biomimetics

Article Three-Dimensional Printed Antimicrobial Objects of Polylactic Acid (PLA)- Nanoparticle Nanocomposite Filaments Produced by an In-Situ Reduction Reactive Melt Mixing Process

Nectarios Vidakis 1, Markos Petousis 1,* , Emmanouel Velidakis 1, Marco Liebscher 2 and Lazaros Tzounis 3

1 Mechanical Engineering Department, Hellenic Mediterranean University, Estavromenos, 71004 Heraklion, Crete, Greece; [email protected] (N.V.); [email protected] (E.V.) 2 Institute of Construction Materials, Technische Universität Dresden, DE-01062 Dresden, Germany; [email protected] 3 Department of Materials Science and Engineering, University of Ioannina, 45110 Ioannina, Greece; [email protected] * Correspondence: [email protected]; Tel.: +30-2810-37-9227

 Received: 14 August 2020; Accepted: 31 August 2020; Published: 2 September 2020 

Abstract: In this study, an industrially scalable method is reported for the fabrication of polylactic acid (PLA)/silver nanoparticle (AgNP) nanocomposite filaments by an in-situ reduction reactive melt mixing method. The PLA/AgNP nanocomposite filaments have been produced initially + reducing silver (Ag ) arising from silver (AgNO3) precursor mixed in the polymer melt to elemental silver (Ag0) nanoparticles, utilizing (PEG) or polyvinyl pyrrolidone (PVP), respectively, as macromolecular blend compound reducing agents. PEG and PVP were added at various concentrations, to the PLA matrix. The PLA/AgNP filaments have been used to manufacture 3D printed antimicrobial (AM) parts by Fused Filament Fabrication (FFF). The 3D printed PLA/AgNP parts exhibited significant AM properties examined by the reduction in Staphylococcus aureus (S. aureus) and (E. coli) bacteria viability (%) experiments at 30, 60, and 120 min duration of contact (p < 0.05; p-value (p): probability). It could be envisaged that the 3D printed parts manufactured and tested herein mimic nature’s mechanism against bacteria and in terms of antimicrobial properties, contact angle for their anti-adhesive behavior and mechanical properties could create new avenues for the next generation of low-cost and on-demand additive manufacturing produced personal protective equipment (PPE) as well as healthcare and nosocomial antimicrobial equipment.

Keywords: Fused Filament Fabrication (FFF); 3D printing; reactive melt processing; antimicrobial (AM) properties; polylactic acid (PLA); silver (Ag) nanoparticles (NPs); polyethylene glycol (PEG); polyvinyl pyrrolidone (PVP); Staphylococcus aureus (S. aureus); Escherichia coli (E. coli)

1. Introduction Poly-Lactic Acid (PLA) which belongs to the family of polyglycolic acid aliphatic polyesters has received an extensive interest the last decades especially as a high-performance polymer for bio-related applications. More specific, PLA has been used in the biomedical sector for various functional objects i.e., implants [1], surgical equipment [2], nanofibrous templates for drug delivery [3], foams for tissue engineering [4], etc. PLA is a thermoplastic in nature polymeric material known also for its biocompatibility, biodegradability, superior mechanical strength compared to other thermoplastics,

