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Biomaterials 34 (2013) 8161e8171

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Biomaterials

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The role of 3D structure and protein conformation on the innateand adaptive immune responses to silk-based biomaterials

Maumita Bhattacharjee a,b,c,d,e, Elke Schultz-Thater b,c, Emanuele Trella b,c, Sylvie Miot b,c,Sanskrita Das a,d,e, Marko Loparic f, Alok R. Ray d,e, Ivan Martin b,c, Giulio C. Spagnoli b,c,**,Sourabh Ghosh a,*

aDepartment of Textile Technology, Indian Institute of Technology, New Delhi 110016, IndiabDepartment of Surgery, University Hospital Basel, University of Basel, SwitzerlandcDepartment of Biomedicine, University Hospital Basel, University of Basel, SwitzerlanddCentre for Biomedical Engineering, IIT Delhi, New Delhi, IndiaeAll India Institute of Medical Sciences, New Delhi, IndiafBiozentrum and Swiss Nanoscience Institute, University of Basel, Switzerland

a r t i c l e i n f o

Article history:Received 7 June 2013Accepted 3 July 2013Available online 26 July 2013

Keywords:Silk-based biomaterialsStructural architecturesBiocompatibilityMonocyte activationCytokines

* Corresponding author.** Corresponding author. Surgical Oncology GrouUniversity Hospital of Basel, University of Basel, Swit

E-mail addresses: gspagnoli@usb.ch (G.C. Spagnol(S. Ghosh).

0142-9612/$ e see front matter Crown Copyright � 2http://dx.doi.org/10.1016/j.biomaterials.2013.07.018

a b s t r a c t

We have investigated monocyte and T cell responsiveness to silk based biomaterials of different physico-chemical characteristics. Here we report that untransformed CD14þ human monocytes respond toovernight exposure to silk fibroin-based biomaterials in tridimensional form by IL-1b and IL-6, but notIL-10 gene expression and protein production. In contrast, fibroin based materials in bidimensional formare unable to stimulate monocyte responsiveness. The elicitation of these effects critically requirescontact between biomaterials and responding cells, is not sustained and becomes undetectable in longerterm cultures. We also observed that NF-kb and p38 MAP kinase play key roles in monocyte activation bysilk-based biomaterials. On the other hand, fibroin based materials, irrespective of their physico-chemicalcharacteristics appeared to be unable to induce the activation of peripheral blood T cells from healthydonors, as evaluated by the expression of activation markers and IFN-g gene.

Crown Copyright � 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Silk protein produced by Bombyx mori silkworm has widelybeen used as biomaterial for centuries. Unique features of silk suchas mechanical strength, ease of chemical modification to impartbiofunctionalization and controlled degradation rate, made it anattractive candidate for biomedical applications. Silk has been usedin different forms such as 2D film, hydrogels, sponges [1] andfibrous scaffolds [2] in tissue engineering and drug delivery,and applications in bio-electronics and biomedical sensing areupcoming [3]. In spite of unprecedented applications of silk asbiomaterial, its biocompatibility has not been fully explored.

Biocompatibility of a biomaterial is generally assessed bystudying immune responses generated upon the interaction of

p, Department of Surgery,zerland.i), sghosh08@textile.iitd.ac.in

013 Published by Elsevier Ltd. All

biomaterials with the cells of immune system including both innateand adaptive immunity. Innate immunity is the non-specific firstline of defense including a variety of cellular and molecular com-ponents, which initiate immune recognition and respond to foreignmaterials. On the other hand, adaptive immunity comes into actionlater, following the initial exposure to the antigen and respondswith a high degree of specificity and the generation of memory.Both pathways are highly interconnected.

Monocytes are important players in innate immunity. They ex-press transmembrane receptors, such as Toll like receptors (TLRs),which facilitate their interaction with the biomaterials as theforeign material and respond to challenges by a variety of mecha-nisms including phagocytosis, generation of reactive oxygen spe-cies and nitrogen intermediates and secretion of pro-inflammatorycytokines including IL1b, IL6 and TNFa. These cytokines in turnmayparticipate in the activation of T lymphocytes.

Silk filaments have been used as surgical sutures since longtime. Recently, however, silk sutures have been replaced by syn-thetic polymeric sutures, due to undesirable immunogenicity anddifference in degradation rates upon implantation at different sites[1,4e7].

rights reserved.

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Native silk fiber has been acknowledged to be an allergenic agent[8,9] causing Type I allergy [10], such as asthma and specific increaseof IgE levels, but also delayed allergic responses. Upon sonication,silk fibers produced by silkworm release soluble factors, whichwerefound to be responsible for induction of pro-inflammatory cytokinesand increased phagocytosis [11]. The outer layer of silk, namelysericin (serine-rich glycoproteins), has been indicated to beresponsible for allergic reactions or pro-inflammatory effects of silk[12,13]. Although a recent report has suggested a possible role offibroin in the induction of Type I allergic reactions [10], most studiesreport minimal immune stimulation by fibroin component. Fibroinmembrane was found to activate macrophages to a lesser extent ascompared to polystyrene and poly(2-hydroxyethyl)methacrylate,since it did not support proper adhesion and spreading of macro-phages, resulting in reduced secretion of IL1b in comparison to theother two polymers [14]. Panilaitis et al. [15] studied the interactionof macrophages with silk fibers, as well as insoluble silk fibroinparticles (10e200 mm), along with collagen and black dyed braidedsilk suture. No significant amount of TNF was found to be releasedfrom a murine leukemia derived macrophage-like cell line (RAW264.7) when cultured with silk fibers both in short term and pro-longed culture, but TNF release was increased in presence of fibroinparticles in a dose dependant manner [15]. Meinel et al. [16] re-ported comparable immunogenic properties of silk films underin vitro and in vivo conditions. In vivo biocompatibility studies ofsilk fibroin reported nominal inflammatory reactions withoutfibrosis and lymphocyte invasion [17].

