Effect of Electrospun Fiber Diameter and Alignment on Macrophage

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Effect of Electrospun Fiber Diameter and Alignment on Macrophage Activation and Secretion of Proinflammatory Cytokines and Chemokines Enrica Saino,†,3 Maria Letizia Focarete,^ Chiara Gualandi,^ Enzo Emanuele,‡ Antonia I. Cornaglia,§ Marcello Imbriani,||,# and Livia Visai*,†,#,3 Department of Biochemistry, ‡Department of Health Sciences, §Department of Experimental Medicine, and Department of Public Health and Neuroscience, University of Pavia, 27100 Pavia, Italy ^ Department of Chemistry “G. Ciamician” and National Consortium of Materials Science and Technology (INSTM, RU Bologna), University of Bologna, Via Selmi 2, 40126 Bologna, Italy # Salvatore Maugeri Foundation IRCCS,Via S. Maugeri 4, 27100 Pavia, Italy 3 Center for Tissue Engineering (C.I.T.), Via Ferrata 1, 27100 Pavia, Italy )

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ABSTRACT: Macrophage activation can be modulated by biomaterial topography according to the biological scale (micrometric and nanometric range). In this study, we investigated the effect of fiber diameter and fiber alignment of electrospun poly(L-lactic) (PLLA) scaffolds on macrophage RAW 264.7 activation and secretion of proinflammatory cytokines and chemokines at 24 h and 7 days. Macrophages were cultured on four different types of fibrous PLLA scaffold (aligned microfibers, aligned nanofibers, random microfibers, and random nanofibers) and on PLLA film (used as a reference). Substrate topography was found to influence the immune response activated by macrophages, especially in the early inflammation stage. Secretion of proinflammatory molecules by macrophage cells was chiefly dependent on fiber diameter. In particular, nanofibrous PLLA scaffolds minimized the inflammatory response when compared with films and microfibrous scaffolds. The histological evaluation demonstrated a higher number of foreign body giant cells on the PLLA film than on the micro- and nanofibrous scaffolds. In summary, our results indicate that the diameter of electrospun PLLA fibers, rather than fiber alignment, plays a relevant role in influencing in vitro macrophage activation and secretion of proinflammatory molecules.

1. INTRODUCTION Medical devices and tissue-engineered constructs may induce an inflammatory reaction, termed foreign body reaction (FBR), after their in vivo implantation. Despite recent advances in material science and tissue engineering, current knowledge of the inflammatory mechanisms associated with FBR remains scanty. Research focusing on the immune response elicited by biomaterials is of paramount importance and should shed more light on their biocompatibility.1
4 The key role played by host macrophages against biomaterials has been increasingly recognized. Of note, it has been shown that exposure to biomaterials can modulate the production of proinflammatory cytokines by macrophages.5
8 The immune response to biomaterials may involve innate and acquired immune responses as well as the production of cytokines and chemokines.9,10 For many years, one type of activated macrophage termed M1 has been recognized. Since the 1990s, studies started to show heterogeneity of macrophages phenotype, indicating its plasticity to polarize into populations with different phenotypes and functions in response to stimuli. Alternatively activated macrophages, or M2, were further r 2011 American Chemical Society

subdivided into three subsets, M2a, M2b, and M2c, based on their phenotype.11 In general, M1 are inflammatory and microbicidal, whereas M2 are immunomodulatory, reparative, and poorly microbicidal. The balance of these differentially activated macrophages plays a crucial role in the phagocytosis of pathogens, the clearance of apoptotic cells, and the remodeling of injured tissues. M1 have decreased phagocytic capability and decreased (FccR)II expression, secrete proinflammatory cytokines, such as TNF, IL-1, IL-6, IL-12, and IL-23, possess antiproliferative functions, and induce Th1 responses, whereas M2 are important in tissue remodeling after inflammation. Mosser et al.12 suggested a spectrum of macrophage phenotypes and characterized macrophage populations based on three fundamental homeostatic activities, including host defense, wound healing, and immune regulation. Macrophages are grouped into three primary phenotypes: classically activated macrophages for microbicidal activity, wound-healing macrophages for tissue repair, and regulatory macrophages for anti-inflammatory Received: February 23, 2011 Published: March 18, 2011 1900

dx.doi.org/10.1021/bm200248h | Biomacromolecules 2011, 12, 1900–1911

Biomacromolecules activity. Macrophages can be stimulated to release a host of cytokines, including interleukins (IL), interferons (IFN), and tumor necrosis factor alpha (TNF-R), which in turn mediate other processes, including chemotaxis and cell activation, tissue repair, and angiogenesis.13 Foreign body giant cells (FBGCs) are observed in the inflammatory reaction against foreign bodies. Formation of these cells is stimulated by macrophage colony stimulating factor (MCSF) and cytokines such as IL-4 and IL-13.14
16 Recent studies performed on polymeric substrates of different type and topography have suggested that macrophage activation and FBGC formation may differ according to surface roughness17,18 and geometry.19,20 Particularly interesting are two studies by Sanders et al. that demonstrated a dependence of the immune response on the diameter of single fiber implants in the micrometric range.21,22 Electrospinning has been recognized as a scaffold fabrication technique with great potential.23
26 From a wide range of polymer solutions and melts,24,27
29 this technique produces continuous polymeric fibers with diameters ranging from a few nanometers to tens of micrometers. 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 orientation.30,31 Accordingly, electrospun substrates have been used for a wide range of tissue engineering applications including neural,32 cardiovascular,33 bone,34 and skin35 tissue engineering. Despite the increased interest in electrospinning, the potential effects of electrospun scaffolds on the immune system have not been fully examined. Bowlin and coworkers36
38 have recently focused on the study of the in vitro innate and acquired immune responses to electrospun blends of poly(p-dioxanone) with collagen and elastin, whereas a recent study by Cao et al.39 investigated the in vivo and in vitro FBR of electrospun polycaprolactone. In the present study, we sought to determine the in vitro immune reactions (macrophage adhesion, morphology, and secretion of proinflammatory molecules) to electrospun scaffolds, focusing on the effects of both fiber diameter (in the micrometric and submicrometric ranges) and fiber alignment. For this aim, murine macrophage-like cells RAW 264.7 were used to investigate the effects of poly-L-lactic acid (PLLA) electrospun scaffolds that differed in fiber alignment and fiber diameter. PLLA is a medical-grade biodegradable polymer that represents a good candidate for scaffold production because it is able not only to support cell growth but also to provide good mechanical properties to the engineered construct. The extensive use of PLLA electrospun scaffolds, for example, for nerve regeneration,40,41 for cardiac tissue repair,42 or in combination with inorganic particles for bone tissue regeneration,43,44 makes the evaluation of the immune response to this kind of scaffold imperative.

2. MATERIALS AND METHODS 2.1. Scaffold and Film Preparation and Characterization. PLLA (Lacea H.100-E) (average molecular weight by GPC = 8.4  104 g/mol, polydispersity index, PDI = 1.7) was supplied by Mitsui Fine Chemicals (Dusseldorf, Germany). Dichloromethane (DCM) and dimethylformamide (DMF) were purchased from Sigma-Aldrich and used without further purification. Electrospun scaffolds were produced by using an in-house electrospinning apparatus composed of a high voltage power supply (Spellman, SL 50 P 10/CE/230), a syringe pump (KDScientific 200 series), a glass syringe, a stainless-steel blunt-ended

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needle (inner diameter = 0.84 mm) connected with the power supply electrode, and a grounded high-speed rotating collector (length = 12 cm, diameter = 5 cm) positioned 15 cm away from the tip of the needle. The polymer solution was dispensed, through a Teflon tube, to the needle vertically placed on the collecting mandrel. Nanometric fibers were produced by dissolving PLLA in a DCM/DMF solvent (65:35 v/v) at a concentration of 13% w/v, whereas micrometric fibers were obtained by using a polymer concentration of 20% w/v. The electrospinning process was carried out under the following conditions: applied voltage = 12 kV, feed rate = 15  10
3 mL/min, at room temperature and relative humidity RH = 40 ÷ 50%. Fibers were collected with a random arrangement on a cylinder rotating at a speed of 5.2 m/s, whereas aligned fibers were obtained by increasing the rotational speed to 16.2 m/s. Electrospun scaffolds, ∼50 μm thick, were kept under vacuum over P2O5 at room temperature overnight to remove residual solvents. A PLLA film (thickness = 0.2 mm) was obtained by compression molding PLLA pellets between Teflon plates with an appropriate spacer at 200 C for 1 min under a pressure of 0.3 ton/m2 using a Carver C12 laboratory press. Fiber morphology was observed with a Philips 515 scanning electron microscope (SEM) at an accelerating voltage of 15 kV. Prior to SEM analysis, samples were sputter-coated with gold. We evaluated fiber diameter distribution by measuring 200 fiber diameters with an image acquisition software (EDAX Genesis), and results were reported as the mean ( standard deviation (SD). The relative degree of fiber alignment was measured by elaborating SEM images with ImageJ 2D fast Fourier transform (2D FFT) function and by applying an oval profile plug-in (designed by Bill O’Connell), as previously described in detail by Ayres et al.45 In brief, grayscale 8-bit images were cropped to 1590  1590 pixels and processed using 2D FFT. The resulting FFT output image was rotated 90 to correct for the inherent rotation of the data induced by 2D FFT analysis. The application of the oval profile plug-in enabled the summing of pixel intensities along the radius for each angle of a circular projection, which was previously placed on the FFT output image by using the ImageJ circular marquee tool. The summed pixel intensities were normalized to a baseline value of 0 and plotted as the function of the corresponding angle in arbitrary units from 0 to 180. 2.2. Cell Culture and Seeding. The murine macrophage-like RAW 264.7 cell line (ATCC, Manassas, VA), kindly provided by Dr. M. C. Bosco (Laboratory of Molecular Biology, Department of Pediatrics, G. Gaslini Institute, Genova, Italy) was cultured on tissue culture plastic (TCP) (75 cm2 flask) (Falcon; Becton Dickinson Labware) in Dulbecco’s modified Eagle’s medium (DMEM, Lonza) supplemented with 10% low-level endotoxin fetal bovine serum (Hyclone) and antibiotics (Biowhittaker) and incubated at 37 C, 5% CO2 in a humidified atmosphere (95% air). Cells were routinely passaged by scraping with a split ratio of 1:5 for assays. PLLA films and electrospun scaffolds (disks of 1.55 cm Ø) were sterilized by treatment with ethanol (EtOH) using the following protocol: under laminar flow, the scaffolds were immersed in 85% v/v EtOH for 15 min, followed by 70% v/v EtOH for 15 min, and then washed three times with phosphatebuffered saline (PBS, pH 7.4) containing 2% penicillin/streptomycin (100 U/ mL) (BioWhittaker-Lonza, Verviers, Belgium), streptomycin (100 μg/mL) (BioWhittaker-Lonza, Verviers), and 0.2% fungizone (Sigma-Aldrich). The scaffolds kept in PBS solution were then sterilized by ultraviolet irradiation (TUV 30W/G30 T8) overnight. RAW cells were then plated onto the different scaffold disks and placed at the bottom of standard 24-well culture plates at a population density of 1  105. All experiments and analyses were performed on PLLA film, and the scaffolds were placed in a fresh well to eliminate cells not seeded on the scaffolds and incubated for 24 h or 7 days. TCP wells and TCP wells plus 10 μg/mL lipopolysaccharide46 (LPS, E. coli; Sigma-Aldrich) served as negative and positive controls, respectively. Supernatants were harvested for assays after 24 h or 7 days and stored at
80 C. 2.3. Scanning Electron Microscopy Analysis. At the end of each incubation time (24 h and 7 days), the scaffolds were fixed with 1901

dx.doi.org/10.1021/bm200248h |Biomacromolecules 2011, 12, 1900–1911

Biomacromolecules 2.5% (v/v) glutaraldehyde solution in 0.1 M Na-cacodylate buffer (pH 7.2) for 1 h at 4 C, washed with Na-cacodylate buffer, and then dehydrated at room temperature in a gradient EtOH series up to 100%.47 The samples were kept in 100% EtOH for 15 min and then critical point-dried with CO2. The specimens were sputter-coated with gold and observed at 200 and 1000 magnification using a Leica Cambridge Stereoscan 440 microscope (Leica Microsystems, Bensheim, Germany) at 8 kV. 2.4. Cell Viability by MTT and LDH Test. MTT Test. To assess the mitochondrial activity of the seeded cells (i.e., the cell viability on each type of scaffold after 24 h or during the 7 day culture period), a test with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich) was performed on days 1, 3, and 7. The culture medium was replaced by a 0.5 mg/mL solution of MTT in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4), and the cell cultures were incubated for 4 h. Viable cells are able to reduce MTT into formazan crystals. After the MTT solution was removed, 500 μL of dimethyl sulphoxide (Sigma-Aldrich) was added to solubilize the formazan products, and the well plate containing the cultured PLLA scaffolds was agitated with shaking for 20 min. We sampled 200 μL aliquots, and the related absorbance values were measured at 570 nm by a microplate reader (BioRad Laboratories, Hercules, CA). A standard curve of cell viability was used to express the results as the percentage of adherence. LDH Test. The cytoxicity of each scaffold was quantitatively determined by measuring the release of LDH by the attached cells. LDH catalyzes the oxidation of lactate to pyruvate with simultaneous reduction of nicotinamide adenine dinucleotide (NAD), resulting in an increase in absorbance to 490 nm. PLLA films and scaffolds were placed inside standard 24-well plates and cultured with RAW 264.7 cells for 1, 3, and 7 days, respectively. At the end of each incubation time, the LDH assay (Sigma-Aldrich) was performed following the manufacturer’s instructions. The absorbance was measured at 490 and 690 nm with a microplate reader (BioRad).48 2.5. Cytokine Antibody Array. Multiple cytokine-expression levels can be simultaneously determined using enzyme-linked immunosorbent assay (ELISA)-based protein array technology49 (mouse cytokine antibody array, Panomics). With this approach, target proteins are captured by the array capture antibody and then detected in a sandwich ELISA format using a cocktail of biotinylated detection antibodies. The signals are visualized by either horseradish peroxidase (HRP)-conjugated streptavidin or enhanced chemiluminescence (ECL) (GE Healthcare). The protein array membranes were incubated with supernatants collected after 24 h and 7 days incubation with RAW 264.7 cells on PLLA films and micro- and nanofibrous scaffolds. For comparison, the supernatant of macrophages cultured on TCPS without (negative control) or with (positive control) LPS was analyzed. The experiment was performed according to the manufacturer’s protocol. The following is a list of cytokines and chemokines detected by the cytokine antibody array: G-CSF (mouse granulocyte colony stimulating factor); RANTES (regulated upon activation normal T cell expressed and presumably secreted); VEGF (vascular endothelial growth factor); IFN-γ (interferon gamma); MIP-1R (macrophage inflammatory protein-1 alpha); IL-6 (interleukin 6); TNF-R (tumor necrosis factor-alpha); M-CSF (macrophage colony-stimulating factor); GM-CSF (granulocyte-macrophage colony-stimulating factor); MIG (monokine induced by interferon gamma); IP10 (interferon gamma-induced protein 10 kDa); IL-1R (interleukin
1alpha); IL-2 (interleukin 2); IL-4 (interleukin 4); IL-5 (interleukin 5); IL-10 (interleukin 10); IL-12 (interleukin 12); IL-13 (interleukin 13).

2.6. ELISA Quantification of Cytokine and Chemokine Release. After screening by cytokine and chemokine antibody array, targeted cytokines were quantified by ELISA (TNF-R, MIP-1R, IL-1R, MCP-1 (monocyte chemoattractant protein-1), and IL-6, BioSource

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International, Camarillo, California; IFN-γ and RANTES, Bender MedSystems, Vienna, Austria; G-CSF and VEGF, Arcus Biologicals, Modena, Italy). The detection limit for each ELISA kit was the following: 0.96 pg/mL for MIP-1R; 1 pg/mL for TNF-R; 1.2 pg/mL for RANTES; 4 pg/mL for IFN-γ and VEGF; 5 pg/mL for G-CSF; 7 pg/ mL for IL-1β; 9 pg/mL for MCP-1; and 3 pg/mL for IL-6. All assays were performed according to the manufacturer’s instructions. In brief, 50 μL of standard, control, or sample was added per well. The tested samples were incubated for 2 h at room temperature. After unbound substances were washed, 100 μL of an enzyme-linked polyclonal antibody was added per well and then incubated for 2 h at room temperature. After a wash to remove any unbound antibody-enzyme reagent, 100 μL of substrate solution was added to each well and then incubated and protected from light for 30 min. The optical density of each well was then determined using a microplate reader (Biorad) set at 490 nm. All samples were assayed in triplicate. For each measurement, a standard concentration curve was generated according to assay kit indications and used to calculate the concentration of released cytokines. The cytokine secretion data were normalized to cell number/scaffold. 2.7. FBGC Formation. Cells grown for 24 h and 7 days on coverslips were fixed with 4% paraformaldehyde and stained with hematoxylin and eosin. TCPS-cultured macrophages were used as negative controls. Samples from each condition were fixed in a mixture of glutaraldehyde (2.5%) and paraformaldehyde (1%) in 0.1 M sodium cacodylate buffer (pH 7.4), dehydrated in graded ethanol, and routinely embedded in Epoxy resin (Fluka, Schweiz). Semithin (0.5 to 1 μm) sections were counterstained with toluidine blue. Samples were examined and photographed with an Axiophot light microscope (Zeiss, Germany). FBGCs were counted from five randomly selected positions on the same stained specimen in a section containing an average of 75 macrophage cells. The counting was performed on three different specimens for each PLLA scaffold. 2.8. Statistical Analysis. RAW 264.7 cells seeded on tissue well plates and stimulated with LPS were used as positive controls. Cytokine levels secreted in positive control experiments were used as the reference (100%). Levels of cytokines in other experiments were expressed as the percentage of cytokine concentrations compared with the positive control. All data are expressed as the mean and SD. Differences in the study variables according to different experimental conditions were calculated using one-way analysis of variance (ANOVA), followed by Bonferroni’s posthoc test. A two-tailed P value