Biomimetics 2020, 5, 42; doi:10.3390/biomimetics5030042 www.mdpi.com/journal/biomimetics Biomimetics 2020, 5, 42 2 of 22 and ease of processing via solution and melt processing methods [5]. Moreover, for biomedical applications as for instance protective personal equipment (PPE), surgical equipment, etc., PLA offers the unique property that it can be sterilized due to its relatively high (typical Tm of PLA ~150–160 ◦C), which is a prerequisite so that PLA based 3D objects can be reusable till the end of their lifetime. 3D printing is a technology in which parts are produced with the sequential addition of material layers. It belongs to the family of the additive manufacturing technologies and has received extensive scientific interest over the years. Additionally, 3D printing is in the forefront amongst other additive manufacturing technologies for on demand product development. In addition to that, 3D printing offers the unique possibility of producing 3D bulk objects consisting of different materials with various physical and chemical properties [6,7]. For these and several other practical reasons, 3D printing and additive manufacturing in general have been adopted with fervor by industry and are increasingly used as production processes. Various techniques are available nowadays for 3D printing solid materials, including Fused Filament Fabrication (FFF), direct metal laser sintering and electron beam fabrication [8]. 3D printing has been employed recently to manufacture surgical equipment [8,9], implants [10], tissue scaffolds [11], 3D bioelectronics [12], etc. One of the most promising applications of 3D printing and especially FFF technology is amongst others in the medical field for the design and development of medical devices and instruments [13,14]. Moreover, surgeons have used patient-specific computed tomography derived 3D prints for the better preoperative planning and the proper design of the surgical approach in complex operations [15–17]. In the same philosophy, 3D printed models have been used also for educational proposes of young surgeons [18,19]. Although the mechanical properties of the 3D printed have been extensively studied in literature [20], there is, however, scant literature to date for the production of multi-functional 3D printed objects, i.e., with enhanced mechanical properties [20] and additionally electrical conductivity [21], thermoelectric property [22], anti-adhesive and antimicrobial properties to avoid biofilm formation [23], etc., utilizing novel and functional materials. Recently, a great scientific focus has been devoted to the development of bulk materials with antimicrobial (AM) properties, specifically for various applications in the health sector so as to increase the environment’s hygiene and avoid the transmission and infections caused by pathogenic microorganisms [24]. Polymers as a big family of plastic materials of which many bulk 3D objects are consisting of in applications such as hospital tables, protective equipment, paints, biomedical products like implants, bandages, catheters, surgery equipment, etc. have been modified by antimicrobial agents in their bulk structure via melt-mixing or solvent mixing methods. Moreover, another promising approach to endow AM properties to bulk 3D plastic components is by depositing AM films onto their outer surface by: (i) either wet chemical methods (dip-coating using AgNP dispersions [25], sonochemical immobilization [26,27], etc.) or (ii) vacuum deposition techniques (magnetron sputtering [28], -beam-assisted deposition process [29], etc.). To that end, a great number of AM agents as for instance nanoparticles (CuNPs) and/or copper complexes, silver NPs (AgNPs) and Ag metal salts, polyhexamethylene , and chitosan based biopolymers, quaternary ammonium compounds, etc. have been reported and proven to prevent the growth of pathogenic microorganisms, such as bacteria, fungi, algae, etc. [30–32] upon being incorporated/blended into the polymer 3D bulk structure or deposited as thin films [33,34]. Amongst others, AgNPs have been in the forefront of research and several times reported for their antibacterial activity, especially due to their broad spectrum of antibacterial activity while at the same time having proven low levels of toxicity against mammalian cells [35]. However, their antimicrobial activity has not yet been fully understood. Relatively, one of the most plausible mechanisms is initially the interaction of AgNPs with the microorganism’s surface that results further to the penetration of AgNPs and/or Ag released ions (Ag+) through the cell walls, allowing them to react with the thiol group of and ending up with the cell’s distortion and death [36]. Silver in its ionized form is highly reactive, as it binds to tissue proteins and endows structural changes to the bacterial cell Biomimetics 2020, 5, 42 3 of 22 wall and nuclear membrane, leading thus to cell lysis and death [37]. A side reaction of AgNPs concomitantly to their interaction with bacteria cells and bactericidal mechanism, is unavoidably the direct interaction with human cells, which leads to cytotoxicity and genotoxicity reported effects [38]. As such, it is of crucial importance AgNPs to be stabilized and/or embedded in the bulk structure of 3D component materials. Therefore, polymer/AgNP nanocomposites can be promising antimicrobial materials, which can be easily processed, i.e., via melt mixing and extrusion processes. Furthermore, polymer/AgNP nanocomposites can be extruded in the form of filaments that can be utilized further to 3D print complex and on-demand antimicrobial 3D printed bulk parts through FFF additive manufacturing technology. In relation to that, the incorporation of AgNPs in a polymer matrix by solvent mixing has been already reported [39]. The production of polymer nanocomposites by well-established industrial and large-scale processing methods as for instance melt compounding using an extruder is very advantageous [40–43]. A very promising approach to fabricate polymer/AgNP nanocomposites is via reactive melt mixing, since preformed AgNPs found as powders tend to agglomerate. The aggregation phenomena are known further to affect the material’s properties, i.e., their antimicrobial efficacy in since the interaction between the bacterial cell and the AgNPs is more intensive if the silver nanoparticles are well dispersed and not agglomerated [44]. In that method, the silver precursor is solubilized in the polymer melt during processing, and either the selected polymer matrix exhibits the functional groups to reduce in-situ the silver salt into elemental silver or some other polymeric material is blended and utilized as the reducing agent. For instance, it has been already reported the generation of AgNPs in a thermoplastic polyurethane matrix (TPU) by in-situ reduction of silver during polymer melt and extrusion processing, while the developed materials have exhibited antimicrobial properties [44]. AgNPs have been formed also via in-situ reduction during melt mixing in a poly(methyl methacrylate) PMMA matrix as well as in different polyamides [45,46]. Finally, Parida et al. [47] recently reported on a solventless in-situ reduction method during the extrusion process to prepare AgNP using different silver precursors and thermoplastic polymers for antimicrobial food packaging application. Moreover, they used the same protocol to synthesize AgNPs in polylactic acid (PLA) and polypropylene (PP), and they found that the surface energy of polymer melt had an effect on the quality and size of AgNPs created. It has been previously reported that the surface topography and roughness may have a great influence on the bacteria attachment and further colonization via the formation of a self-produced polysaccharide based biofilm [48]. The factors that dictate the underlying mechanism of “attachment” include: (i) surface hydrophobicity, (ii) electrostatic interactions, (iii) van der Waals forces, and (iv) steric hindrance [49]. In specific, the hydrophobicity is a surface property that can be engineered via mimicking the micro- and nanostructure of naturally occurring surfaces such as cicada and dragonfly wings, lotus leaves, and shark skin [48]. Moreover, the nano and micro-scale hierarchical structure on lotus leaves are responsible for its unique superhydrophobic and self-cleaning properties [50]. 3D FFF printing offers the unique opportunity to create microstructured surfaces, i.e., down to 50 µm printed object layer thickness, while incorporated nanoparticles in the case of using nanocomposite thermoplastic filaments that appear onto the surface of the 3D printed object could result in hierarchical nano-/and micro-scale structures like the structure of lotus leaves. Herein, the main purpose of this research article is to fabricate high quality PLA/AgNP nanocomposite 3D printing filaments via reactive melt mixing which is a facile, scalable and industrially viable method, while the filaments have been utilized further to manufacture 3D printed samples of different printed layer thickness that have been tested in terms of hydrophillicity, mechanical and antibacterial performance. Specifically, PLA/AgNP antimicrobial filaments have been produced by reactive melt mixing blending: (i) PLA, AgNO3 and PEG, as well as (ii) PLA, AgNO3 and PVP, while PEG and PVP have been utilized as the reducing agent polymeric material, the PLA as the polymer matric and AgNO3 as the AgNP salt precursor. AgNPs have been generated in situ in the PLA polymer matrix, yielding extremely efficient antimicrobial (AM) objects as determined by BiomimeticsBiomimetics2020 2020, 5,, 425, x FOR PEER REVIEW 5 of4 22 of 22

All powder mixtures prepared for the four different recipes appearing in white color were fed theinto reduction a Noztek in ProStaphylococcus (Shoreham, UK) aureus single(S. aureusscrew )extruder and Escherichia preheated coli at (230E. coli°C )and bacteria processed viability at the (%) aftersame being temperature exposed for 30,5 min 60, prior and 120to being min, extruded respectively. to 3D Di printingfferent characterizationfilaments (Figure techniques1c). It is worth have beenmentioning carried out that i.e., a Ramandirect proof spectroscopy for the andsuccessfu thermogravimetricl reactive melt analysismixing (TGA)process for and the the PLA in-situ/AgNP nanocompositegeneration of extrudedAgNPs in filaments,the PLA matrix while was water the contactobserved angle change of a in sessile color drop,of all extruded optical and filaments, scanning electronnamely, microscopy from white (SEM), colour antibacterial of the powder tests, mixtures and tensile fed into and micro-hardnessthe extruder to typical tests of silver the 3D colour, printed respectivecharacteristic samples. colour of silver “in-bulk” and/or AgNPs at a high concentration. The same procedure was followed for all PLA/Ag/PEG and PLA/Ag/PVP extruded filaments (filaments were dried at 50 2. Materials°C for four andhours) Methods (Figure 1d), while the process flow for the different recipes is schematically shown in TheFigure methodology 1. Samples from followed the PLA/AgNP in this work nanocomposites for the development filaments’ resulting of the nanocomposites,from the four theirdifferent characterization, recipes followed the studywere initially of their analyzed antimicrobial (diameter, and discontinuities, their mechanical etc.) properties before being is used shown further for 3D printing (Figure 2). Regarding the measured filaments diameter, the average value schematically in Figure1 and described in detail further below. was 1.74 mm and the standard deviation was ±0.04mm.

FigureFigure 1. Schematic1. Schematic illustration illustration for thefor process the process followed follo forwed the creationfor the ofcreation poly-Lactic of poly-Lactic Acid (PLA) Acid/silver (PLA)/silver nanoparticle (AgNP) nanocomposite filaments, for their characterization as well as the nanoparticle (AgNP) nanocomposite filaments, for their characterization as well as the study of their study of their antimicrobial and mechanical properties: (a) PLA, AgNO3 and PEG or PVP (reducing antimicrobial and mechanical properties: (a) PLA, AgNO3 and PEG or PVP (reducing agents) powders agents) powders used for the reactive melt mixing, (b) the drying process of the powders/ materials used for the reactive melt mixing, (b) the drying process of the powders/ materials before the reactive before the reactive melt mixing process, (c) the PLA/AgNP filament fabrication via melt extrusion melt mixing process, (c) the PLA/AgNP filament fabrication via melt extrusion process, (d) the drying process, (d) the drying process of the produced PLA/AgNP nanocomposite filaments, (e) process of the produced PLA/AgNP nanocomposite filaments, (e) measurement of the produced measurement of the produced filament diameter to be used for the FFF 3D printing process, (f) 3D filament diameter to be used for the FFF 3D printing process, (f) 3D printing FFF process for the printing FFF process for the manufacturing of the samples, (g) SEM image of the 3D printed samples’ manufacturing of the samples, (g) SEM image of the 3D printed samples’ side surface showing the side surface showing the additively 3D printed layers of the samples, (h) SEM typical image of h additivelybacteria attached 3D printed onto layers the samples of the surface samples, after ( )di SEMfferent typical durations image in contac of bacteriat with attachedthe antibacterial onto the samples3D printed surface samples after diusedfferent in durationsthe antibact inerial contact tests withcarried the out antibacterial in this work, 3D printed(i) 3D printed samples dog used inbone-shaped the antibacterial sample tests during carried tensile out testing. in this work, (i) 3D printed dog bone-shaped sample during tensile testing.

Biomimetics 2020, 5, 42 5 of 22

2.1. Materials The polymer matrix used in this work was industrial grade PLA (PLA 3052D) fine powder received from Plastika Kritis S.A (Heraklion, Crete, Greece). This specific PLA grade has a 3.3 relative , 200 ◦C melting temperature, 55–60 ◦C glass transition temperature, and a of 1240 kg/m3. Silver nitrate (AgNO , 99%), Polyethylene glycol (PEG) with average molecular weight 3 ≥ Mn 4600 g/mol and Polyvinyl Pyrrolidone (PVP) with average molecular weight Mn 10,000 g/mol ≈ ≈ were supplied by Sigma Aldrich (Steinheim, Germany). All the chemical reagents in this study were used as received without further purification. In this work, PEG has been used as a melt blended additive material co-compounded with the PLA + in order to function as a reducing agent of Ag ions (Ag ) stemming from mixed AgNO3. In specific, the reduction of Ag+ by PEG has been proposed to occur through the oxidation of the PEG hydroxyl (-OH) terminal groups to groups and the concomitant creation of Ag0 NPs, as it has been previously reported in literature [51,52]. On the other hand, PVP that has been utilized also in this work as a melt blended reducing agent material has been reported to function as a reducing agent for the creation of AgNPs via different Ag precursor compounds as well as suitable colloidal stabilizer, surfactant, shape-directing agent and dispersant [53]. The possible interactions of PEG and PVP polymeric additives with the Ag+ and further the reduction process occurring by the melt mixing operational temperature (T = 230 ◦C), which resulted into “in-situ” creation and incorporation of AgNPs within the PLA matrix could be seen in the graphical abstract figure of this work.

2.2. Fabrication of PLA/AgNP Nanocomposites by Reactive Melt-Mixing Extrusion Process and 3D Printing of PLA/AgNP Nanocomposite Filaments

Initially, the PLA matrix material was mixed with AgNO3 and PVP (Figure1a), as well as AgNO 3 and PEG powders, respectively, utilizing a mechanical homogenizer (the Silverson L5M-A laboratory mixer was used as a homogenizer for about 10 min for each batch of the polymers produced.). The powder mixtures were dried then in a vacuum oven (Figure1b), at 70 ◦C for 48 h. Specifically, the following mixtures were prepared prior to the reactive melt mixing/compounding extrusion process for the reduction of Ag+ to metallic Ag0 NPs formed in-situ within the PLA matrix during melt mixing:

1. Recipe-01: 100 g (PLA): 20 g (AgNO3): 10 g (PEG), hereafter denoted as PLA/Ag/PEG (rec-01); 2. Recipe-02: 100 g (PLA): 10 g (AgNO3): 5 g (PEG), hereafter denoted as PLA/Ag/PEG (rec-02); 3. Recipe-03: 100 g (PLA): 20 g (AgNO3): 10 g (PVP), hereafter denoted as PLA/Ag/PVP (rec-03); and 4. Recipe-04: 100 g (PLA): 10 g (AgNO3): 5 g (PVP), hereafter denoted as PLA/Ag/PVP (rec-04).

The selection of AgNO3 and PEG/PVP respectively masses has been done based on a protocol of Nam et al. [54], without “in-detail” calculation in this work of the molar ratio of OH− groups (PEG) or Pyrrolidinone groups (PVP) to the Ag+, since it is not the main aim/focus of this research study. All powder mixtures prepared for the four different recipes appearing in white color were fed into a Noztek Pro (Shoreham, UK) single screw extruder preheated at 230 ◦C and processed at the same temperature for 5 min prior to being extruded to 3D printing filaments (Figure1c). It is worth mentioning that a direct proof for the successful reactive melt mixing process and the in-situ generation of AgNPs in the PLA matrix was the observed change in color of all extruded filaments, namely, from white colour of the powder mixtures fed into the extruder to typical silver colour, characteristic colour of silver “in-bulk” and/or AgNPs at a high concentration. The same procedure was followed for all PLA/Ag/PEG and PLA/Ag/PVP extruded filaments (filaments were dried at 50 ◦C for four hours) (Figure1d), while the process flow for the di fferent recipes is schematically shown in Figure1. Samples from the PLA/AgNP nanocomposites filaments’ resulting from the four different recipes followed were initially analyzed (diameter, discontinuities, etc.) before being used further for 3D printing (Figure2). Regarding the measured filaments diameter, the average value was 1.74 mm and the standard deviation was 0.04 mm. ± Biomimetics 2020, 5, x FOR PEER REVIEW 6 of 22

2.3. 3D Printing of PLA/AgNP Nanocomposite Filaments The commercially available desktop 3D printer Intamsys Funmat HT was used in all cases. All specimens were 3D printed in the horizontal orientation and American Society for Testing and Materials (ASTM) D638-02a standard (type V specimens with 3.2 mm thickness) dog-bone shaped specimens were manufactured. As the standard requires, five specimens were produced for neat PLA and for each one of the four different recipes. The specimens were manufactured with the following 3D printing parameters: 100% solid infill, Biomimetics45 degrees2020, 5deposition, 42 orientation angle, 0.2 mm layer height, and 230 °C 3D printing nozzle6 of 22 temperature.

Figure 2. (a) Determining the wt. concentration for the creation of the different recipes (b) Neat PLA Figure 2. (a) Determining the wt. concentration for the creation of the different recipes (b) Neat PLA powder and the filament produced with this powder (c) 100 g (PLA): 20 g (AgNO3): 10 g (PEG), powder and the filament produced with this powder (c) 100 g (PLA): 20 g (AgNO ): 10 g (PEG), hereafter hereafter denoted as PLA/Ag/PEG (rec-01) powder and the filament produced 3with this powder (d) denoted as PLA/Ag/PEG (rec-01) powder and the filament produced with this powder (d) 100 g (PLA): 100 g (PLA): 10 g (AgNO3): 5 g (PEG), hereafter denoted as PLA/Ag/PEG (rec-02) powder and the 10 g (AgNO3): 5 g (PEG), hereafter denoted as PLA/Ag/PEG (rec-02) powder and the filament produced filament produced with this powder, (e) 100 g (PLA): 20 g (AgNO3): 10 g (PVP), hereafter denoted as with this powder, (e) 100 g (PLA): 20 g (AgNO ): 10 g (PVP), hereafter denoted as PLA/Ag/PVP (rec-03) PLA/Ag/PVP (rec-03) powder and the filament3 produced with this powder, (f) 100 g (PLA): 10 g powder and the filament produced with this powder, (f) 100 g (PLA): 10 g (AgNO ): 5 g (PVP), hereafter (AgNO3): 5 g (PVP), hereafter denoted as PLA/Ag/PVP (rec-04) powder and the3 filament produced denotedwith this as PLApowder./Ag/ PVP (rec-04) powder and the filament produced with this powder. 2.3. 3D Printing of PLA/AgNP Nanocomposite Filaments 2.4. Characterisation Techniques The commercially available desktop 3D printer Intamsys Funmat HT was used in all cases. Raman spectroscopy was performed with a Labram HR-Horiba (Horiba Scientific, Kyoto, AllJapan) specimens scientific were micro-Raman 3D printed insystem. the horizontal All spectra orientation were acquired and Americanin the back-scattering Society for Testinggeometry and Materialswith a 514.5 (ASTM) nm D638-02aline of an Ar standard+ ion laser (type operating V specimens at 1.5 mW with power 3.2 mm at thickness)the focal plane. dog-bone In order shaped to specimensfacilitate werethe excitation manufactured. light onto As the the standard sample’s requires, surface fiveas well specimens as collecting were produced the back-scattering for neat PLA andRaman for each activity, one of a the 50× four long di ffworkingerent recipes. distance objective has been utilized as part of an optical microscopeThe specimens set-up. were manufactured with the following 3D printing parameters: 100% solid infill, 45 degreesThermogravimetric deposition orientation analysis angle, (TGA) 0.2 studies mm layer were height, carried and out 230 using◦C 3D a printingNETZSCH nozzle STA temperature. 409C/CD (NETZSCH Gerätebau GmbH, Selb, Germany). The experiments have been conducted from ambient 2.4.(25 Characterisation °C) up to 800 °C Techniques in oxygen atmosphere with a heating rate of 10 K/min, while for the temperature calibration,Raman spectroscopy Curie point standards was performed were utilized. with a Labram HR-Horiba (Horiba Scientific, Kyoto, Japan) scientificThe micro-Raman sessile drop method system. was All empl spectraoyed wereto investigate acquired the in wettability the back-scattering of 3D printed geometry PLA/AgNP with a 514.5with nm different line of anlayer Ar +thicknession laser (100 operating and 300 at µm 1.5 mWlayer powerthickness) at the by focal H2O plane.using the In orderDataphysics to facilitate OCA the excitation20 (Dataphysics, light onto theFilderstadt, sample’s surfaceGermany) as well contact as collecting angle theanalyser back-scattering system. RamanThe environment activity, a 50 experimental conditions for all measurements were 65% relative humidity at 25 ± 1 °C. All samples × long working distance objective has been utilized as part of an optical microscope set-up. were kept for drying at 60 °C under vacuum overnight before performing the contact angle Thermogravimetric analysis (TGA) studies were carried out using a NETZSCH STA 409C/CD measurements. A droplet of deionized water with 2 µL volume was dispensed onto the surface of (NETZSCH Gerätebau GmbH, