used as

pro-angiogenic reservoir to overcome one of the major issues in TE namely proper vascularization or to promote a pheno-type shift of macrophages from M1 to M2. Moreover, co-culture models need to be adjusted for specific applications due to the scaffold-dependence of macrophage immunophenotype and MSC multipotency.

5. Summary and Outlook

A key challenge in tissue engineering is to immune-modulate inflammatory reactions to implanted scaffolds so as to pro-mote to improve neo-tissue formation and engraftment within the host. Although implantation of scaffolds in vivo induces significant immune response, often hampering regenera-tion, complete immunosuppression would be extremely detri-mental to the scaffold success. In fact, proper modulation of innate response and possibly suppression of humoral/adaptive response would be more beneficial. The beneficial aspects of the inflammatory response need to be harnessed while limiting the detrimental aspects. The 3-way interaction between scaf-fold, immune cells and stem cells suggests that the immune response may be modulated via proper design of scaffolds, which will in turn influence the regenerative potential of stem cells, vascularization and resolve inflammation. Several strat-egies may be used to control the initial immune response to scaffolds. Firstly, the choice of material for scaffold fabrication should be judiciously made. The possibility to vary electrospun scaffold characteristics such as fiber diameter, pore size, fiber alignment, porosity, topography opens a new frontier in the field of immunology. Coating of scaffold surfaces with non-immunogenic materials is another way to modulate inflamma-tion and enhance tissue regeneration. Surface functionalization of the electrospun scaffolds with specific molecules was shown to be an efficient tool to increase MSC recruitment and tissue regeneration. Additionally, pharmacological compounds such as anti-inflammatory drugs, or anti-inflammatory cytokines

Adv. Biosys. 2017, 1700093

Table 2. The impact of physical features of electrospun fibers on macrophage functions.

Polymer Physical feature Inflammatory reaction/ cell response and polarization Secretion Ref

PLA Topography (fiber v/s film) Stronger inflammatory reaction on film MIP-1α, RANTES, INF- α (proinflammatory) ↑ on films [125]

VEGF (proangiogenic) ↑ on fibers

PCL Topography (fiber v/s film) Monocytes exhibited a round shape (poor cell attachment)

on the fiber surface vs well spread on filmFibrotic response ↓ on fibers [126]

PDX Fiber diameter Arginase 1 (M2 marker) ↑ and inducible nitric oxide synthase

(M1 marker) ↓ on thicker fibers

VEGF, TGF-β1 and bFGF (po-angiogenic) ↑ on thicker fibers [90]

PCL Fiber diameter Towards M2 (CD206+) phenotype on thicker fibers No data [72]

PU Fiber diameter Mannose receptor, CD206 (M2 marker) ↑ on thicker fibers Fibrotic response ↓ on thicker fibers [127]

PDX Pore size Arginase 1 (M2 marker) ↑ and inducible nitric oxide synthase

(M1 marker) ↓ on thicker fibers

VEGF, TGF-β1 and bFGF (po-angiogenic) ↑ on thicker fibers [90]

PCL Fiber alignment Monocytes exhibited a round shape (poor cell attachment)

on the fiber surface vs well spread on random fiberFibrotic response ↓ on aligned fibers [126]

PLA Fiber alignment No influence on macrophage activation No change in secretion of proinflammatory cytokines

and chemokines

[125]

PLA Surface roughness RAW 264.7 cells were less elongated on nanostructured fibers

compared to those cultured on smooth fibers

No data [134]


1700093 (11 of 13) © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimAdv. Biosys. 2017, 1700093

could be encapsulated or conjugated to the electrospun scaf-folds. Another possibility is the in situ delivery of specific cells for instance Treg or MSCs to immune-modulate the reaction against TE scaffolds. The use of immuno-modulation in TE is still in its infancy and better understanding of the biomaterial-immune system interactions will help in the development of more effective immune-engineered scaffolds.

6. Abbreviations

Arg, arginase; bFGF, basic fibroblast growth factor; DA, degree of acetylation; DAMPs: danger associated molecular patterns; Dex, dexamethasone; ECM, extracellular matrix; HA, hyalu-ronic acid; IL, interleukin; MMP, matrix metalloproteinase; MSC, mesenchymal stem cells; NK, natural killer; Nrg, neureg-ulin; OEC, outgrowth endothelial cells; PBMC, peripheral blood mononuclear cells; PCL, poly(ε-caprolactone); PDGF: platelet-derived growth factor; PDX, polydioxanone; PEG, polyethylene glycol; PEI, poly(ethylene imine); PEO, poly(ethylene oxide); PET, polyethylene terephthalate; PLGA: poly(lactide-co-gly-colide); PU: polyurethane; PMNs: polymorphonuclear neutro-phils; PPAAm: plasma-polymerized allylamine; PPy: polypyrole; ROS, reactive oxygen species; TE, tissue engineering; TGF, transforming growth factor; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.

AcknowledgementsThe author acknowledges generous financial support from the Alexander von Humboldt Foundation (Georg Forster postdoc stipend) and the University of Siegen, Germany.

Conflict of InterestThe author declares no conflict of interest.

Keywordsimmunology, inflammation, polymeric scaffolds, tissue engineering

Received: May 11, 2017Published online:

[1] M. J. Smith, D. C. Smith, K. L. White, G. L. Bowlin, J. Eng. Fibers Fabr. 2007, 2, 41.

[2] A. R. Amini, C. T. Laurencin, S. P. Nukavarapu, Crit. Rev. Biomed. Eng. 2012, 40, 363.

[3] R. M. Boehler, J. G. Graham, L. D. Shea, Biotechniques 2011, 51, 239.[4] F. Berthiaume, T. J. Maguire, M. L. Yarmush, Annu. Rev. Chem.

Biomol. Eng. 2011, 2, 403.[5] A. Crupi, A. Costa, A. Tarnok, S. Melzer, L. Teodori, Eur. J. Immunol.

2015, 45, 3222.[6] D. F. Williams, Biomaterials 2008, 29, 2941.[7] R. Londono, S. F. Badylak, Ann. Biomed. Eng. 2014, 43, 577.[8] S. Browne, A. Pandit, Front. Bioeng. Biotechnol. 2015, DOI: 10.3389/

fbioe.2015.00067.

[9] B. Jiang, R. Liao, J. Cardiovasc. Transl. Res. 2010, 3, 410.[10] Y. K. Kim, R. Que, S. W. Wang, W. F. Liu, Adv. Healthc. Mater. 2014,

3, 989.[11] M. Dobaczewski, N. G. Frangogiannis, Front. Biosci., Scholar Ed.

2008, 22, 391.[12] M. J. Redd, L. Cooper, W. Wood, B. Stramer, P. Martin, Philos.

Trans. R Soc. Lond. B Biol. Sci. 2004, 359, 777.[13] D. D. Lo, A. S. Zimmermann, A. Nauta, M. T. Longaker,

H. P. Lorenz, Birth Defects Res. C Embryo Today 2012, 96, 237.[14] Y. Liu, L. Wang, T. Kikuiri, K. Akiyama, C. Chen, X. Xu, R. Yang,

W. Chen, S. Wang, S. Shi, Nat. Med. 2011, 17, 1594.[15] J. Chang, F. Liu, M. Lee, B. Wu, K. Ting, J. N. Zara, Proc. Natl.

Acad. Sci. USA 2013, 110, 9469.[16] F. E. Ahmed, B. S. Lalia, R. Hashaikeh, Desalination 2015, 356, 15.[17] W. E. Teo, S. Ramakrishna, Nanotechnology 2006, 17, 89.[18] Q. P. Pham, U. Sharma, A. G. Mikos, Tissue Eng. 2006, 12, 1197.[19] A. Rogina, Appl. Surf. Sci. 2014, 296, 221.[20] J. D. Schiffman, C. L. Schauer, Polym. Rev. 2008, 48, 317.[21] J. M. Anderson, A. Rodriguez, D. T. Chang, Semin. Immunol. 2008,

20, 86.[22] D. T. Luttikhuizen, M. C. Harmsen, M. J. V. Luyn, Tissue Eng. 2006,

12, 1955.[23] D. F. Williams, Biomaterials 2008, 29, 2941.[24] C. Gretzer, L. Emanuelsson, E. Liljensten, P. J. Thomsen,

Biomater. Sci. Polym. Ed. 2006, 17, 669.[25] J. M. Anderson, Curr. Opin. Hematol. 2000, 7, 40.[26] J. M. Anderson, Ann. Rev. Mater. Res. 2001, 31, 81.[27] P. Rajesh, S. Verma, V. Verma, K. Balani, A. Agarwal, R. Narayan,

in Biosurfaces: A Materials Science and Engineering Perspective, Wiley, Miami, FL, USA, 2015, pp. 106–125.

[28] C. J. Wilson, R. E. Clegg, D. I. Leavesley, M. J. Pearcy, Tissue Eng. 2005, 11, 1.

[29] L. Tang, J. W. Eaton, J. Exp. Med. 1993, 178, 2147.[30] J. Vitte, A. M. Benoliel, A. Pierres, P. Bongrand, Eur. Cell.Mater.

2004, 7, 52.[31] L. Zhang, Z. Cao, T. Bai, L. Carr, J. R. Ella-Menye, C. Irvin,

B. D. Ratner, S. Jiang, Nat. Biotechnol. 2013, 31, 553.[32] Y. Liu, R. Medda, Z. Liu, K. Galior, K. Yehl, J. P. Spatz,

E. A. Cavalcanti-Adam, K. Salaita, Adv. Health. Mater. 2014, 3, 989.[33] K. Anselme, M. Bigerelle, B. Noel, E. Dufresne, D. Judas, A. Lost,

P. Hardouin, J. Biomed. Mater. Res. 2000, 49, 155.[34] R. A. Latour, Encycl. Biomater. Biomed. Eng. 2005, 28, 1.[35] C. J. Wilson, R. E. Clegg, D. I. Leavesley, M. J. Pearcy,. Tissue Eng.

2005, 11, 1.[36] R. L. Diegelmann, M. C. Evans, Front. Biosci. 2004, 9, 283.[37] C. Esche, C. Stellato, L. A. Beck, J. Invest. Dermatol. 2005, 125, 615.[38] W. M. Nauseef, N. Borregaard, Nat. Immunol. 2014, 15, 602.[39] J. Bocsi, M. Richter, J. Hambsch, M. J. Barten, I. Dahnert,

P. Schneider, A. Tarnok, Cytometry A 2006, 69, 165.[40] A. Tarnok, J. Bocsi, M. Pipek, P. Osmancik, G. Valet, P. Schneider,

J. Hambsch, Cytometry 2001, 46, 247.[41] A. Tarnok, P. Schneider, Shock 2001, 16(Suppl 1), 24.[42] A. Diegeler, A. Tarnok, T. Rauch, D. Haberer, V. Falk, R. Battellini,

R. Autschbach, J. Hambsch, P. Schneider, F. W. Mohr, Thorac. Cardiovasc. Surg. 1998, 46, 327.

[43] C. A. Feghali, T. M. Wright, Front Biosci. 1997, 2, 12.[44] S. A. Eming, P. Martin, M. Tomic-Canic, Sci. Transl. Med. 2014, 6,

265.[45] J. M. Anderson, A. Rodriguez, D. T. Chang, Sem. Immunol. 2008,

20, 86.[46] J. M. Zeller, AORN J. 1983, 37, 1284.[47] A. Ortega-Gomez, M. Perretti, O. Soehnlein, EMBO Mol. Med.

2013, 5, 661.[48] L. Dong, C. Wang, Trends Biotechnol. 2013, 31, 342.[49] J. M. Anderson, Ann. Rev. Mater. Res. 2001, 31, 81.



[50] G. Wick, A. Backovic, E. Rabensteiner, N. Plank, C. Schwentner, R. Sgonc, Trends Immunol. 2010, 31, 110.

[51] M. M. Tiemessen, A. L. Jagger, H. G. Evans, M. J. van Herwijnen, S. John, L. S. Taams, Proc. Natl. Acad. Sci. USA 2007, 104, 19446.

[52] G. Liu, H. Ma, L. Qiu, L. Li, Y. Cao, J. Ma, Y. Zhao, Immunol. Cell Biol. 2011, 89, 130.

[53] Y. Kharraz, J. Guerra, C. J. Mann, A. L. Serrano, P. Munoz-Canoves, Mediators Inflamm. 2013, 491497.

[54] D. O. Freytes, J. W. Kang, I. Marcos-Campos, G. Vunjak-Novakovic, J. Cell Biochem. 2013, 114, 220.

[55] F. Mac Gabhann, A. A. Qutub, B. H. Annex, A. S. Popel, Wiley Interdiscip. Rev. Syst. Biol. Med. 2010, 2, 694.

[56] D. L. Laskin, V. R. Sunil, C. R. Gardner, J. D. Laskin, Annu. Rev. Pharmacol. Toxicol. 2011, 51, 267.

[57] S. M. Hindi, J. Shin, Y. Ogura, H. Li, A. Kumar, PLoS One 2013, 8, e72121.

[58] A. Mantovani, S. Sozzani, M. Locati, P. Allavena, A. Sica, Trends Immunol. 2002, 23, 549.

[59] A. Vishwakarma, N. S. Bhise, M. B. Evangelista, J. Rouwkema, M. R. Dokmeci, A. M. Ghaemmaghami, N. E. Vrana, A. Khademhosseini, Trends Biotechnol. 2016, 34, 470.

[60] Z. Chen, X. Mao, L. Tan, T. Friis, C. Wu, R. Crawford, Y. Xiao, Biomaterials 2014, 35, 8553.

[61] R. Tang, F. Wei, L. Wei, S. Wang, G. Ding, J. Tissue Eng. Regen. Med. 2014, 8, 226.

[62] Z. Yan, Y. Zhuansun, G. Liu, R. Chen, J. Li, P. Ran, Immunol. Lett. 2014, 162, 248.

[63] J. D. Glenn, K. A. Whartenby, World J. Stem Cells 2014, 6, 526.[64] Y. Li, Y. H. Qu, Y. F. Wu, L. Liu, X. H. Lin, K. Huang, J. Wei,

Cell Biol. Int. 2014, 39, 435.[65] K. C. Rustad, G. C. Gurtner, Adv Wound Care 2012, 1, 147.[66] D. J. Prockop, J. Y. Oh, Mol. Ther. 2012, 20, 14.[67] D. I. Cho, M. R. Kim, H. Jeong, H. C. Jeong, M. H. Jeong,

S. Ho Yoon, Y. S. Kim, Y. Ahn, Exp. Mol. Med. 2014, 46, 70.[68] S. Adutler-Lieber, T. Ben-Mordechai, N. Naftali-Shani, E. Asher,

D. Loberman, E. Raanani, J. Leor, J. Cardiovasc. Pharmacol. Ther. 2013, 18, 78.

[69] A. Mantovani, S. Sozzani, M. Locati, P. Allavena, A. Sica, Trends Immunol. 2002, 23, 549.

[70] K. Movahedi, D. Laoui, C. Gysemans, M. Baeten, G. Stangé, J. Van den Bossche, M. Mack, D. Pipeleers, P. In’t Veld, P. De Baetselier, J. A. Van Ginderachter, Cancer Res. 2010, 70, 5728.

[71] K. L. Spiller, R. R. Anfang, K. J. Spiller, J. Ng, K. R. Nakazawa, J. W. Daulton, G. Vunjak-Novakovic, Biomaterials 2014, 35, 4477.

[72] Z. Wang, Y. Cui, J. Wang, X. Yang, Y. Wu, K. Wang, X. Gao, D. Li, Y. Li, X. L. Zheng, Y. Zhu, D. Kong, Q. Zhao, Biomaterials 2014, 35, 5700.

[73] L. Teodori, A. Costa, R. Marzio, B. Perniconi, D. Coletti, S. Adamo, B. Gupta, A. Tarnok, Front Physiol. 2014, 5, 218.

[74] U. Galili, Tissue Eng. Part B Rev. 2015, 21, 231.[75] T. J. Keane, R. Londono, N. J. Turner, S. F. Badylak, Biomaterials

2012, 33, 1771.[76] D. Castellano, M. Blanes, B. Marco, I. Cerrada, A. Ruiz-Saurí,

B. Pelacho, M. Araña, J. A. Montero, V. Cambra, F. Prosper, P. Sepúlveda, Stem Cells Dev. 2014, 23, 1479.

[77] K. Nakamura, S. Yokohama, M. Yoneda, S. Okamoto, Y. Tamaki, T. Ito, M. Okada, K. Aso, I. Makino, J. Gastroenterol. 2004, 39, 346.

[78] S. Hirabara, T. Kojima, N. Takahashi, M. Hanabayashi, N. Ishiguro, Biochem. Biophys. Res. Commun. 2013, 430, 519.

[79] J. E. Rayahin, J. S. Buhrman, Y. Zhang, T. J. Koh, R. A. Gemeinhart, ACS Biomater. Sci Eng. 2015, 1, 481.

[80] B. Veleirinho, D. S. Coelho, P. F. Dias, M. Maraschin, R. Pinto, E. Cargnin-Ferreira, A. Peixoto, J. A. Souza, R. M. Ribeiro-do-Valle, J. A. Lopes-da-Silva, PLoS ONE 2014, 9, e95293.

[81] J. N. Barbosa, I. F. Amaral, A. P. Aguas, M. A. Barbosa, J. Biomed. Mater. Res A. 2010, 93, 20.

[82] M. J. Smith, K. L. White Jr, D. C. Smith, G. L. Bowlin, Biomaterials 2009, 30, 149.

[83] R. J. Schutte, A. Parisi-Amon, W. M. Reichert, J. Biomed Mater Res A 2009, 88, 128.

[84] C. R. Almeida, T. Serra, M. I. Oliveira, J. A. Planell, M. A. Barbosa, M. Navarro, Acta Biomaterialia 2014, 10, 613.

[85] K. S. Stankevich, A. Gudima, V. D. Filimonov, H. Klüter, E. M. Mamontova, S. I. Tverdokhlebov, J. Kzhyshkowska, Mater. Sci. Eng. C Mater. Biol. Appl. 2015, 51, 117.

[86] A. Scislowska-Czarnecka, E. Pamula, A. Tlalka, E. Kolaczkowska, J. Biomater. Sci. 2012, 23, 715.

[87] V. E. Wagner, J. D. Bryers, J. Biomed. Mater. Res A. 2003, 66, 62.[88] A. D. Lynn, T. R. Kyriakides, S. J. Bryant, J. Biomed. Mater. Res. A.

2010, 93, 941.[89] V. Ballotta, A. Driessen-Mol, C. V. C. Bouten, F. P. T. Baaijens,

Biomaterials 2014, 35, 4919.[90] K. M. Ainslie, S. L. Tao, K. C. Popat, H. Daniels, V. Hardev,

C. A. Grimes, T. A. Desai, J. Biomed. Mater. Res. A. 2009, 91, 647.[91] K. Garg, N. A. Pullen, C. A. Oskeritzian, J. J. Ryan, G. L. Bowlin,

Biomaterials 2013, 34, 4439.[92] L. M. Delgado, Y. Bayon, A. Pandit, D. I. Zeugolis, Tissue Eng. Part

B Rev. 2015, 21, 298.[93] K. L. Spiller, R. R. Anfang, K. J. Spiller, J. Ng, K. R. Nakazawa,

J. W. Daulton, G. Vunjak-Novakovic, Biomaterials 2014, 35, 4477.[94] S. B. Orenstein, Y. Qiao, U. Klueh, D. L. Kreutzer, Y. W. Novitsky,

Hernia 2010, 14, 401.[95] P. R. Umashankar, P. V. Mohanan, T. V. Kumari, Toxicol. Int. 2012,

19, 51.[96] F. Y. McWhorter, T. Wang, P. Nguyen, T. Chung, W. F. Liu, Proc.

Natl. Acad. Sci. USA 2013, 110, 17253.[97] M. Bhattacharjee, E. Schultz-Thater, E. Trella, S. Miot, S. Das,

M. Loparic, A. R. Ray, I. Martin, G. C. Spagnoli, S. Ghosh, Biomaterials 2013, 34, 8161.

[98] X. Cui, J. Wen, X. Zhao, X. Chen, Z. Shao, J. J. Jiang, J. Biomed. Mater. Res. Part A 2013, 101, 1511.

[99] B. V. Fearing, M. E. Van Dyke, Acta Biomater. 2014, 10, 3136.[100] M. I. Oliveira, S. G. Santos, M. J. Oliveira, A. L. Torres,

M. A. Barbosa, Eur. Cells Mater. 2012, 24, 136.[101] A. W. Bridges, A. J. Garcia, J. Diabetes Sci. Technol. 2008, 2, 984.[102] M. Tanaka, A. Mochizuki, T. Motomura, K. Shimura, M. Onishim,

Y. Okahata, Colloids Surf. A 2001, 193, 145.[103] M. Tanaka, A. Mochizuki, N. Ishii, T. Motomura, T. Hatakeyama,

Biomacromolecules 2002, 3, 36.[104] S. Terada, K. Suzuki, M. Nozaki, T. Okano, N. Takemura,

J. Reconstr. Microsurg. 1997, 13, 9.[105] T. H. Zhang, R. E. Marchant, Macromolecules 1994, 27, 7302.[106] M. A. Ruegsegger, R. E. Marchant, J. Biomed. Mater. Res. 2001, 56,

159.[107] S. L. Willis, J. L. Court, R. P. Redman, J. H. Wang, S. W. Leppard,

V. J. O’Byrne, S. A. Small, A. L. Lewis, S. A. Jones, P. W. Stratford, Biomaterials 2001, 22, 3261.

[108] S. Y. Kim, Y. M. Lee, Biomaterials 2002, 23, 1697.[109] F. Zhang, E. T. Kang, K. G. Neoh, P. Wang, K. L. Tan,

J. Biomed. Mater. Res. 2001, 56, 324.[110] M. Schnabelrauch, R. Wyrwa, H. Rebl, C. Bergemann, B. Finke,

M. Schlosser, U. Walschus, S. Lucke, K. D. Weltmann, J. B. Nebe, Int. J. Polym. Sci. 2014, 439784.

[111] J. Kang, H. S. Yoo, Biomacromolecules 2014, 15, 2600.[112] N. M. Vacanti, H. Cheng, P. S. Hill, J. D. Guerreiro, T. T. Dang,

M. Ma, S. Watson, N. S. Hwang, R. Langer, D. G. Anderson, Biomacromolecules 2012, 13, 3031.

[113] Y. F. Li, M. Rubert, Y. Yu, F. Besenbacher, M. Chen, RSC Adv. 2015, 5, 34166.



[114] J. Xue, M. He, Y. Niu, H. Liu, A. Crawford, P. Coates, D. Chen, R. Shi, L. Zhang, Int. J. Pharmaceutics 2014, 475, 566.

[115] T. Simón-Yarza, A. Rossi, K. H. Heffels, F. Prósper, J. Groll, M. J. Blanco-Prieto, Tissue Eng. Part. A. 2015, 21, 1654.

[116] H. T. Bui, O. H. Chung, J. D. Cruz, J. S. Park, Macromol. Res. 2014, 22, 1288.

[117] S. Buddhiranon, L. A. DeFine, T. S. Alexander, T. Kyu, Biomacromolecules 2013, 14, 1423.

[118] A. Zlotnik, O. Yoshie, Immunity 2012, 36, 705.[119] R. M. Boehler, J. G. Graham, L. D. Shea, Biotechniques 2011, 51,

239.[120] J. R. Potas, F. Haque, F. L. Maclean, D. R. Nisbet, J. Immunol.

Methods 2015, 420, 38.[121] A. Gelmi, A. Cieslar-Pobuda, Ebo de Muinck, M. Los, M. Rafat,

E. W. H. Jager, Adv. Healthcare Mater. 2016, 5, 1471.[122] A. F. Quigley, J. M. Razal, M. Kita, R. Jalili, A. Gelmi, A. Penington,

R. Ovalle-Robles, R. H. Baughman, G. M. Clark, G. G. Wallace, R. M. I. Kapsa, Adv. Healthcare Mater. 2012, 1, 801.

[123] J. Y. Lee, C. A. Bashur, A. S. Goldstein, C. E. Schmidt, Biomaterials 2009, 30, 4325.

[124] G. M. Xiong, S. Yuan, J. K. Wang, A. T. Do, N. S. Tan, K. S. Yeo, C. Choong, Acta Biomater. 2015, 23, 240.

[125] E. Saino, M. L. Focaret, C. Gualandi, E. Emanuele, A. I. Cornaglia, M. Imbriani, L. Visai, Biomacromolecules 2011, 12, 1900.

[126] H. Cao, K. McHugh, S. Y. Chew, J. M. Anderson, J. Biomed. Mater. Res A. 2010, 93, 1151.

[127] K. Wang, W. D. Hou, X. Wang, C. Han, S. N. Vuletic, W. X. Zhang, Q. S. Ren, L. Chen, Y. Luo, Biomaterials 2016, 102, 249.

[128] B. Wójciak-Stothard, A. Curtis, W. Monaghan, K. Macdonald, C. Wilkinson, Exp. Cell Res. 1996, 223, 426.

[129] S. N. Christo, A. Bachhuka, K. R. Diener, A. Mierczynska, J. D. Hayball, K. Vasilev, Adv Healthcare Mater. 2016, 5, 956.

[130] S. Chen, J. A. Jones, Y. Xu, H. Y. Low, J. M. Anderson, K. W. Leong, Biomaterials 2010, 31, 3479.

[131] T. U. Luu, S. C. Gott, B. W. K. Woo, M. P. Rao, W. F. Liu, ACS Appl. Mater. Interfaces 2015, 7, 28665.

[132] K. A. Barth, J. D. Waterfield, D. M. Brunette, J. Biomed. Mater. Res. A. 2013, 101, 2679.

[133] B. Wójciak-Stothard, A. Curtis, W. Monaghan, K. MacDonald, C. Wilkinson, Exp. Cell Res. 1996, 223, 426.

[134] N. J. Schaub, T. Britton, R. Rajachar, R. J. Gilbert, ACS Appl. Mater. Interfaces 2013, 5, 10173.

[135] J. T. Xia, Z. Triffitt, Biomed. Mater. 2006, 1, 1.[136] A. Luzardo-Alvarez, N. Blarer, K. Peter, J. F. Romero, C. Reymond,

G. Corradin, B. Gander, J. Controlled Release 2005, 109, 62.[137] E. Dohle, I. Bischoff, T. Böse, A. Marsano, A. Banfi, R. E. Unger,

C. J. Kirkpatrick, Eur. Cell Mater. 2014, 27, 149.[138] M. A. Haniffa, X. N. Wang, U. Holtick, M. Rae, J. D. Isaacs,

A. M. Dickinson, C. M. Hilkens, M. P. Collin, J. Immunol. 2007,179, 1595.

[139] N. Wada, P. M. Bartold, S. Gronthos, Stem Cells Dev. 2011, 20, 647.[140] R. A. Ferrer, A. Saalbach, M. Grünwedel, N. Lohmann,

I. Forstreuter, S. Saupe, E. Wandel, J. C. Simon, S. Franz, J. Invest. Dermatol. 2016, DOI: 10.1016/j.jid.2016.11.035.

[141] D. Polchert, J. Sobinsky, G. Douglas, M. Kidd, A. Moadsiri, E. Reina, K. Genrich, S. Mehrotra, S. Setty, B. Smith, A. Bartholomew, Eur. J. Immunol. 2008, 38, 1745.

[142] W. T. Tse, J. D. Pendleton, W. M. Beyer, M. C. Egalka, E. C. Guinan, Transplantation 2003, 75, 389.

[143] H. Nakajima, K. Uchida, A. R. Guerrero, S. Watanabe, D. Sugita, N. Takeura, A. Yoshida, G. Long, K. T. Wright, W. E. Johnson, H. Baba, J. Neurotrauma. 2012, 29, 1614.

[144] R. Yañez, M. L. Lamana, J. García-Castro, I. Colmenero, M. Ramírez, J. A. Bueren, Stem Cells 2006, 24, 2582.

[145] J. Kim, P. Hematti, Exp. Hematol. 2009, 37, 1445.[146] C. N. Manning, C. Martel, S. E. Sakiyama-Elbert, M. J. Silva,

S. Shah, R. H. Gelberman, S. Thomopoulos, Stem Cell Res. Ther. 2015, 6, 74.

[147] D. I. Cho, M. R. Kim, H. Y. Jeong, H. C. Jeong, M. H. Jeong, S. Ho Yoon, Y. S. Kimand, Y. Ahn, Exp. Mol. Med. 2014, 46, e70.

[148] D. A. Cantu, P. Hematti, W. J. Kao, Stem Cells Transl. Med. 2012, 1, 740.

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