Incorporation of graphene oxide into poly(ɛ-caprolactone) 3D printed fibrous scaffolds improves their antimicrobial properties

Journal Pre-proof Incorporation of graphene oxide into poly(ɛ-caprolactone) 3D printed fibrous scaffolds improves their antimicrobial properties

Sofia F. Melo, Sara C. Neves, Andreia T. Pereira, Inês Borges, Pedro L. Granja, Fernão D. Magalhães, Inês C. Gonçalves PII:

S0928-4931(19)32160-5

DOI:

https://doi.org/10.1016/j.msec.2019.110537

Reference:

MSC 110537

To appear in:

Materials Science & Engineering C

Received date:

11 June 2019

Revised date:

8 December 2019

Accepted date:

9 December 2019

Please cite this article as: S.F. Melo, S.C. Neves, A.T. Pereira, et al., Incorporation of graphene oxide into poly(ɛ-caprolactone) 3D printed fibrous scaffolds improves their antimicrobial properties, Materials Science & Engineering C (2018), https://doi.org/ 10.1016/j.msec.2019.110537

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© 2018 Published by Elsevier.

Journal Pre-proof Incorporation of graphene oxide into poly(ɛ-caprolactone) 3D printed fibrous scaffolds improves their antimicrobial properties Sofia F Melo1,2,3,4,5,*, Sara C Neves1,2,*, Andreia T Pereira1,2,4, Inês Borges1, Pedro L Granja1,2,3,4, Fernão D Magalhães5, Inês C Gonçalves1,2,# 1

i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

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INEB—Instituto de Engenharia Biomédica, Universidade do Porto, Portugal

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FEUP—Faculdade de Engenharia da Universidade do Porto, Portugal

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ICBAS—Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Portugal 5

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LEPABE—Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia, Faculdade de Engenharia da Universidade do Porto, Portugal *

Equal contribution of both authors.

#

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ABSTRACT

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Corresponding author: Tel.: +351 220408800; Email: [email protected] (Inês C Gonçalves).

Implantable medical devices infection and consequent failure is a severe health issue,

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which can result from bacterial adhesion, growth, and subsequent biofilm formation at the implantation site. Graphene-based materials, namely graphene oxide (GO), have been described as potential antibacterial agents when immobilized and exposed in polymeric matrices. This work focuses on the development of antibacterial and biocompatible 3D fibrous scaffolds incorporating GO. Poly(ε-caprolactone) scaffolds were produced, with and without GO, using wet-spinning combined with additive manufacturing. Scaffolds with different GO loadings were evaluated regarding physicalchemical characterization, namely GO surface exposure, antibacterial properties, and ability to promote human cells adhesion. Antimicrobial properties were evaluated through live/dead assays performed with Gram-positive and Gram-negative bacteria. 2 h and 24 h adhesion assays revealed a time-dependent bactericidal effect in the presence of GO, with death rates of adherent S. epidermidis and E. coli reaching ~80% after 24 h of contact with scaffolds with the highest GO concentration. Human fibroblasts cultured 1

Journal Pre-proof for up to 14 days were able to adhere and spread over the fibers, independently of the presence of GO. Overall, this work demonstrates the potential of GO-containing fibrous scaffolds to be used as biomaterials that hinder bacterial infection, while allowing

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human cells adhesion.

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Journal Pre-proof INTRODUCTION One of the main challenges associated to implantable devices is to find efficient strategies to face bacterial adhesion and consequent infection [1]. Device colonization by bacteria can lead to its malfunction, as it may result in biofilm formation at the implantation site [2]. This represents a serious health problem, worsened by growing antibiotic resistance, and causing the loss of the implanted device or even sepsis [3]. This has been subject of extensive research, but the desirable successful prevention or effective solution are yet to be achieved. organisms

associated

to

polymeric

meshes

infection

are

of

Common

Staphylococcus spp [4], [5] with Staphylococcus epidermidis on the leading positions

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[6], but also Gram-negative Enterobacteriaceae, such as the rod-shaped Escherichia

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coli [7], [8]. These are both biofilm-forming bacteria which produce extracellular polysaccharides when proliferating on a surface, enhancing their survival efficiency [8],

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[9]. S. epidermidis has a naturally high resistance to antimicrobials, which generates great concerns. E. coli also resists to antibiotics such as penicillin, since the outer

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membrane surrounding the cell wall provides an additional barrier [10]. Bacterial adhesion to device’s surface is not a one-time phenomenon, but rather an evolving

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process. Initially, there is a rapid attachment to the surface, mediated either by nonspecific factors (such as surface tension, hydrophobicity, and electrostatic forces) or

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by specific adhesins [11], followed by an accumulation phase, during which bacteria adhere to each other and form the biofilm [12]. Alternatives to antibiotics are being thoroughly explored, and carbon-derived materials are receiving growing attention [13]. Since graphene’s (G) first isolation in 2004 [14], its derivatives have been developed and investigated, commonly conjugated with polymers to produce composites or used to modify their surface [15]–[17]. In 2010, the antibacterial properties of graphene-based materials (GBMs) were explored for the first time [18], leading to a growing number of reports that describe some GBMs as antimicrobial nanomaterials [19]–[24]. The interaction between GBMs and biological systems has also been studied [25]–[28], giving some insights regarding their effect on different types of organisms. Nevertheless, these interactions need to be explored in more detail regarding GBMs immobilized in polymeric matrices since their characteristics may vary. Antimicrobial properties, for instance, are known to be different when comparing GBMs in suspension with GBMs immobilized on a surface 3

Journal Pre-proof [24], [29]–[31]. Moreover, direct physical contact of bacteria with GBMs at a surface (either with sharp edges or basal planes) is a requirement for GBMs-containing biomaterials to have an antibacterial action, with no effect being observed when no direct contact is established [31], [32]. In this work, graphene oxide (GO) was selected among GBMs to be produced and incorporated in polymeric fibrous scaffolds, since smaller and more oxidized forms of GBMs have been associated with higher biocompatibility towards mammalian cells [33]. Furthermore, stronger bactericidal properties have been described in oxidized forms of graphite (Gt) and graphene nanoplatelets (GNPs) [21], [24], [34]. It is

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described that the orientation and exposure of GO sheets on the fibers’ surface are important parameters, being essential factors for antibacterial properties [31]. The

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chosen method for fiber-based composite scaffolds production was a combination of

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additive manufacturing (AM) with wet-spinning, a non-solvent induced phase separation (NIPS) technique. This allows the production of fibers with diameter in the

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range of 100 µm, which is sufficiently wide to allow incorporation of GO sheets but narrow enough to expose them at the fibers surface. This process has been previously

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described for the manufacturing of polymeric scaffolds for tissue engineering [35]–[37]. Briefly, a dispensing tip is fed with a polymeric solution and submerged in a non-

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solvent of the polymer that causes its precipitation and filament formation. In this work, GO exposure was expected to be achieved in micrometric wet-spun polymeric fibers.

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Furthermore, the combination of wet-spinning with controlled spatial deposition of the filaments via AM presents the additional advantage of 3D-organized fabrication, instead of the random deposition of the fibers. To create 3D-structured scaffolds, poly(ε-caprolactone) (PCL) is currently among the most popular synthetic polymers used [38]. PCL fibers assembled into random or organized 3D structures have been broadly studied in the scope of tissue engineering approaches [39]–[42]. PCL is highly appealing due to its physical-chemical and mechanical characteristics [43], [44], and non-toxic degradation products. It received Food and Drug Administration (FDA) approval and European Conformity (CE) marking for a number of drug delivery and medical device applications [43]. Furthermore, this polymer presents additional advantages, namely availability, relatively low cost, suitability for modification [45], and relatively long biodegradation time, which makes it widely used also in long-term implants [46].

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Journal Pre-proof Improvements in PCL performance have been attempted through its modification, namely by adding new components like GO [42]. Although few studies are available [47], GO incorporation in PCL matrices led to improvements in terms of hydrophilicity, mechanical and thermal properties, and biocompatibility [48]–[50]. Ultimately, this work aimed to assess whether the use of GO represents a step forward in the antimicrobial 3D printed biomaterials field, similarly to what has been observed for GBMs in so many distinct areas [51]. In order to do so, this work comprised the production of GO-containing 3D PCL fibrous scaffolds and the

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assessment of their antimicrobial and biocompatible properties.

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Journal Pre-proof MATERIALS AND METHODS 1. Materials production 1.1. Graphene Oxide

GO was prepared according to the Modified Hummer’s Method (MHM) [52], adjusting reagents volume/mass for larger scale production (2000 mL flasks). Briefly, 320 mL of H2SO4 (VWR, Germany) were mixed with 80 mL of H3PO4 (Chem-Lab, Belgium) in a 4:1 ratio, and stirred at room temperature (RT) for an improved oxidation. 8 g of graphite (carbon graphite micropowder, American Elements, USA, purity above

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99%, diameter between 7 and 11 µm) were added to this solution and then cooled down

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to 0 ºC using an ice bath before gradual addition of 48 g of KMnO4 (JMGS, Portugal). The solution was heated up to 35 ºC and stirred for 2 hours. After lowering the

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temperature to 0 ºC, 1200 mL of distilled water were slowly added. This was followed

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by careful addition of H2O2 35% (WWR, Germany) until oxygen release stopped. After overnight resting, the solution was decanted to separate the solid deposit

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from the acidic supernatant. The remaining product was washed with dH2O and this aqueous solution was centrifuged (Eppendorf 5810R) at 4000 rpm for 20 minutes at RT.

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This step was repeated until the washing water pH was equal to dH2O pH. By the end of this process, sonication was performed for 6h to exfoliate the oxidized material into

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graphene oxide.

1.2. PCL/GO fibrous scaffolds

In order to prepare PCL (Sigma-Aldrich, average Mn 80 000 g mol−1) and GOcontaining PCL solutions, appropriate solvents were studied. These should efficiently disperse GO and dissolve PCL. The solvents tested were chloroform (VWR, Germany), acetone

(JMGS,

Portugal)

and

tetrahydrofuran

(THF,

VWR,

Germany)

(Supplementary Table 1). After this, PCL non-solvents, namely isopropanol (VWR, Germany) and ethanol (VWR, Germany), were screened, to select the best coagulation bath to obtain the polymer filaments (Supplementary Table 2). Different PCL and GO concentrations were tested, ranging from 7.5% to 15% w/v (weight of PCL per volume of solvent) and from 0% to 10% w/w (weight of GO per weight of PCL), respectively (Supplementary Table 3).

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Journal Pre-proof Once optimal conditions were found, GO was dispersed in THF at final concentrations of 5% w/w and 7.5% w/w. PCL was then dissolved overnight at RT either in these dispersions or in pure THF, in a final polymer concentration of 7.5% w/v. From here on, the prepared dispersions will be referred to as 0%, 5% and 7.5%, corresponding to the GO concentration present. Given their suitable viscosity, these formulations allowed the use of a needle with internal diameter of 184 µm (28G) for scaffold plotting. Solutions were loaded into a glass syringe and extruded into an ethanol coagulation bath using a syringe pump (New Era Pump Systems). The layer-by-layer fabrication of the scaffolds was performed using a 3D

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plotting machine (custom-made xyz plotter, INEGI – University of Porto, Portugal), adapting a previously described setup [35]. Printing parameters such as flow rate (F)

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(syringe pump) and plotting speed (Vdep) (xyz plotter) (defined as relative percentage to

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the plotter firmware parameters, Supplementary Information), were adjusted, ranging from 0.5 mL/h to 1.0 mL/h and from 50% to 120%, respectively (Supplementary

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Table 4). The chosen parameters were defined as F = 0.5 mL/h and Vdep = 80%. 3D design was then optimized in terms of xyz inter-fiber distances and staggering. To

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evaluate the best fit for a well-defined, precisely spaced structure, tested xy distances ranged from 200 μm to 400 μm, z-steps from 20 μm to 80 μm, and staggering between

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layers from 50 μm to 200 μm (Supplementary Table 5). The final 3D design of the scaffolds is displayed bellow in Figure 1, including top-view and cross-section schemes

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that represent the virtual model used.

Figure 1. 3D model of the produced scaffolds, showing the top view (where d x and dy are displayed) and the cross-section view (where dz is shown and the staggering between layers is visible).

Following plotting, PCL and PCL/GO scaffolds were rinsed 3 - 5 times with ethanol and dried in the fume hood. After this, cylinders of 4-mm diameter were coredout from the additive manufactured structures using a stainless steel biopsy puncher 7

Journal Pre-proof (Integra® Miltex®) and used for the biological in vitro studies. All samples were sterilized with ethylene oxide at Hospital de São João (Porto, Portugal), following the established sterilization protocol for medical devices.

2. Materials characterization 2.1. X-ray photoelectron spectroscopy (XPS)

The success of graphite oxidation using the MHM was verified by X-ray photoelectron spectroscopy (XPS) analysis, which was performed at CEMUP (Centro

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de Materiais da Universidade do Porto), with the equipment Kratos Axis Ultra HSA. A monochromatic Al X-ray source (anode) operating at 15 kV (90 W) was used. A 300

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m x 700 m square was the analyzed area.

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80 eV was the energy used for survey spectra, whereas 40 eV were used for O 1s and C 1s high-resolution spectra acquisition. The effect of the electric charge was

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corrected by setting the reference of the C 1s peak to 285.0 eV. Spectral deconvolution

2.2. Optical microscopy

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was performed with CasaXPS software.

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Printed PCL and PCL/GO fibers were first observed by stereomicroscopy (Olympus, Japan), with a magnification of 6.3x. Fibers organization, diameter and

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possible printing defects were visualized, allowing a fast screening of preferable printing parameters.

2.3. Scanning electron microscopy (SEM)

Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) analysis of final PCL/GO scaffolds was performed using a high resolution (Schottky) environmental scanning electron microscope, with X-Ray microanalysis and backscattered electrons diffraction patterns analysis (Quanta 400 FEG ESEM/EDAX Genesis X4M), requiring a 15 kV voltage for the desired quality images. This analysis was performed at CEMUP (Centro de Materiais da Universidade do Porto). Scaffolds were fixed on conductive carbon tape strips, both for top views and cross-section observations. Cross-section images acquisition required scaffolds’ cryofracturing after fast-freezing them with liquid nitrogen. 8

Journal Pre-proof Before SEM/EDS analysis, all samples were sputter-coated with Au/Pd, using the SPI Module Sputter Coater equipment for 100 seconds with a 15 mA current, to improve samples conductivity and consequent imaging quality. 3. Antibacterial effect assessment 3.1. Bacterial strains and growth conditions

Antibacterial activity was evaluated towards Staphylococcus epidermidis (ATCC 35984) and Escherichia coli (ATCC 25922). Bacteria were grown in Trypticase Soy Agar (TSA, Merck, USA) plates overnight at 37 ºC, and colonies were hand-picked

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with an inoculation loop immediately after incubation or after plate storage at 4 ºC (1-

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week shelf life). Two colonies were collected, inoculated in Trypticase Soy Broth (TSB, Merck, USA) and cultured overnight at 37 ºC in an orbital shaker oven (Raypa, Spain),

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at 150 rpm. Bacteria overnight inoculum was centrifuged (2700 rpm for 10 min) and the

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3.2. Bacterial adhesion assays

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pellet was rinsed with fresh TSB. This procedure was repeated three times.

The concentration of bacteria overnight cultures was determined by optical density (OD) measurement at 600 nm, using an UV-Vis Spectrophotometer (Perkin

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Elmer, USA). Initial inoculum concentration was adjusted to 1 × 107 Colony-Forming Units (CFUs)/mL in TSB, which was further supplemented with 10% v/v human

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plasma, to mimic the protein adsorption to the scaffolds that naturally occurs in physiological conditions. A lower initial bacteria concentration was also tested for S. epidermidis (6 × 105 CFUs/mL) and is presented as Supplementary Information (Figure S1).

Bacteria seeding was performed by placing a drop of 10 μL of bacterial suspension on top of the sterilized scaffolds and incubation in 24-well plates was performed for 2 h or 24 h at 37 ºC under static conditions in an incubator (Binder, Germany). 3.3. Visualization of adherent bacteria

After 2 h or 24 h of incubation, scaffolds with adherent bacteria were rinsed three times with NaCl 0.85% w/v to remove non-adherent bacteria. Samples were stained with BacLightTM LIVE/DEAD® Kit (Molecular Probes, USA) to investigate 9

Journal Pre-proof adherent bacteria viability using fluorescence microscopy. The component that identifies live cells (SYTO9) has its emission intensity wavelength centered at about 530 nm and the component that identifies dead cells (propidium iodide) has its emission intensity wavelength centered at about 630 nm Both stock solutions were mixed (1:1 ratio) and 10 μL drops were placed on top of each scaffold, followed by a 15-minute incubation time, protected from light. After staining, the scaffolds were transferred to an imaging support (Ibidi, Germany), and imaged using a Laser Scanning Confocal Microscope Leica TCS SP5 II (Leica Microsystems, Germany). Scans of a total z-height of at least 150 μm were

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performed, in order to include at least one complete scaffold layer, which has 100 μm in height (detailed information can be found in Supplementary Figure S2). A step size of

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1.5 μm was used, considering S. epidermidis and E. coli dimensions (coccoid

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morphology with 0.8 - 2 μm diameter, and rod-shaped morphology, 2 μm long, respectively).

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Representative scanned z-series of the samples were projected onto a single plane and pseudo-colored using ImageJ software [53].

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For SEM analysis (Supplementary Figure S3), 24 h-incubated S. epidermidis was rinsed with PBS and fixed using paraformaldehyde (PFA, Merck, USA) 4% w/v in

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PBS for 15 minutes. After rinsing with PBS, samples were dehydrated in a series of ethanol solutions (50, 60, 70, 80, 90, 96 and 99% v/v in water), dried overnight in

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hexamethyldisilazane and Au/Pd sputtered before visualization. Image acquisition was performed as described in section 2.3.

4. In vitro biocompatibility assessment 4.1. HFF-1 cell line and culture conditions

In vitro biocompatibility assays were performed using human foreskin fibroblasts (HFF-1) cell line (ATCC, SCRC1041). HFF-1 where expanded in Dulbecco's modified Eagle's medium (DMEM+, Gibco, Thermo Fisher Scientific, USA) supplemented with 10% v/v fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, USA) and 1% v/v penicillin/streptomycin (Pen/Strep, Biowest, France) at 37 ºC, in a humidified atmosphere, containing 5% v/v CO2. Media was refreshed every two days. When reaching 90% confluence, cells were rinsed with 5 mL of PBS (37 ºC) and 10

Journal Pre-proof detached from culture flasks using 2 mL of 0.25% w/v trypsin solution (Sigma Aldrich, USA) in PBS. In all passages, HFF-1 cells were seeded at an approximate density of 15 000 cells/cm2, as recommended by the supplier. 4.2. Indirect contact assay

The cytocompatibility of the scaffolds was initially evaluated using the indirect contact assay, by incubation of HFF-1 with extracts of materials (Figure S4), which were prepared as described in ISO 10993−12:2004. Briefly, the scaffolds were extracted with Dulbecco's modified Eagle's medium (DMEM+, Gibco, Thermo Fisher Scientific,

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USA) supplemented with 10% v/v fetal bovine serum (FBS, Gibco, Thermo Fisher

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Scientific, USA) and 1% v/v penicillin/streptomycin (Pen/Strep, Biowest, France) for 24 h at 37 ºC in an orbital shaker at 100 rpm. HFF-1 cells were seeded in 96-well plates

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at density of 1×105 cells/mL and kept in culture for 24 h in DMEM+ at 37 °C with 5 % v/v CO2. The medium was then replaced by material extracts, according to ISO 10993-

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5:2009(E) and incubated for 24 h at 37 ºC with 5 % v/v CO2. Extracts of tissue culture

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plastic coverslips (TC-PET, Sarstedt, Germany) were used as positive control of in vitro biocompatibility and a solution of DMEM with 0.1% (v/v) Triton (X-100) was used as

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negative control.

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4.2.1. Metabolic activity

Mitochondrial metabolic activity of cells was quantified by resazurin assay. For that, culture medium was removed from the wells and fresh media containing 10% v/v resazurin was incubated with the cells for 4 h at 37 °C with 5 % v/v CO2. Afterwards, media was transferred to a black 96-well plate and the relative fluorescence units (RFUs) were measured (λex ≈ 530 nm, λem ≈ 590 nm) using a micro-plate reader (Synergy MX, BioTek).

4.3. Direct contact assay

Before cell seeding, scaffolds were incubated in culture medium overnight to promote protein adsorption. Cells were then seeded on the polymeric scaffolds by placing a droplet of 10 μL of cell suspension (60 000 cells) on top of each scaffold. Fibroblasts were allowed to adhere for 1 h before 500 μL of supplemented DMEM+ per

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Journal Pre-proof well were added. Incubation was performed up to 14 days, with media being refreshed every other day. 4.3.1. Metabolic activity After 1, 7 and 14 days of culture, mitochondrial metabolic activity of cells was quantified by resazurin assay as described above (section 4.2.1). Before incubation with DMEM+ with 10% v/v resazurin, the seeded scaffolds were transferred to new 48-well plates to ensure that only cells adherent to the scaffolds were being measured.

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4.3.2. Visualization of adherent cells

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After 1, 7 and 14 days of culture, fibroblasts seeded scaffolds were rinsed with

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PBS and fixed using paraformaldehyde (PFA, Merck, USA) 4% w/v in PBS for 15

fluorescent microscopy and SEM.

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minutes. Samples were again rinsed with PBS and prepared for visualization by

For fluorescence microscopy, the adherent cells were stained for the identification of

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filamentous actin (F-ACTIN) and DNA, using phalloidin conjugated with Alexa Fluor® 488 (1:100, Molecular Probes®) and 6-diamidino-2-phenylindole dihydrochloride

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(DAPI, 3 µg/mL, Merck, USA), respectively. Incubation with phalloidin was performed for 1 hour, under mild agitation, and afterwards cells were rinsed and kept at 4 ºC in

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PBS until microscopy observation, to avoid drying. DAPI was added approximately 10 minutes before confocal imaging. Representative scanned z-series of the samples were projected onto a single plane and pseudo-colored using ImageJ software [53]. For SEM analysis, fixed samples were dehydrated in a series of ethanol solutions (50, 60, 70, 80, 90, 96 and 99% v/v in water), treated with hexamethyldisilazane, and dried overnight before being Au/Pd sputtered. Image acquisition was performed as described in section 2.3.

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Journal Pre-proof RESULTS 1. Graphene Oxide

XPS analysis revealed that the produced GO was constituted by 70.9% of carbon atoms and 29.1% of oxygen atoms (Figure 2A), while commercial graphite typically has a much lower percentage of oxygen atoms (8% of O 1s, as described for pristine graphite by Pinto et al. [54]). After peak deconvolution (Figure 2C), C-O groups, namely epoxy and hydroxyl groups, which emerge on the basal planes of GO sheets [55], were found to be the most prevalent oxygen-containing functional groups on GO

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structure, with an occurrence of 40.9% of all C bonds (Figure 2B). Carbonyl (C=O) and carboxylic groups (O-C=O), which appear on GO edges [33], [55], were less

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predominant, representing 12.3% and 2.5%, respectively. Overall, XPS results showed

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that, after performing MHM, graphite was efficiently oxidized, with the introduction of

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several oxygenated groups.

Figure 2. XPS analysis of GO. (A): Differences in atomic percentages (at %) of carbon 1s and oxygen 1s between GO and commercial graphite. *XPS results for graphite were obtained from Pinto et. al. [54]; (B): Contents of chemical groups resulting from deconvolution of carbon 1s high resolution spectra of GO; (C): Carbon 1s high-resolution spectra of GO.

2. PCL/GO fibrous scaffolds

As shown in Figure 3, PCL and composite PCL/GO fibrous scaffolds with approximately 1.2 cm x 1.2 cm were plotted, according to the 3D model previously disclosed in Figure 1. The obtained 0% GO scaffolds (pristine PCL) are white, and the incorporation of GO within the polymeric matrix can be verified by the change in color, as GO-containing scaffolds present a light brown (for 5% GO) and dark brown (for 13

Journal Pre-proof 7.5% GO) color. As can be observed, fibers are well defined and precisely plotted, following the virtual design. 3D-structured and stable matrices were obtained, with no

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dragging or fibers fusion (in the xx or yy axis) being observed.

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Figure 3. Stereomicroscope images of obtained PCL scaffolds with 0%, 5% and 7.5% GO. The lower lane represents zoomed regions of the upper lane images. Scale bar: top – 5 mm; bottom (zoom) – 500 μm.

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SEM observation of the scaffolds (Figure 4) corroborates the precise plotting of the filaments, showing the staggering between layers, visible in both top view and

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cross-section images (at lower magnifications). Fibers’ average diameter was measured along different fibers, with 0% GO scaffolds presenting average diameters of 107 ± 11 µm, 5% GO scaffolds of 102 ± 9 µm and 7.5% GO scaffolds of 103 ± 13 µm. From the SEM analysis, it was also possible to confirm GO incorporation in the fibers and its exposure. Top views of the fibers surface show GO exposure for both 5% and 7.5% GO concentrations (third row of images in Figure 4). GO sheets protrude from the surface, creating a wrinkled topography, with surface roughness and irregularity increasing when GO is incorporated. Smooth-surfaced small round particles present at the fibers surfaces appeared to be non-dissolved polymer. EDS analysis was performed to these regions, indicating the presence of carbon and oxygen. Hypothetical contamination with other elements was rejected, but the exact nature of those aggregates remains unclear. Focusing on the cross-sections, it is possible to observe that the inner porosity of the fibers changed with the presence of GO within the PCL matrix. The size and the 14

Journal Pre-proof irregularity of the combs increased in a direct proportion to the amount of incorporated

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Figure 4. SEM analysis of the top views and cross-sections of PCL scaffolds with 0%, 5% and 7.5% GO. Scale bar (from the top to the bottom row): top view - 400 μm, 50 μm, 2 μm; bottom - 200 μm, 50 μm.

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Journal Pre-proof 3. Antibacterial properties of PCL/GO fibrous scaffolds

PCL/GO scaffolds antibacterial effect was assessed after 2 h and 24 h incubation with S. epidermidis and E. coli. Adherent bacteria were stained and visualized by confocal microscopy. PCL and composite PCL/GO fibers presented some autofluorescence at the 495/518 nm excitation/emission wavelengths combination. Regarding S. epidermidis (Figure 5A), the total number of bacteria adhered to the fibers after 2 h of incubation was similar in all scaffolds (no statistically significant

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differences), independently of the presence of GO. However, higher amounts of dead bacteria were found in 5% GO (1.10 ± 0.93 bacteria/104 µm2 of fiber) and 7.5% GO

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(1.94 ± 1.10 bacteria/104 µm2 of fiber), comparing to 0% GO scaffolds (0.07 ± 0.10 bacteria/104 µm2 of fiber) (p = 0.0014 and p < 0.0001, respectively). As such, the

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incorporation of GO promotes bacterial death, with the percentages of dead bacteria

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increasing from only 2.9% in 0% GO to 41.8% in 5% GO and 53.8% in 7.5% GO. 24 h adhesion assays confirmed the bactericidal effect of GO-containing

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scaffolds. Similarly to what was observed for the 2 h assay, the number of total bacteria found in the fibers was not significantly different between the samples,

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independently of the presence of GO. Interestingly, the death rates were higher comparing to the ones obtained after 2 h incubation, being 13.7% in 0% GO, and

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reaching 71.9% in 5% GO and 77.8% in 7.5% GO scaffolds. The number of dead bacteria was again higher in 5% and 7.5%, when compared to 0% GO scaffolds (p < 0.0001 in both concentrations). Moreover, the number of live bacteria that adhered to the scaffolds was significantly lower in GO-containing scaffolds (p ≤ 0.0001), with only around 1.7 bacteria/104 µm2 of fiber, comparing to around 5.3 bacteria/104 µm2 of fiber in PCL with 0% GO scaffolds. Additional preliminary antimicrobial assays were performed with a lower S. epidermidis initial inoculum (Figure S1). It has been shown that independently of the concentration of the initial inoculum, the tendency for the increase of the percentage of dead bacteria in GO-containing scaffolds is kept. Both confocal microscopy and SEM (Figure S3) images reveal that biofilm formation was not observed in any of the scaffolds, even after 24 h. Bacteria were scattered across the surface, either individually or in small clusters.

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Journal Pre-proof In the E. coli adhesion assays (Figure 5B), the results revealed the same timedependent bactericidal potential of GO, together with a decreased number of total adherent bacteria after 24 h of contact with GO-containing scaffolds. In the 2 h adhesion assay, the number of adherent bacteria on the 7.5% GO scaffolds (4.13 ± 3.01 bacteria/104 µm2 of fiber) was significantly lower than the total amount found in 0% GO scaffolds (11.81 ± 9.56 bacteria/104 µm2 of fiber) (p = 0.001), suggesting that this concentration of GO was influencing initial bacterial adhesion. In 5% GO scaffolds, despite no differences were found in the total number of adherent bacteria when compared to the control, the number of dead E. coli was

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significantly higher (1.43 ± 0.80 bacteria/104 µm2 of fiber) when compared to 0% GO scaffolds (0.77 ± 0.57 bacteria/104 µm2 of fiber), which corresponds to a higher death

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rate in the presence of GO.

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After 24 h of contact, the effect of GO was more pronounced on preventing bacterial adhesion and compromising the viability of adhered ones. The total number

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of bacteria was significantly higher in the absence of GO when compared to both 5% and 7.5% GO scaffolds (p ≤ 0.0035). The death rates found were 16.4% in 0% GO,

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60.9% in 5% GO and 76.2% in 7.5% GO scaffolds, following the same tendency found for S. epidermidis. Moreover, the number of live E. coli was significantly lower

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(p ≤ 0.0001) in GO-containing scaffolds (< 6 bacteria/104 µm2 of fiber) than in 0% GO scaffolds (27.37 ± 13.46 bacteria/104 µm2 of fiber).

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These interesting results also demonstrate that while the number of live bacteria increases in PCL with 0% GO scaffolds from 2 h to 24h, this does not occur in GO-containing scaffolds. In 5% and 7.5% GO, the number of live bacteria is either maintained (with S. epidermidis) or decreased (with E. coli) over the 24h, corroborating the bactericidal effect of these scaffolds over time.

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Figure 5. S. epidermidis (A) and E. coli (B) adhesion to PCL scaffolds with 0%, 5% and 7.5% GO, after 2 h and 24 h incubation in 10% v/v plasma supplemented TSB, stained with the LIVE/DEAD Baclight kit. Left panel: Representative confocal microscopy images of live and dead adherent bacteria. Scale bar: 100 μm. Right panel: Stacked bars graph with adherent live and dead bacteria counting per 104 μm2 of fiber § displayed in green and red, respectively. Statistically significant differences on the number of total ( ), # dead (*) and live ( ) bacteria compared to 0% GO scaffolds are indicated on top of the stacked bars (p ≤ 0.05; non-parametric Kruskal-Wallis test).

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Journal Pre-proof 4. In vitro biocompatibility of PCL/GO fibrous scaffolds

The in vitro biocompatibility of the scaffolds was evaluated by both indirect and direct contact assays. Indirect contact assay (Figure S4) performed after contact of materials extracts with HFF-1 cells revealed that cells metabolic activity is around 100 % after contact with all materials extracts, confirming that the leaching products of the scaffolds are not cytotoxic. In the direct contact assay, performed to evaluate PCL and composite PCL/GO scaffolds ability to promote mammalian cells attachment and growth, HFF-1 cells

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were seeded on the scaffolds (Figure 6). The metabolic activity was monitored

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throughout the 14 days culture time, and samples were imaged after 1, 7 and 14 days of culture.

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The metabolic activity of the cells during direct contact with the scaffolds (Figure 6A) confirm that the scaffolds are biocompatible. Statistically significant

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differences were found between the metabolic activity of HFF-1 cells in GO-

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containing scaffolds between day 1 and day 7 of culture (p ≤ 0.0001), and in all scaffolds between day 7 and day 14 (p ≤ 0.0001), indicating that cells were active

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during the two weeks of culture.

Representative confocal microscopy and SEM images of the scaffolds are presented in Figure 6B, where it is possible to observe that cells adhere at the surface

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of the fibers just after 1 day of culture. Furthermore, cells were able to proliferate and colonize the scaffolds, secreting extracellular matrix (supported by the SEM images) for up to 14 days of culture in all scaffolds formulations. Furthermore, the morphology of the cells on the scaffolds corroborate the trends observed on the metabolic activity measurements, since fibroblasts were adhered and spread along the fibers, with evident F-actin stretching and spindle-like morphology (Figure S5) during the 14 days of culture, in all the produced scaffolds.

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Figure 6. (A): Metabolic activity of HFF-1 during direct contact, assessed by resazurin assay after 1, 7 and 14 days of culture in 0%, 5% and 7.5% GO scaffolds. Results are displayed as fluorescence arbitrary # units (AU). Statistically significant differences between each time point and day 1 (*) or day 7 ( ) are indicated on top of the bars (p ≤ 0.0001); 2way ANOVA with Tukey's multiple comparisons test). (B): Representative confocal microscopy and scanning electron microscopy (lower lane) images of HFF-1 cells behavior in PCL scaffolds with 0%, 5% and 7.5% GO, after 1, 7 and 14 days of culture (DNA in blue and F-actin in green). Confocal images represent single plane projections of at least 70 μm height zstack. Scale bars: 200 μm.

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Journal Pre-proof DISCUSSION In recent years, GBMs have been proposed as potential materials for biomedical applications, namely in medical devices and implants. They represent relatively lowcost raw materials, with proven low cytotoxicity for mammalian cells and described antimicrobial capacity. In the current work, GO-containing 3D PCL fibrous scaffolds were successfully produced by wet-spinning combined with additive manufacturing, and further evaluated in terms of GO exposure, antimicrobial activity and ability to adhere human cells.

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The production of GO by Gt oxidation and exfoliation was verified by XPS analysis (Figure 2). The percentage of oxygen in GO close to 30% was in accordance to

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what is expected for oxidation through MHM [56], [57].

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The method chosen for fiber-based composite scaffolds production was selected in order to enhance GO exposure. On one hand, fibers had to be wide enough to

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incorporate GO sheets presenting diameters ranging from 2 to 10 μm. This excluded nanometric fibers production techniques, such as electrospinning [49], [58]–[60]. On the

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other hand, GO exposure in composites produced by melt-dependent techniques is known to be difficult to obtain [31], since GO sheets are usually covered or

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encapsulated by the polymer in the obtained fibers [61]. When using techniques as fused deposition modeling (FDM), fibers with several hundred micrometers (> 300 μm)

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are produced [61], [62]. Therefore, an intermediate technique was chosen, by combining AM with wet-spinning (a non-solvent induced phase separation (NIPS) technique), allowing fiber production with a 50 - 150 µm diameter range [35], [63]. The introduction of GO into a polymeric matrix can have several physicalchemical effects. Accordingly, the analysis of the obtained scaffolds revealed that GO had an impact on the morphological features of PCL filaments obtained by NIPS. The scaffolds cross-sections revealed the formation of wide, irregular and heterogeneously arranged pores caused by the presence of GO (Figure 4). The distribution of hydrophilic GO dispersed in a hydrophobic matrix (PCL) might be a key factor affecting the morphology of polymer precipitation. This was also described by Dinescu et al. [64] in chitosan/GO composite scaffolds, corroborating our data. Regarding the surface of the composite fibers, and in accordance with our results (top-views on Figure 4), the presence of grooves and protuberances was previously described, whereas pristine PCL fibers were described as relatively smooth [49]. Independently of the GO 24

Journal Pre-proof concentration used, fibers measurements revealed similar diameters among the produced scaffolds (approximately 100 μm in all cases). Taken together, SEM analysis allow to state that 0%, 5% and 7.5% GO scaffolds with a 3D network of macropores were successfully fabricated and GO exposure at the fibers surface, necessary to confer the antimicrobial profile, was achieved in 5% and 7.5% GO scaffolds. When evaluating the antimicrobial activity of the developed structures (Figure 5), the effect of GO was noticed after a relatively short period (2 h) towards S. epidermidis and E. coli, especially when incorporated in higher concentrations (7.5%

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GO scaffolds). This rapid effect of GO was previously described by Hong et al. [65] towards E. coli after 2 h in GO-containing polyvinylidene fluoride (PVDF) fibers. A

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99.6% disinfection effectiveness was found for the highest GO concentration tested

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[65]. Interestingly, in our study, this initial interaction between bacteria and GO was different for S. epidermidis and E. coli. For the first, death rates in 7.5% GO scaffolds

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were already high after 2 h of contact, whereas for E. coli a decrease in the number of total adherent bacteria was observed, despite low death percentage. This can be due to

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the different morphology of both microorganisms, since Gram-negative bacteria have an outer membrane, which can prevent or delay direct piercing, influencing the first cell-to-

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surface contact upon adhesion. As S. epidermidis is more susceptible to membrane destabilization, bactericidal effect of GO can be identified earlier.

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Our antimicrobial assays also allowed to conclude that the presence of GO has a noticeable time-dependent effect on bacterial activity, since both bacteria were mostly dead (almost 80%) after 24 h of contact with our 7.5% GO scaffolds. Moreover, the total amount of adherent E. coli was significantly lower in 7.5% GO scaffolds, revealing that the ability of GO in preventing bacterial adhesion was also intensified over time. Several authors described GO antibacterial effect as being time-dependent, particularly when GO is incorporated in a composite polymeric matrix. An et al. [66] found significant differences in the antibacterial effect of GO present in a PU/PLA/GO composite against E. coli and S. aureus, when comparing incubation times of 4h and 24h. In 4h incubation assays, 54% - 91% growth inhibition percentages were found, depending on the GO concentration, whereas in 24 h assays 99% - 100% growth inhibition was found, for both bacterial species [66]. Although GO has been recurrently referred to as the most promising GBM when pursuing an antibacterial effect, the majority of the studies assessed its effect in 25

Journal Pre-proof dispersions, rather than immobilized [21], [23], [27], [31], [67]. Nevertheless, there are few papers regarding GO antibacterial activity when immobilized in PCL. PCL/GO composite discs were produced by Kumar et. al. [68], and used to study antibacterial activity against E. coli, showing significantly lower colony count (56%) with respect to pristine PCL (100%). Bacterial studies revealed that interaction with functionalized GO induced bacterial cell death by membrane damage [68]. Murugan et. al. [69] produced mineralized hydroxyapatite (M-HAP)/PCL/GO composite coatings, which were tested for bactericidal properties against S. aureus (inhibition zone with 16 mm) and E. coli (inhibition zone with 20.5 mm). In the absence

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of GO, both inhibition zones measured approximately 11 mm [69].

Our data demonstrated that antimicrobial action of GO is maintained when this

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nanomaterial is incorporated in PCL in 3D-organized scaffolds, composed by fibers

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with a diameter of approximately 100 µm. Furthermore, an antibacterial effect is unraveled towards different bacteria, S. epidermidis and E. coli, which are

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microorganisms commonly found in implant infections. Moreover, in our data, supplementation of bacterial growing media with human plasma did not affect

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experimental readouts, since the acquired images and sequential bacteria counts were similar to the ones obtained without TSB supplementation (data not shown).

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Antibacterial properties of GBMs are believed to be caused by chemical and physical interactions upon the direct contact of graphene sheets with bacteria [70], [71],

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in which the bacterial cell membrane seems to be the main target [72]. These antibacterial effects have been associated with either induced oxidative stress or bacteria physical disruption [66], [71], which are mechanisms that can be activated after only 2 h and exacerbated after 24 h of contact. When bacteria are exposed only to basal planes, mainly two effects have been reported: electron transference between GBMs and bacteria membrane [73] and O2 adsorption on defect sites and edges of the GBMs [24], also followed by electron transference, both resulting in oxidative stress and bacteria death. When sharp edges are protruding, besides the mentioned events, also physical insertion of the GBMs sharp edges through the bacteria membrane (nano-knife effect) [74], [75], protein-protein bonding disruption and pore formation may occur [20], [71], [76], disrupting the membrane and killing bacteria. Since our composite scaffolds enclose both individualized and aggregated exposed GO sheets (supported by SEM observation), all these phenomena can occur. However, in oxidized graphene based materials, the oxidation process typically leads to folding of the sharp edges, making 26

Journal Pre-proof physical membrane disruption less likely, and indicating oxidative stress caused by reactive oxygen species (ROS) production and electronic destabilization as the main antimicrobial mechanism of action of the herein developed GO-containing scaffolds. Regarding the in vitro biocompatibility assessment (Figure 6, Figure S4, and Figure S5), both the morphology of HFF-1 cells and their metabolic activity during the 14 days of culture in all conditions indicate that scaffolds not only are non-cytotoxic but also promote fibroblasts proliferation and ability to secrete endogenous extracellular matrix. As our intention was to assess cellular adhesion to the fibers, we performed short-period experiments – up to 14 days. Several studies are described in the literature

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using GBMs to ensure biocompatibility and even promote cell adhesion and growth [25], [26], [33], [77], [78]. Jalaja et al. [32] incorporated GO in electrospun gelatin to

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form GO-decorated gelatin nanofibers. Cytocompatibility was assessed using mouse

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fibroblasts (L-929) and the results indicated that the GO-containing gelatin nanofibers support cell adhesion and proliferation [32]. Similar results were obtained by Yoon et

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al. [79], who cultured neuronal cells for 2 days using a different composite (PLGA/GO), revealing that the hydrophilic nature of GO enhanced wettability and

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interaction with proteins and, therefore, biocompatibility of PLGA was maintained [79]. Different authors also suggest binding of the GO surface with serum proteins in culture

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media via electrostatic interactions, due to the presence of oxygenated groups. Hence, cell attachment to GO-containing surfaces may be enhanced due to a higher density of

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surface-bound molecules [80], [81].

The GO incorporation in PCL scaffolds herein suggested significantly improved the antibacterial activity of the pristine polymer, while allowing human cells adhesion. Since bacterial infection represents a constant threat when implantable medical devices are used, our data suggests promising performance of PCL/GO scaffolds in several biomedical applications, namely as surgical sutures or 3D scaffolds for tissue engineering, in which PCL is currently used [40], [82], [83]. This study clarified several aspects regarding GO containing scaffolds fabrication techniques, GO antimicrobial potential when incorporated in 3D fibrous scaffolds, and GO biocompatible character. Despite the very promising results, extrapolation of these conclusions to scaffolds made from other polymers has to be carefully considered since the interaction of GO with other polymers may induce different exposure of the graphene platelets and therefore exert different antimicrobial properties. Indeed, the incorporation of graphene based materials with different degrees 27

Journal Pre-proof of oxidation, thickness and lateral size could be explored, as well as different patterns of the 3D printed scaffolds. Furthermore, even though a Gram-positive and a Gram-negative bacteria have been used and antibacterial action verified in both, it would be interesting to challenge the surfaces with other bacteria involved in implantable device-related infections,

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namely S. aureus or P. aeruginosa, either alone or in a mixed inoculum.

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Journal Pre-proof CONCLUSION Wet-spinning combined with additive manufacturing allowed the production of well-defined PCL and composite PCL/GO fibrous scaffolds with average fiber diameters of 100 µm. A concentration of 5% GO was apparently enough to expose GO sheets at the surface of the composite fibers. Antimicrobial properties of PCL and composite PCL/GO 3D-organized fibrous scaffolds were assessed for the first time, revealing GO time-dependent bactericidal effect and an increase in death rate from less than 20% in neat PCL scaffolds to nearly 80% in composite scaffolds with 7.5% GO,

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after 24 h of contact with both Gram-positive and Gram-negative bacteria. In vitro biocompatibility evaluation showed that PCL and composite PCL/GO scaffolds allowed

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human fibroblasts adhesion, spreading and colonization during 14 days of culture. As such, GO-containing fibrous scaffolds developed in this work promoted bacteria death,

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while allowing human cells activity. These features demonstrate the potential of GO

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incorporation in polymer fibrous scaffolds for antimicrobial medical implantation

CONFLICTS OF INTEREST

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purposes.

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There are no conflicts of interest to declare.

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ACKNOWLEDGMENTS

The authors acknowledge the support of the i3S Scientific Platform Bioimaging, member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBIPOCI-01-0145-FEDER-022122), under the coordination of María Lázaro (PhD), for the training in the Spectral Confocal Laser Scanning Microscope. The work described was financially supported by FEDER through COMPETE2020 - Programa Operacional Competitividade e Internacionaliza o (POCI), Programa Operacional Regional do Norte (NORTE2020) - and by national funds through FCT (Funda o para a Ci ncia e Tecnologia): POCI-01-0145-FEDER-007274 (i3S), UID/EQU/00511/2019 (LEPABE), Projects (SkinPrint)

PTDC/CTM-BIO/4033/2014 and

(NewCat),

NORTE‐01‐0145‐FEDER‐000012,

PTDC/BBB-ECT/2145/2014 Research

position

contract

IF/01479/2015 and PhD grant PD/BD/114156/2016.

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Journal Pre-proof REFERENCES

[8] [9] [10] [11]

[12]

[13] [14] [15]

[16]

[17] [18] [19] [20] [21]

[22] [23]

[24]

of

[7]

ro

[6]

-p

[5]

re

[4]

lP

[3]

na

[2]

J. S. VanEpps and J. G. Younger, ―Implantable Device-Related Infection,‖ SHOCK, vol. 46, no. 6, pp. 597–608, Dec. 2016. R. O. Darouiche, ―Device‐ Associated Infections: A Macroproblem that Starts with Microadherence,‖ Clin. Infect. Dis., vol. 33, no. 9, pp. 1567–1572, Nov. 2001. L. Rimondini, M. Fini, and R. Giardino, ―The microbial infection of biomaterials: A challenge for clinicians and researchers. A short review.,‖ J. Appl. Biomater. Biomech., vol. 3, no. 1, pp. 1–10, 2005. M. E. Falagas and S. K. Kasiakou, ―Mesh-related infections after hernia repair surgery,‖ Clin. Microbiol. Infect., vol. 11, no. 1, pp. 3–8, Jan. 2005. J. J. Jkss et al., ―Outcome of the patients with chronic mesh infection following open inguinal hernia repair.,‖ J. Korean Surg. Soc., vol. 84, no. 5, pp. 287–91, May 2013. O. Guillaume et al., ―Infections associated with mesh repairs of abdominal wall hernias: Are antimicrobial biomaterials the longed-for solution?,‖ Biomaterials, vol. 167, pp. 15–31, Jun. 2018. R. Narkhede, N. M. Shah, P. R. Dalal, C. Mangukia, and S. Dholaria, ―Postoperative Mesh Infection—Still a Concern in Laparoscopic Era,‖ Indian Journal of Surgery, vol. 77, no. 4. Springer, pp. 322–326, 01-Aug-2015. G. Sharma et al., ―Escherichia coli biofilm: development and therapeutic strategies,‖ Journal of Applied Microbiology, vol. 121, no. 2. Blackwell Publishing Ltd, pp. 309–319, 01-Aug-2016. G. O’Toole, H. B. Kaplan, and R. Kolter, ―Biofilm Formation as Microbial Development,‖ Annu. Rev. Microbiol., vol. 54, no. 1, pp. 49–79, Oct. 2000. J. Tortora, Gerard, B. R. Funke, and C. L. Case, ―Microbioloy - an introduction,‖ p. 960, 2010. M. E. Rupp, J. S. Ulphani, P. D. Fey, K. Bartscht, and D. Mack, ―Characterization of the importance of polysaccharide intercellular adhesin/hemagglutinin of Staphylococcus epidermidis in the pathogenesis of biomaterial-based infection in a mouse foreign body infection model.,‖ Infect. Immun., vol. 67, no. 5, pp. 2627–32, May 1999. J. Galdbart, J. Allignet, H. Tung, C. Rydèn, and N. El Solh, ―Screening for Staphylococcus epidermidis Markers Discriminating between Skin‐ Flora Strains and Those Responsible for Infections of Joint Prostheses,‖ J. Infect. Dis., vol. 182, no. 1, pp. 351–355, Jul. 2000. A. Al-Jumaili, S. Alancherry, K. Bazaka, and M. V. Jacob, ―Review on the antimicrobial properties of Carbon nanostructures,‖ Materials (Basel)., vol. 10, no. 9, pp. 1–26, 2017. K. S. Novoselov et al., ―Electric Field Effect in Atomically Thin Carbon Films,‖ Science (80-. )., vol. 306, no. 5696, pp. 666–669, Oct. 2004. R. Feng, G. Guan, W. Zhou, C. Li, D. Zhang, and Y. Xiao, ―In situ synthesis of poly(ethylene terephthalate)/graphene composites using a catalyst supported on graphite oxide,‖ J. Mater. Chem., vol. 21, no. 11, p. 3931, Mar. 2011. A. E. Jakus, E. B. Secor, A. L. Rutz, S. W. Jordan, M. C. Hersam, and R. N. Shah, ―Threedimensional printing of high-content graphene scaffolds for electronic and biomedical applications,‖ ACS Nano, vol. 9, no. 4, pp. 4636–4648, Apr. 2015. T. K. Das and S. Prusty, ―Graphene-Based Polymer Composites and Their Applications,‖ Polym. Plast. Technol. Eng., vol. 52, no. 4, pp. 319–331, Mar. 2013. W. Hu et al., ―Graphene-Based Antibacterial Paper,‖ ACS Nano, vol. 4, no. 7, pp. 4317–4323, Jul. 2010. H. Tashan et al., ―Antibacterial Properties of Graphene Based Materials: Emphasis on Molecular Mechanisms, Surface Engineering and Size of Sheets,‖ Mini. Rev. Org. Chem., vol. 15, Jul. 2018. V. T. H. Pham et al., ―Graphene Induces Formation of Pores That Kill Spherical and Rod-Shaped Bacteria,‖ ACS Nano, vol. 9, no. 8, pp. 8458–8467, Aug. 2015. S. Liu et al., ―Antibacterial Activity of Graphite, Graphite Oxide, Graphene Oxide, and Reduced Graphene Oxide: Membrane and Oxidative Stress,‖ ACS Nano, vol. 5, no. 9, pp. 6971–6980, Sep. 2011. T. I. Kim et al., ―Antibacterial Activities of Graphene Oxide–Molybdenum Disulfide Nanocomposite Films,‖ ACS Appl. Mater. Interfaces, vol. 9, no. 9, pp. 7908–7917, Mar. 2017. S. Gurunathan, J. Woong Han, A. Abdal Daye, V. Eppakayala, and J. Kim, ―Oxidative stressmediated antibacterial activity of graphene oxide and reduced graphene oxide in Pseudomonas aeruginosa,‖ Int. J. Nanomedicine, vol. 7, p. 5901, Nov. 2012. F. Perreault, A. F. De Faria, S. Nejati, and M. Elimelech, ―Antimicrobial Properties of Graphene Oxide Nanosheets: Why Size Matters,‖ ACS Nano, vol. 9, no. 7, pp. 7226–7236, Jul. 2015.

Jo ur

[1]

30

Journal Pre-proof

[32]

[33] [34] [35]

[36] [37] [38] [39]

[40] [41]

[42] [43] [44] [45]

[46] [47] [48]

[49]

of

[31]

ro

[30]

-p

[29]

re

[28]

lP

[27]

na

[26]

Y. Yang, A. M. Asiri, Z. Tang, D. Du, and Y. Lin, ―Graphene based materials for biomedical applications,‖ Mater. Today, vol. 16, no. 10, pp. 365–373, Oct. 2013. A. M. Pinto, I. C. Gon alves, and F. D. Magalh es, ―Graphene-based materials biocompatibility: A review,‖ Colloids Surfaces B Biointerfaces, vol. 111, pp. 188–202, Nov. 2013. L. Pang, C. Dai, L. Bi, Z. Guo, and J. Fan, ―Biosafety and Antibacterial Ability of Graphene and Graphene Oxide In Vitro and In Vivo.,‖ Nanoscale Res. Lett., vol. 12, no. 1, p. 564, Oct. 2017. Q. Chen, J. D. Mangadlao, J. Wallat, A. De Leon, J. K. Pokorski, and R. C. Advincula, ―3D printing biocompatible polyurethane/poly(lactic acid)/graphene oxide nanocomposites: Anisotropic properties,‖ ACS Appl. Mater. Interfaces, vol. 9, no. 4, pp. 4015–4023, 2017. N. Yadav et al., ―Graphene Oxide-Coated Surface: Inhibition of Bacterial Biofilm Formation due to Specific Surface–Interface Interactions,‖ ACS Omega, vol. 2, no. 7, pp. 3070–3082, Jul. 2017. J. D. Mangadlao, C. M. Santos, M. J. L. Felipe, A. C. C. de Leon, D. F. Rodrigues, and R. C. Advincula, ―On the antibacterial mechanism of graphene oxide (GO) Langmuir–Blodgett films,‖ Chem. Commun., vol. 51, no. 14, pp. 2886–2889, Feb. 2015. P. C. Henriques, I. Borges, A. M. Pinto, F. D. Magalh es, and I. C. Gon alves, ―Fabrication and antimicrobial performance of surfaces integrating graphene-based materials,‖ Carbon N. Y., vol. 132, pp. 709–732, Jun. 2018. K. Jalaja, V. S. Sreehari, P. R. A. Kumar, and R. J. Nirmala, ―Graphene oxide decorated electrospun gelatin nanofibers: Fabrication, properties and applications,‖ Mater. Sci. Eng. C, vol. 64, pp. 11–19, Jul. 2016. A. Pinto et al., ―Smaller particle size and higher oxidation improves biocompatibility of graphene-based materials,‖ Carbon N. Y., vol. 99, pp. 318–329, 2016. R. N. Gomes et al., ―Antimicrobial graphene nanoplatelets coatings for silicone catheters,‖ Carbon N. Y., vol. 139, pp. 635–647, Nov. 2018. C. Mota, D. Puppi, D. Dinucci, M. Gazzarri, and F. Chiellini, ―Additive manufacturing of star poly(ε-caprolactone) wet- spun scaffolds for bone tissue engineering applications,‖ J. Bioact. Compat. Polym., vol. 28, no. 4, pp. 320–340, Jul. 2013. F. Dini et al., ―Tailored star poly (ε-caprolactone) wet-spun scaffolds for in vivo regeneration of long bone critical size defects,‖ J. Bioact. Compat. Polym., vol. 31, no. 1, pp. 15–30, Jan. 2016. D. Puppi et al., ―Additive manufacturing of wet-spun polymeric scaffolds for bone tissue engineering,‖ Biomed. Microdevices, vol. 14, no. 6, pp. 1115–1127, Dec. 2012. S. Stratton, N. B. Shelke, K. Hoshino, S. Rudraiah, and S. G. Kumbar, ―Bioactive polymeric scaffolds for tissue engineering,‖ Bioact. Mater., vol. 1, no. 2, pp. 93–108, Dec. 2016. Y. Qian et al., ―3D Fabrication with Integration Molding of a Graphene Oxide/Polycaprolactone Nanoscaffold for Neurite Regeneration and Angiogenesis,‖ Adv. Sci., vol. 5, no. 4, p. 1700499, Apr. 2018. B. Azimi, P. Nourpanah, M. Rabiee, and S. Arbab, ―Poly (ε-caprolactone) Fiber: An Overview,‖ J. Eng. Fiber. Fabr., vol. 9, 2014. I. Sousa, A. Mendes, R. F. Pereira, and P. J. Bártolo, ―Collagen surface modified poly(εcaprolactone) scaffolds with improved hydrophilicity and cell adhesion properties,‖ Mater. Lett., vol. 134, pp. 263–267, Nov. 2014. D. Ege, A. R. Kamali, and A. R. Boccaccini, ―Graphene Oxide/Polymer-Based Biomaterials,‖ Adv. Eng. Mater., vol. 19, no. 12, Dec. 2017. M. A. Woodruff and D. W. Hutmacher, ―The return of a forgotten polymer—Polycaprolactone in the 21st century,‖ Prog. Polym. Sci., vol. 35, no. 10, pp. 1217–1256, Oct. 2010. A. Sarasam and S. Madihally, ―Characterization of chitosan–polycaprolactone blends for tissue engineering applications,‖ Biomaterials, vol. 26, no. 27, pp. 5500–5508, Sep. 2005. E. Malikmammadov, T. E. Tanir, A. Kiziltay, V. Hasirci, and N. Hasirci, ―PCL and PCL-based materials in biomedical applications,‖ J. Biomater. Sci. Polym. Ed., vol. 29, no. 7–9, pp. 863–893, Jun. 2018. H. Sun, L. Mei, C. Song, X. Cui, and P. Wang, ―The in vivo degradation, absorption and excretion of PCL-based implant,‖ Biomaterials, vol. 27, no. 9, pp. 1735–1740, Mar. 2006. W. K. Chee, H. N. Lim, N. M. Huang, and I. Harrison, ―Nanocomposites of graphene/polymers: a review,‖ RSC Adv., vol. 5, no. 83, pp. 68014–68051, Aug. 2015. R. Scaffaro, F. Lopresti, A. Maio, L. Botta, S. Rigogliuso, and G. Ghersi, ―Electrospun PCL/GOg-PEG structures: Processing-morphology-properties relationships,‖ Compos. Part A Appl. Sci. Manuf., vol. 92, pp. 97–107, Jan. 2017. J. Song, H. Gao, G. Zhu, X. Cao, X. Shi, and Y. Wang, ―The preparation and characterization of polycaprolactone/graphene oxide biocomposite nanofiber scaffolds and their application for directing cell behaviors,‖ Carbon N. Y., vol. 95, pp. 1039–1050, Dec. 2015.

Jo ur

[25]

31

Journal Pre-proof

[57] [58]

[59]

[60] [61] [62]

[63]

[64]

[65]

[66] [67] [68]

[69]

[70] [71]

[72]

of

[56]

ro

[55]

-p

[54]

re

[53]

lP

[52]

na

[51]

C. Wan and B. Chen, ―Poly(ε-caprolactone)/graphene oxide biocomposites: mechanical properties and bioactivity,‖ Biomed. Mater., vol. 6, no. 5, p. 055010, Oct. 2011. J. R. Potts, D. R. Dreyer, C. W. Bielawski, and R. S. Ruoff, ―Graphene-based polymer nanocomposites,‖ Polymer (Guildf)., vol. 52, no. 1, pp. 5–25, Jan. 2011. W. S. Hummers and R. E. Offeman, ―Preparation of Graphitic Oxide,‖ J. Am. Chem. Soc., vol. 80, no. 6, p. 1339, 1958. J. Schindelin et al., ―Fiji: an open-source platform for biological-image analysis,‖ Nat. Methods, vol. 9, no. 7, pp. 676–682, Jul. 2012. A. M. Pinto, S. Moreira, I. C. Gonçalves, F. M. Gama, A. M. Mendes, and F. D. Magalhães, ―Biocompatibility of poly(lactic acid) with incorporated graphene-based materials,‖ Colloids Surfaces B Biointerfaces, vol. 104, pp. 229–238, Apr. 2013. D. R. Dreyer, S. Park, C. W. Bielawski, and R. S. Ruoff, ―The chemistry of graphene oxide,‖ Chem. Soc. Rev., vol. 39, no. 1, pp. 228–240, Dec. 2010. L. Stobinski et al., ―Graphene oxide and reduced graphene oxide studied by the XRD, TEM and electron spectroscopy methods,‖ J. Electron Spectros. Relat. Phenomena, vol. 195, pp. 145–154, Aug. 2014. Y. Geng, S. J. Wang, and J.-K. Kim, ―Preparation of graphite nanoplatelets and graphene sheets,‖ J. Colloid Interface Sci., vol. 336, no. 2, pp. 592–598, Aug. 2009. I. Jun, H.-S. Han, J. R. Edwards, and H. Jeon, ―Electrospun Fibrous Scaffolds for Tissue Engineering: Viewpoints on Architecture and Fabrication.,‖ Int. J. Mol. Sci., vol. 19, no. 3, Mar. 2018. P. Bala Balakrishnan, L. Gardella, M. Forouharshad, T. Pellegrino, and O. Monticelli, ―Star poly(ε-caprolactone)-based electrospun fibers as biocompatible scaffold for doxorubicin with prolonged drug release activity,‖ Colloids Surfaces B Biointerfaces, vol. 161, pp. 488–496, Jan. 2018. K. Jansen et al., ―Fabrication of Kidney Proximal Tubule Grafts Using Biofunctionalized Electrospun Polymer Scaffolds,‖ Macromol. Biosci., p. 1800412, Dec. 2018. X. Wei et al., ―3D Printable Graphene Composite,‖ Sci. Rep., vol. 5, no. 1, pp. 1–7, Sep. 2015. W. Wang et al., ―Enhancing the Hydrophilicity and Cell Attachment of 3D Printed PCL/Graphene Scaffolds for Bone Tissue Engineering.,‖ Mater. (Basel, Switzerland), vol. 9, no. 12, Dec. 2016. S. C. Neves, C. Mota, A. Longoni, C. C. Barrias, P. L. Granja, and L. Moroni, ―Additive manufactured polymeric 3D scaffolds with tailored surface topography influence mesenchymal stromal cells activity,‖ Biofabrication, vol. 8, no. 2, pp. 1–15, May 2016. S. Dinescu et al., ―In vitro cytocompatibility evaluation of chitosan/graphene oxide 3D scaffold composites designed for bone tissue engineering.,‖ Biomed. Mater. Eng., vol. 24, no. 6, pp. 2249– 56, 2014. B. Hong, H. Jung, and H. Byun, ―Preparation of Polyvinylidene Fluoride Nanofiber Membrane and Its Antibacterial Characteristics with Nanosilver or Graphene Oxide,‖ J. Nanosci. Nanotechnol., vol. 13, no. 9, pp. 6269–6274, Sep. 2013. X. An, H. Ma, B. Liu, and J. Wang, ―Graphene Oxide Reinforced Polylactic Acid/Polyurethane Antibacterial Composites,‖ J. Nanomater., vol. 2013, pp. 1–7, Sep. 2013. H. E. Karahan et al., ―Graphene Materials in Antimicrobial Nanomedicine: Current Status and Future Perspectives,‖ Adv. Healthc. Mater., vol. 7, no. 13, p. 1701406, Jul. 2018. S. Kumar, S. Raj, E. Kolanthai, A. K. Sood, S. Sampath, and K. Chatterjee, ―Chemical Functionalization of Graphene To Augment Stem Cell Osteogenesis and Inhibit Biofilm Formation on Polymer Composites for Orthopedic Applications,‖ ACS Appl. Mater. Interfaces, vol. 7, no. 5, pp. 3237–3252, Feb. 2015. N. Murugan, C. Murugan, and A. K. Sundramoorthy, ―In vitro and in vivo characterization of mineralized hydroxyapatite/polycaprolactone-graphene oxide based bioactive multifunctional coating on Ti alloy for bone implant applications,‖ Arab. J. Chem., vol. 11, no. 6, pp. 959–969, Sep. 2018. X. Zou, L. Zhang, Z. Wang, and Y. Luo, ―Mechanisms of the Antimicrobial Activities of Graphene Materials,‖ J. Am. Chem. Soc., vol. 138, no. 7, pp. 2064–2077, 2016. Z. Jia et al., ―From Solution to Biointerface: Graphene Self-Assemblies of Varying Lateral Sizes and Surface Properties for Biofilm Control and Osteodifferentiation,‖ ACS Appl. Mater. Interfaces, vol. 8, no. 27, pp. 17151–17165, Jul. 2016. H. M. Hegab, A. ElMekawy, L. Zou, D. Mulcahy, C. P. Saint, and M. Ginic-Markovic, ―The controversial antibacterial activity of graphene-based materials,‖ Carbon N. Y., vol. 105, pp. 362– 376, Aug. 2016.

Jo ur

[50]

32

Journal Pre-proof

[80] [81] [82]

[83]

of

[79]

ro

[78]

-p

[77]

re

[76]

lP

[75]

na

[74]

B. Lu et al., ―Graphene-based composite materials beneficial to wound healing,‖ Nanoscale, vol. 4, no. 9, p. 2978, Apr. 2012. O. Akhavan and E. Ghaderi, ―Toxicity of Graphene and Graphene Oxide Nanowalls Against Bacteria,‖ ACS Nano, vol. 4, no. 10, pp. 5731–5736, Oct. 2010. F. Zou et al., ―Wrinkled Surface-Mediated Antibacterial Activity of Graphene Oxide Nanosheets,‖ ACS Appl. Mater. Interfaces, vol. 9, no. 2, pp. 1343–1351, Jan. 2017. G. Oberdörster, E. Oberdörster, and J. Oberdörster, ―Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles.,‖ Environ. Health Perspect., vol. 113, no. 7, pp. 823– 39, Jul. 2005. S. Gurunathan and J.-H. Kim, ―Synthesis, toxicity, biocompatibility, and biomedical applications of graphene and graphene-related materials,‖ Int. J. Nanomedicine, vol. 11, p. 1927, May 2016. S. Sayyar, E. Murray, B. C. Thompson, S. Gambhir, and D. L. Officer, ―Covalently linked biocompatible graphene/ polycaprolactone composites for tissue engineering Publication Details.‖ O. J. Yoon et al., ―Nanocomposite nanofibers of poly(d, l-lactic-co-glycolic acid) and graphene oxide nanosheets,‖ Compos. Part A Appl. Sci. Manuf., vol. 42, no. 12, pp. 1978–1984, Dec. 2011. W. C. Lee et al., ―Origin of enhanced stem cell growth and differentiation on graphene and graphene oxide.,‖ ACS Nano, vol. 5, no. 9, pp. 7334–41, Sep. 2011. J.-T. Jeong et al., ―Effect of graphene oxide ratio on the cell adhesion and growth behavior on a graphene oxide-coated silicon substrate,‖ Sci. Rep., vol. 6, no. 1, p. 33835, Dec. 2016. L. R. Manea, L. Hristian, A. L. Leon, and A. Popa, ―Recent advances of basic materials to obtain electrospun polymeric nanofibers for medical applications,‖ IOP Conf. Ser. Mater. Sci. Eng., vol. 145, no. 3, p. 032006, Aug. 2016. D. Mondal, M. Griffith, and S. S. Venkatraman, ―Polycaprolactone-based biomaterials for tissue engineering and drug delivery: Current scenario and challenges,‖ Int. J. Polym. Mater. Polym. Biomater., vol. 65, no. 5, pp. 255–265, Mar. 2016.

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3D printed scaffolds containing graphene oxide are antibacterial and biocompatible 84/85

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3D fibrous scaffolds were fabricated by wet-spinning and additive manufacturing 81/85

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Composite scaffolds are bactericidal (~80% death) against Gram+ and Grambacteria 84/85

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Human fibroblasts fully colonized both poly(ɛ-caprolactone) and composite scaffolds 85/85

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The developed composites are promising materials to be used in tissue engineering 83/85

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