In the above studies the possible roles of secondary conforma-tion of silk protein, and overall architecture of the biomaterials havebeen largely overlooked. However, major factors governingimmunogenicity of any biomaterial could also include molecularsize, shape, morphology, surface chemistry, surface topography,surface stiffness, implantation site and processing induced degra-dation products [18e22]. For instance, Poly-vinyl pyrrolidone (PVP)with a 10,000 Da MW is a weak immunogen, whereas 360,000 DaMW PVP is a strong immunogen [23]. The role of total surface areato volume ratio of the biomaterial may also be crucial, as a higherinflammatory reaction was reported to be induced by particles<20 mm in diameter as compared with particles >50 mm [18,19].Bartneck et al. [21] studied the effect of material morphology inthree-dimensional (3D) nanofibers with different porosity andsurface chemistry in comparison with 2D substrate on the macro-phage phenotype and observed the expression of anti-inflammatory markers upon stimulation in 2D system and pro-inflammatory markers in 3D system.

Here we explored innate and adaptive immune responsesgenerated in vitro against silk biomaterials used in well docu-mented different physico-chemical forms.

Table 1Dimensions of scaffolds used.

Samples Weight Size

Sericin film 1.8 mg 0.3� 0.3 cm2

Fibroin film 1.8 mg 0.3� 0.2 cm2

3D fibroin 1.8 mg 0.2� 0.1� 0.1 cm3

2. Materials and methods

2.1. Preparation of silk biomaterials

B. mori silk cocoons were cut into small pieces and degummed by boiling in0.02 M Na2CO3 for 60 min to remove the outer sericin layer of silk. The supernatantcontaining sericin was collected and dialyzed for 48 h against deionized distilledwater to obtain 13% w/v sericin solution. Sericin solution was cast on teflon coatedplates and dried overnight in oven at 40 �C. The dried films were made insoluble byovernight treatment with 70% ethanol.

After degumming, the insoluble fibroin fibers were rinsed in distilled water andthen dried for at least 12 h and dissolved in 9.3 M LiBr at 60 �C for 4 h to obtain a 20%w/v pure fibroin solution of dried silk. The silkeLiBr solution was dialyzed in waterusing dialysis cassette (Pierce, M.W.C.O. 3.5 kDa) to obtain 8% w/v pure silk fibroinsolution. The silk solution was then concentrated against 15% PEG for 4 h to obtain13% w/v silk fibroin solution. Bidimensional (2D) fibroin films were prepared bycasting the fibroin solution on teflon coated plates and slowly dried overnight in anoven at 40 �C. 3D fibroin scaffolds were prepared by lyophilizing the silk fibroinsolution in 24well plates. The silk solutionwas frozen in liquid nitrogen (�196 �C) for

24 h. The frozen membranes were then lyophilized for 24 h in a (LABCONCOLyophilizer, Kansas City,MO, USA) and treatedwith 70% ethanol for overnight. Biopsypunches were used to cut cylindrical scaffold of particular dimensions (Table 1).

2.2. Scanning electron microscopy (SEM)

Silk fibroin and sericin films and 3D fibroin scaffolds were analyzed for thepresence of pores by SEM. The cultured silk samples were fixed with 4% formalde-hyde, dehydrated using alcohol gradients and sputter coated with gold. All sampleswere examined by a Zeiss EVO 50 Scanning electron microscope (Oberkochen,Germany). Pore size of the scaffolds was calculated (n¼ 5) using Image J software(NIH, USA).

2.3. Attenuated total reflectance-Fourier transformed infrared spectroscopy (ATR-FTIR)

Infrared spectra for silk fibroin films, 3D fibroin scaffolds and sericin films weremeasured on Attenuated Total Reflectance FTIR model Alpha-P, Bruker (Ettlingen,Germany). All spectra were taken in the spectral range of 4000e500 cm�1 byaccumulation of 264 scans and with a resolution of 4 cm�1. The IR spectra of fibroinfilms, 3D fibroin and sericin films covering the amide I region 1600e1700 cm�1 weredeconvoluted using Peakfit v4.12 software (Systat Software Inc. San Jose, CA, USA).After normalizing the spectra, relative area under each peak was used to identifysecondary structures in the sample. Peak absorption bands were assigned as follows:1605e1615 cm�1 Tyr-enriched chains or aggregated strands, 1610e1625 and 1696e1704 cm�1 as b-sheet crystal structures; 1640e1650 cm�1 as random coil confor-mation; 1650e1662 cm�1 as a-helices; and 1660e1695 cm�1 as b-turns [24].

2.4. X-ray diffraction (XRD)

X-ray diffraction was performed by (X’Pert PRO PANanalytical e DY2022,Almelo, The Netherlands) with CuKa radiation (1.5405�A) from 0� to 50� (2q). The Xray source was operated at 40 kV and 30 mA.

2.5. Contact angle measurement

The wettability of the samples was measured by adding a water droplet of 30 mlon 20 mm� 15 mm films (sericin and fibroin) and cylindrical 3D fibroin having adiameter of 4 mm and 2 mm height. The contact angle was measured by using atangent placed at the intersection of the liquid and solid phase. The angles weremeasured at 5 different areas for each sample to calculate means and SD.

2.6. Atomic force microscopy (AFM)

The elastic moduli at the nanometer scale were mapped quantitatively using aFlexAFM ARTIDIS (Nanosurf AG, Liestal, Switzerland) and a Nanowizard I (JPK In-struments, Berlin, Germany) atomic force microscopes as described earlier [25].Briefly, standard rectangular cantileverswith sharppyramidal tips (nominal tip radius20 nm, nominal k¼ 6.33 N/m; custom made cantilever, University of Neuchâtel,Switzerland) were used for stiffness measurements. The spring constant of eachcantilever was determined using the thermal noise method [26]. Each sample wasmeasured at 18 or more random spots comprising a scan area of 1800 mm2, with anindentation force of 1 mN at a frequency of 3 Hz. Generatedmapswith forceedistancecurves of at least 16�16 points were analyzed using the Oliver & Pharr method forcalculating the dynamic elastic modulus (stiffness) in a custom-developed Labviewsoftware (National Instruments, Austin, TX, USA), as reported earlier [2,27].

2.7. Surface area measurement

The specific area of the silk biomaterials was calculated using a surface areaanalyzer (Micromeritics, ASAP 2010, USA). Nitrogen gas was used as adsorbate andliquid nitrogen as the cooling media for adsorption/desorption isotherms of sam-ples. The surface areas of the 2D fibroin and 2D sericin film and 3D fibroin scaffoldswere calculated as single point from the BrunauereEmmetteTeller (BET) isothermusing adsorption points.

2.8. Isolation of human peripheral blood cells

Peripheral blood was collected from consenting healthy donors, with ethicalapproval from University Hospital of Basel, Switzerland. Fifty milliliters of blood

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were diluted in PBS at a ratio of 1:2, layered onto Histopaque 1077 (Sigma, St. Louis,MO, USA) and centrifuged at 2400 rpm for 20 min to obtain peripheral bloodmononuclear cells (PBMC). Cells were then washed once with PBS and once withMACS buffer (PBS, 2% FCS, 2 mM EDTA). The CD14þ monocytes were separated bypositive cell sorting using anti-CD14-conjugated microbeads (Miltenyi Biotech,Bergisch Gladbach, Germany) according to techniques recommended by themanufacturer. Monocytes were then suspended at a 4�105/ml cell concentration inRPMI 1640 supplemented with Kanamycin (100 mg/ml), Hepes buffer (10 mM), so-dium pyruvate (1 mM), Glutamax (1 mM), non-essential amino acids and 10% FCS(all from Invitrogen, Basel, Switzerland). CD4þ and CD8þ T cells were also immu-nomagnetically separated from the CD14� fractions of the PBMC.

2.9. Culture of CD14þ cells with silk biomaterials

The biomaterials, namely, 2D sericin film, 2D fibroin film, 3D fibroin, collagenUltrafoam (microfibrillar collagen sponge, bovine source, Bard Davol, Inc., Zurich,Switzerland) and tissue culture plates (TCP, Falcon, Allschwil, Switzerland) asnegative control, and LPS (from Escherichia coli 0111.B4 100 ng/ml, Invitrogen, SanDiego, CA, USA) as positive control, were placed in the 24 well plates and incubatedwith CD14þmonocytes (4�105 cells/well) in 1ml of RPMI 1640 supplementedwithKanamycin, Hepes, sodium pyruvate, Glutamax and non-essential amino acids, 10%FCS, at 37 �C up to 18 h.

The biomaterials were then cultured with CD14þ cells under similar conditionsfor up to 6 days and cells and culture supernatants were sampled at 1, 3 and 6 days ofculture to evaluate cytokine gene expression and protein release (see below).

2.10. Transwell experiments

For transwell experiments silk based biomaterials were sterilized using 70%ethanol and washed with PBS and along with Ultrafoam and LPS were put in the topwells/inserts of transwell plates with filters of 3.0 mm pores (Corning, Corning NJ)with 200 ml media in it and CD14þ cells were added to the bottom wells with4 � 105 cells/well in presence of 800 ml of RPMI 1640 at 37 �C up to 18 h. Cells andsupernatants were collected for the analysis of cytokine gene expression and release,as detailed above.

2.11. Analysis of pro-inflammatory signal transduction pathways

Different inhibitors of inflammatory signal transduction pathways were used toobtain insights into mechanisms underlying biomaterials recognition by monocytesand the generation of immune reactivity. Polymyxin B (PMB) (Calbiochem, Merck,Darmstadt, Germany) was used at 10 mg/ml, PDTC (Pyrrolidine dithiocarbamate)(Sigma, St. Louis, MO, USA) at 300 nM, LY294002 (PI3 Kinase Inhibitor; Sigma, St.Louis, MO, USA) at 10 mM and SB202190 (MAP Kinase Inhibitor; Sigma, St. Louis, MO,USA) at 10 mM. Signal transduction inhibitors were added at the beginning of culturesand supernatants were collected at different time points, as detailed above andtested for cytokine protein release.

2.12. Quantitative real time PCR (qRT-PCR)

Total RNA was isolated by using Nucleospin RNA II kit (MachereyeNagel, Düre,Germany) following manufacturer’s protocol. Purified mRNA was reverse tran-scribed into cDNA by priming 11 ml of total RNA with 1 ml (200 mg/ml) of Oligo dT(Roche Diagnostics, Mannheim, Germany) at 65 �C for 10 min followed by imme-diate transfer in ice. A mixture of 1 ml dNTPmix (10mM), 4 ml first-strand buffer (5�),2 ml DTT (0.1 M) and 1 ml M-MLV reverse transcriptase (200 units) (all by InvitrogenLtd., Paisley, UK) was added and incubated at 37 �C for 1 h.

Expression of IL1b and IL6 genes was analyzed by 7300 Real Time PCR system(Applied Biosystems, Rotkreuz, Switzerland) according to manufacturer’s protocol,using specific primers and probes (Assays on Demand, Applied Biosystems). Theexpression of genes of interest was normalized to GAPDH house keeping gene.Expression levels were calculated using the 2�(Dct) method with GAPDH as areference gene [28].

2.13. Cytokine release

Culture supernatants were collected for cytokine release analysis by quantitativeELISA assays, using antibody pairs and standards specific for IL6 (BD PharMingen,San Diego, CA, USA) and IL1b (eBiosciences, San Diego, CA, USA) detection accordingto instructions from manufacturer. Plates were read at 405 nm in a Spectramax190plate reader (Molecular Devices, Germany) and analyzed using SOFTmax software(Molecular Devices, Sunnyvale, CA, USA).

2.14. T cell cultures

Silk biomaterials were incubated with CD4þ or CD8þ T cells in the presence orabsence of autologous CD14þ at a 5:1 ratio (5�105:1�105 cells/well) in RPMI 1640supplemented with Kanamycin, Hepes, sodium pyruvate, Glutamax and non-

essential amino acids supplemented with 5% human serum (Blutbank Basel,Switzerland) at 37 �C for 18 h.

Cells were then collected and CD69 and CD25 expressionwas evaluated by flow-cytometry (FACScalibur, Becton-Dickinson, Allschwil, Switzerland) by using specificfluorochrome labeled monoclonal antibodies. IFNg gene expression (IFNg) wasanalyzed by qRT-PCR (7300 Real Time PCR system, Applied Biosystems, Rotkreuz,Switzerland) in the presence of specific primers and probes (Assays on Demand,Applied Biosystems), as detailed above.

2.15. Statistics

Statistical significancewas calculated by independent t tests using SPSS software(SPSS Inc., an IBM Company, Chicago, USA) and was considered significant atp< 0.05.

3. Results

3.1. Monocyte responsiveness to 3D fibroin scaffolds

Pre-clinical “in vitro” assessment of biocompatibility of newlydeveloped biomaterials is frequently performed using establishedhuman cell lines derived from myeloid leukemia cells [15,29].However, due to their malignant nature and long term culture,these cells might fail to reliably mirror functional features ofnormal, untransformed cells of the innate immune system. There-fore, we used freshly purified peripheral blood monocytes to assesstheir responsiveness upon incubation with a lyophilized 3D fibroinscaffold generated for regenerative medicine applications. LPS,Ultrafoam collagen gel (UF) and standard tissue culture plates (TCP)were used as positive and negative controls, respectively.

Monocyte activation is typically associated with the expressionof genes encoding pro-inflammatory cytokines, including IL1b andIL6 specific genes, as well as with their protein secretion. Weobserved that overnight incubation in the presence of a 3D fibroinscaffold was able to induce a significant expression of genesencoding IL1b and IL6 pro-inflammatory cytokines (Fig. 1A,B) andthe secretion of the corresponding proteins (Fig. 1C,D).

3.2. Generation and physical characterization of silk biomaterials

Since “in vitro” data indicated that a fibroin based 3D scaffoldwas able to activate human monocytes, we hypothesized thatbiomaterial architectures, and their inherent biochemical compo-sition might be responsible for immune activation. To test thishypothesis we generated silk fibroin biomaterials as 2D film and in3D form, and sericin films. These materials were characterized bydifferent techniques to gain insights into their morphology, surfacechemistry, topography and wettability with potential relevance inthe activation of immune competent cells.

SEM images showed that sericin and fibroin films have smoothsurface and no pores (Fig. 2A,B)whereas the 3Dfibroin scaffolds haveroughsurfaces, consistingofflats andgrooves, (Fig. 2C)andareporousin nature with non-uniform pore diameter (129.88� 29.22 mm;n¼ 5). The BET surface area analysis for the silk based biomaterialsindicated that they were all in the same range (Fig. 2H). Indeed, thesurface areawas found to be 0.0578m2/g for 2D sericin, 0.0529m2/gfor 2D fibroin and 0.0531 m2/g for 3D fibroin.

ATR-FTIR was used to characterize the silk biomaterials in termsof macromolecular conformation. The infrared spectral absorptionby the peptide backbones of amide I (1700e1600 cm�1) and amideII (1600e1500 cm�1) are usually assigned for the determination ofsilk protein conformation [24,30]. Sericin film in spectra a (Fig. 3A)showed its characteristic amide I and amide II peaks at 1622 cm�1

and 1521 cm�1, respectively. These bands however, may be attrib-uted to the characteristic b-sheet structure induced by methanoltreatment. 2D silk fibroin film in spectra b showed the presence ofcharacteristic C]O stretching for amide I as observed at 1644 cm�1,

Fig. 1. Monocyte activation by 3D fibroin scaffold. Peripheral blood CD14þ monocytes from healthy donors were purified and cultured in the presence of the indicated materials orleft untreated on tissue culture plates (TCP) overnight. Total cellular RNAwas then extracted, reverse transcribed and amplified in the presence of primers and probes specific for IL-1b (panel A), IL-6 (panel B) or GAPDH house-keeping gene. Data were analyzed by using GAPDH gene expression as reference. Cytokine release in 24 h culture supernatants wasanalyzed by ELISA (panels C/D). Data refer to duplicate samples from one representative experiment out of four independently performed with cells from different healthy donors.

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secondary NeH bending for amide II at 1519 cm�1, and CeNstretching for amide III at 1228 cm�1. Spectra c for 3D silk fibroinshowed that the presence of characteristic C]O stretching foramide I is observed at 1622 cm�1, the secondary NeH bending foramide II at 1506 cm�1, and CeN stretching for amide III is visible at1229 cm�1 (Fig. 3A). Taken together, the IR spectra indicate that 2Dsilk fibroin film predominantly exists in silk I and 3D silk fibroinexists in silk II conformational state. The percentage of the sec-ondary components was then calculated using Fourier self-deconvolution (FSD) of the infrared spectra of the amide I regionto determine the percentage of secondary components. In sericinthe percent of b-sheet content is 37.6% with only 3.1% of randomcoils (Fig. 3B). The fraction of secondary structure within 2D fibroinfilm is 27.1% a-helices, 12.2% random coils, 8.7% b-sheet (weak) oraggregated b-strands and 7.9% b-turns (Fig. 3C). The 3D fibroinscaffold includes a 51.6% strong b-sheet crystal structure showingmolecular aggregation with more packed structure and highercrystallinity and 28.9% aggregated strands (Fig. 3D).

XRD analysis confirmed the existence of different macromo-lecular conformations in 2D fibroin film and 3D fibroin scaffold.Indeed, the silk fibroin film made by casting the solution in tefloncoated plates and slow drying at 40 �C exhibited silk I conformationshowing the characteristic peak at 12.2�, 19.7� and 24.7� [31e33].However, upon lyophilization and alcohol treatment the 3D fibroinscaffold matrix showed b-sheet macromolecular organization asevident by displacement from the silk I peak (12.2�, 19.7�) to the silkII characteristic peak at 20.7� (Fig. 3E). XRD data of sericin filmrevealed the appearance of the diffraction peaks at 20.7� with astrong spacing of 4.27�A and 26.8� and a medium spacing of 3.32�A.Furthermore, according to Bragg law (l¼2d sin q) the average

distance (d) for 2D silk film was observed to be at 4.1 �A, consistentwith silk I structure, and at 4.2 �A (silk II) in 3D fibroin [31e33].

The contact angle measured for sericin film and fibroin filmwasfound to be 78.3� � 2.4� and 67.3� � 4.16� (Fig. 3F,G) respectively.Notably, the random coiled conformation of 2D fibroin film made itmore hydrophilic as compared to sericin. The contact angle for 3Dfibroin matrix could not be measured, due to its porous nature, thismaterial absorbed the water droplets immediately upon contact.However, the presence of b-sheet conformation in 3D fibroinwouldmake it comparatively more hydrophobic.

AFM analysis further validated SEM observations. Silk filmspossessed smooth surface, whereas 3D fibroin scaffold consisted ofaggregated and flat features (Fig. 4A). Furthermore, AFM analysisshowed that the2Dfibroinfilmexhibited thehighest stiffnesshavingmoduli of 26�105 Pa as compared to sericin and 3D fibroin, whosestiffness were 18� 105 Pa and 6�105 Pa, respectively (Fig. 4B).

3.3. Monocyte response to silk materials of different architectureand conformation: cytokine gene expression and cytokine release

In order to address differential responsiveness of untransformedhuman cells to silk based materials of different architecture andconformation, we first explored monocyte adhesion by SEM. SEMimages showed that purified peripheral blood CD14þ monocytesadhered well to the sericin film (Fig. 2D,E) and to the 3D fibroinscaffold (Fig. 2F,G) but minimal adhesion to the fibroin film wasobserved as compared to other matrices. The monocytes attachedto non-porous sericin film displayed spread shape with extendedfilopodia (as shown by the arrows), whereasmonocytes adhering tothe porous 3D fibroin matrix appeared to be spherical in shape.

Fig. 2. Surface morphology of silk-based biomaterials and morphological features of monocyte attachment. (A) SEM of 2D sericin. (B) SEM of 2D fibroin, showing non-poroussmooth surface. (C) SEM of 3D fibroin showing non-uniform pores. SEM of monocytes cultured on (D, E) 2D sericin, showing spread shaped cells with extended filopodia (asindicated by the arrows). (F,G) 3D fibroin scaffold showing spherical shaped monocytes attached to the porous surface. (H) BET surface area measurement.

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Silk derived materials were then incubated overnight in thepresence of CD14þ monocytes, in order to assess their differentialimmunostimulatory ability. Ultrafoamdid not induce cytokine geneexpression levels significantly higher than those detectable in cellsfrom unstimulated cultures. On the other hand, as expected, highIL1b gene expression was detectable in cells stimulated with LPSpositive control (Fig. 5A). Sericin film also induced a relevant IL1bgene expression. Minimal IL1b gene expressionwas observed in 2Dfibroin film stimulated cells. Most importantly, we observed thatIL1b gene expression in 3D fibroin stimulated cells was significantlyhigher (p< 0.05) as compared to that detectable in cells culturedover 2D fibroin film (Fig. 5A).

Similarly, minimal levels of IL6 gene expression were observedin 2D fibroin film stimulated cells whereas sericin showed thehighest immunostimulatory capacity, followed by 3D fibroin(Fig. 5B). The expression levels of both cytokines were found to bedramatically decreased after 3 days cultures in all samplesincluding LPS stimulated cells and reached baseline levels after day6 (Fig. 5A,B).

Gene expression data were corroborated at the protein level byquantifying IL1b and IL6 production by ELISA after overnight culture.IL1b levels were found to be 1.7 ng/ml in supernatants from sericinstimulated cultures, as compared to 0.6 ng/ml for 3D fibroin andnegligible levels for cultures performed in the presence of 2D fibroinfilm (p< 0.05). Accordingly, IL6 release of>6 ng/mlwas detectable in

supernatants of sericin stimulated cultures, as compared to 2.3 ng/mlfor 3Dfibroin and 0.007 ng/ml for 2Dfibroin film (p< 0.05, Fig. 6A,B).However, cytokine release after day 3 was found to be reduced in allexperimental groups and after day 6 cytokine levels in culture su-pernatants were virtually negligible (Fig. 6A,B). Taken together, thesedata indicate that architecture and protein conformation of silk-based biomaterials play important roles in monocyte activation.

3.4. Transwell experiments

To verify whether the ability of the biomaterials under investi-gation could be due to the presence of soluble factors, transwellexperiments were performed. Biomaterials were placed on theupper chamber of 3.0 mm transwell filters, and the CD14þ cellswere seeded in the lower chamber. In these culture conditions,expression of IL1b and IL6 at both gene and protein level was onlyobserved upon LPS stimulation (Fig. 7AeD). These data indicatethat a direct contact is required between silk-based biomaterialsand the immune cells to evoke responses.

3.5. Signaling pathways involved in monocyte response to silkbiomaterials

To gain insights into specific signal transduction mechanismsunderlying monocyte responsiveness to 3D fibroin scaffold we

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Fig. 3. Spectral analysis of silk-based biomaterials. (A) ATR-FTIR spectra: (a) 2D sericin (b) 2D fibroin (c) 3D fibroin. Fourier self-deconvolution of (B) 2D sericin, (C) 2D fibroin, (D) 3Dfibroin, providing evidence of Silk I conformation in 2D fibroin film and Silk II conformation in 3D fibroin. (E) XRD spectra showing different protein conformation in (a) 2D sericin(b) 2D fibroin (c) 3D fibroin. (F) Contact angle measurement showing wettability of 2D sericin. (G) Contact angle showing wettability of 2D fibroin.

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added a panel of defined inhibitors to our cultures. Polymyxin B is acyclic cationic polypeptide antibiotic which blocks the biologicaleffects of gram negative lipopolysaccharide (LPS) through bindingto its toxic component, negatively charged lipid A. PDTC inhibitsactivation of NF-kb, a transcription factor mediating a variety ofinflammatory responses. LY294002 is a potent, cell permeable in-hibitor of phosphatidylinositol 3-kinase (PI3K) known to regulateTLR-mediated inflammatory responses, whereas SB202190 is a p38MAPK inhibitor. Since reagents were diluted in DMSO or water,both products were used as negative control.

In the presence of PDTC, IL6 production by CD14þ cells inducedby all stimuli was inhibited, consistent with the involvement of NF-kb in the production of pro-inflammatory cytokines induced bysilk-based biomaterials. Polymyxin B inhibited IL6 production inLPS stimulated cultures, but failed to affect IL6 production inducedby sericin and 3D fibroin scaffold. SB202190 p38 MAP kinase in-hibitor significantly decreased IL6 production in all cultures,whereas LY294002 PI3K inhibitor was always ineffective (Fig. 8).

3.6. T cell responsiveness to silk materials

To extend our investigation to cells from the adaptive immunesystem, we cultured purified CD4þ and CD8þ T cells together with

silk based biomaterials in the presence of autologous CD14þ cells asantigen presenting cells. T cell activation was monitored by testingthe expression of CD69 and CD25 lymphocyte activation markersand the expression of IFNg gene. None of the biomaterials underinvestigation was able to induce a significant upregulation ofphenotypic markers or cytokine gene expression (data not shown).

4. Discussion

Silk has been extensively used as a biopolymer in tissue engi-neering and regenerative medicine. However, the critical issue ofsilk protein’s ability to stimulate the immune system remainslargely unexplored. Cells of the adaptive immune system areresponsible for the long term responsiveness to antigenic chal-lenges and for the establishment of immunological memory. Incontrast, cells belonging to the innate immune system representthe first line defense against microorganisms and effectivelyrespond to exogenous and endogenous “danger” signals. Interest-ingly, immune response has been shown to be dictated by surfaceproperties intrinsic to biomaterials including size, shape, surfacechemistry and topography [16e21].

In the present study we evaluated the capacity of silk bio-materials in different secondary conformations to activate human

Fig. 5. Monocyte responsiveness to silk-based biomaterials with different physic-chemical characteristics: cytokine gene expression. Peripheral blood CD14þ mono-cytes from healthy donors were purified and cultured for the indicated times asdetailed in “Materials and methods” in the presence of different silk-based bio-materials or control biomaterials, or left untreated in tissue culture plates (TCP). Totalcellular RNA was then extracted, reverse transcribed and amplified in the presence ofprimers and probes specific for IL-1b (panel A), IL-6 (panel B) and GAPDH house-keeping gene, used as reference. Data refer to duplicate samples from one represen-tative experiment out of two independently performed with cells from differenthealthy donors.

Fig. 4. AFM imaging. (A) AFM image of 3D fibroin showing flat and “groove” features.(B) Stiffness of silk biomaterials, as measured by AFM.

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monocytes and T cells. Importantly, in this study we did not useestablished human cell lines derived from myeloid leukemia cells[15,29] as responder cells. Instead, freshly purified peripheral bloodmonocytes from healthy donors were used to generate data moreclosely mirroring interactions presumably occurring “in vivo”.Furthermore, to explore the role played by conformational andstructural features of silk-based biomaterials, we developed a 2Dsilk fibroin film, which was mainly in silk I state (a-helices), and a3D fibroin scaffold, which was predominantly in silk II b-sheetconformation. ATR-FTIR studies and the deconvolution of theamide I region showed the predominance of silk I conformationwithin the 2D fibroin film along with some fractions of otherconformations, whereas the strong absorption band of 3D fibroin at1622 cm�1 was consistent with the b-sheet conformation (silk II)[24,30]. These findings were further validated by XRD studies, asthe diffraction peak and the spacings obtained in XRD confirmedthe existence of amorphous structure (silk I) in 2D silk fibroin filmand the transition of silk I to silk II (b-sheet conformation withcrystalline structure) in 3D silk fibroin scaffold. Our data areconsistent with earlier reports showing that fibroin films formedby slow drying at low temperature (40 �C) are mainly in silk Iconformation [34,35]. However, presence of silk I conformationmay generate concern regarding stability of the matrix in water ormedia. Within the gland of silkworm, fibroin protein solution as-sumes random coil conformation and it is soluble in water. Justbefore extrusion silk assumes insoluble silk I conformation (a-he-lices, b-turn and weak b-sheet crystal structure), whereas afterextrusion it is insoluble in water due to formation of crystallineb-sheet (silk II conformation) [35]. In order to address the issue of

water insolubility of fibroin film, we conducted FTIR and DSCstudies before and after immersing silk biomaterials in PBS for 6days. No significant difference in the fractions of random coil,b-sheet, a-helices, and b-turns was observed in the fibroin filmafter 6 days as detectable by Fourier self deconvoluted spectralanalysis of amide I peak (data not shown). Secondly, we conductedDSC study to investigate difference in stability of silk I and silk IIcrystals, using 2D fibroin film before or after PBS treatment for 6days. DSC curve of 2D fibroin film showed an endothermic peak at100 �C due to the presence of silk I crystals (which is a hydratedstructure) (Supplementary figure) [35]. Consistent with earlier re-ports the 2D fibroin film showed two degradation peaks at around252 �C and 259 �C [35]. The degradation peak around 257e260 �C isrelated to the stability induced by the b-sheet crystals, while theother degradation peak around 247e252 �C is due to the silk Istructure. These findings are consistent with a reasonable stability

Fig. 6. Monocyte responsiveness to silk-based biomaterials with different physico-chemical characteristics: cytokine production. Peripheral blood CD14þ monocytesfrom healthy donors were purified and cultured for the indicated times (panels A andB), as detailed in “Materials and methods” in the presence of different silk-basedbiomaterials or control biomaterials, or left untreated (tissue culture plates: TCP).Culture supernatants were then harvested and their cytokine content was quantifiedby ELISA. Data refer to duplicate samples from one representative experiment out oftwo independently performed with cells from different healthy donors.

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of silk I crystals. Thirdly, previous reports indicate that a fibroin filmgenerated by using the same water annealing and slow dryingmethod utilized in this study, and displaying a similar silk I content,was water stable [34,35].

Furthermore, we calculated the surface area of all the silk bio-materials using BET isotherm based surface area analyzer. Thisstudy confirmed that all three materials were having comparableranges of surface area.

We then focused on the response of freshly isolated CD14þmonocytes to different silk-based biomaterials developed. Mono-cytes cultured with silk biomaterials adhered to sericin film and 3Dfibroin but minimal attachment to fibroin film was observed.Monocytes attached to sericin film maintained spread morphologywith extended filopodia while they maintained a roundedmorphology on the 3D fibroin substrate. This finding is largelyconsistent with data from previous studies where non-porous sub-strate supported the spread shape of monocytes, eventually forminga sheet on the substrate, whereas porous substrate supported cir-cular cellular morphology similar to that observed in our sericin film

[20]. McBane et al. [36] also reported spread monocyte morphologyon 2D polyurethane films whereas more rounded appearance wasdetectable on the 3D polyurethane scaffolds. Young et al. [20] alsoshowed that non-porous hydrophobic surface supported lessermonocyte attachment but higher amount of cytokines were pro-duced as compared to porous hydrophilic surface. Thus, our findingsare also consistent with a partial role of porosity and wettability ofthe matrices in the modulation of monocyte behavior.

Stiffness of underlying matrix could be another important fac-tor to influence monocyte responses such as adhesion, spreadingand activation [37,38]. Earlier studies reported that stiffer matricesfavored macrophage attachment and spreading as compared to thelower moduli matrices [37]. In our study we observed thatalthough 2D fibroin film was the stiffest material, followed bysericin film and 3D fibroin scaffold, cell attachment was maximalin sericin film and 3D fibroin with nominal attachment to fibroinfilm. However, the cell morphology on the silk matrices closelyreminds studies by Blakney et al. [37], who reported similarrounded morphology of macrophages on lower moduli substratewhereas spread shape morphology with protruding filopodia uponculture on stiffer matrices. Furthermore, the expression of thethree major pro-inflammatory cytokines TNFa, IL1b and IL6 waslowest for the softest material. On the contrary, in our study IL1band IL6 gene expression and protein release were maximal uponstimulation by 3D fibroin, having lower moduli as compared to 2Dfibroin film. This suggests that stiffness of the biomaterial playsminimal roles in the induction of immune response by silk-basedbiomaterials.

We then addressed the role of biomaterial surface propertiesand protein conformation on immune cell activation. Activation ofmonocytes by silk biomaterials led to the expression of IL1b and IL6genes and to the production of the corresponding cytokines.However, the level of expression significantly varied with the typeof biomaterials and was found to be the highest in CD14þ cellsstimulated by sericin film, followed by 3D fibroin and then 2Dfibroin film. The reason for the weaker immune response to 2Dfibroin film and strong response to 3D fibroin is likely to be rep-resented by differential protein conformation. Earlier studies re-ported that peptides are poor immunogens but protein aggregatesor supramolecular assembly of proteins are strongly immunogenic[39,40]. The strong immunogenic properties of peptides assembledby fibrillizing in the generation of strong antibody responses werereported by Rudra et al. [39]. Furthermore, prolonged T cell inde-pendent B cells response was observed to be induced by highmolecular weight polymers multiple repeat epitope units [41].Thus, in our study 2D fibroin film having mainly silk I conformationwith reduced b-sheet was a poor immuno-stimulator as comparedto the 3D fibroin with higher b-sheet content, e.g. more assembledstructure acting as a strong immunostimulus. This indicates thatcytokine induction is more governed by the protein conformationrather than by other properties such as wettability, porosity andstiffness of the material.

Most remarkably, the elicitation of these effects critically requiredphysical contact between monocytes and silk materials, and wasnot detectable in cultures performed under trans-well conditions.These data clearly indicate that the well documented physico-chemical differences between the materials under investigation arecritical to their capacity to stimulate innate immune responses.

A recent study [21] has reported that macrophages culturedover 2D substrate express CD163, an M2 anti-inflammatory marker[42] and 27E10, an M1 pro-inflammatory marker upon culture on a3D substrate. However cells cultured on the 2D substrate releasedpro-inflammatory cytokines whereas cells cultured on 3D substratereleased M2-like factors with a decreased production of pro-inflammatory cytokines. Importantly, in our study we have

Fig. 7. Direct contact requirement for monocyte activation by silk-based biomaterials. Peripheral blood CD14þmonocytes from healthy donors were purified and cultured overnightin the presence of different silk-based biomaterials or control biomaterials in transwell plates with chambers separated by 3.0 m pore membranes, preventing direct cell/biomaterialcontact. Total cellular RNAwas then extracted, reverse transcribed and amplified in the presence of primers and probes specific for IL-1b (panel A), IL-6 (panel B) and GAPDH house-keeping gene, used as reference. Culture supernatants were also harvested and their cytokine content was quantified by ELISA. Data refer to duplicate samples from an experimentout of two independently performed with cells from different healthy donors.

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observed that none of the materials under investigation, regardlessof its conformationwas able to induce expression or release of IL10,a typical M2 cytokine (data not shown).

We found that the immunostimulatory effects of silk basedmaterials were characterized by a characteristic kinetic, with highinduction of pro-inflammatory cytokines during the first day ofculture, rapidly declining in the following days, and becomingundetectable by day 6 of culture. Notably macrophage or bonemarrow stem cell reactivity to silk materials was reported to bepoor in prolonged cultures [16]. Accordingly, silk induced IL1bproduction was reported to be increased upon up to 3 day cultureand then gradually decreased as compared to poly-glycolic acid(PGA) [43]. Our observations largely correlate with these studiesregarding the poor long term pro-inflammatory potential of silkbiomaterial.

We further analyzed the signaling pathways involved in theactivation of monocytes upon interactionwith the silk biomaterials.NF-kb transcription factor is largely involved in regulating a num-ber of pro-inflammatory pathways. Indeed, by performing our

experiments in the presence of specific inhibitors, we found thatNF-kb and p38 MAP kinase critically contribute to monocyte acti-vation stimulated by silk materials. Importantly, Polymyxin B, ahighly effective inhibitor of endotoxin-mediated monocyte activa-tion was unable to inhibit IL6 production induced by 3D Fibroin,thus ruling out the possibility of endotoxin contamination behindour observations. On the other hand, NF-kb and p38 MAP kinaseplay key roles in monocyte activation driven by Toll-like receptors(TLR) triggering. Therefore, future research is required to clarifywhether the immunostimulatory ability of 3D silk-based bio-materials also resides in their capacity to trigger selected TLR.

Most interestingly, in our studies we were unable to detect asignificant T cell responsiveness to silk-based biomaterials. How-ever, the slow kinetics of the induction of T cell responses, mighthave failed to be adequately mirrored in our experiments.Furthermore, we cannot exclude that T cell responsiveness mightonly be detectable in healthy donors with an important previousexposure to the materials under investigation. Additional in-vestigations are warranted to address these issues.

Fig. 8. Signal transduction pathways involved in monocyte response to silk biomaterials. Peripheral blood CD14þ monocytes from a healthy donor were purified and culturedovernight in the presence of the indicated silk-based or control biomaterials and Polymyxin B (PMB), PDTC, LY294002 (LY) or SB202190 (SB) or DMSO or water, used as solvents.Signal transduction inhibitors were added at the beginning of cultures and supernatants were collected and tested by ELISA for IL-6 content. Data refer to triplicate cultures fromone healthy donor.

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5. Conclusion

Our data strongly support the notion that physical characteristicsand protein conformation play decisive roles in the induction of pro-inflammatory responses in monocytes upon stimulation with silk-based biomaterials. The presence of silk II conformation withhigher b-sheet content in 3D fibroin proved to be more immuno-genic as compared to 2D fibroin film with silk I conformation.Furthermore they provide a simple set of experimental approachessuitable for the initial pre-clinical evaluation of the bio-compatibilityof materials of potential use in tissue regeneration.

Acknowledgments

We thank Elisabetta Padovan (Basel, Switzerland) for advice onmonocyte and T cell activation assays and for critical reading of themanuscript. This work was supported by the Indo-Swiss JointResearch Programme (ISJRP 123 003) and Indian Council of MedicalResearch project (09/NCD-1).

Appendix A. Supplementary data

Supplementary data related to this article can be found online athttp://dx.doi.org/10.1016/j.biomaterials.2013.07.018.

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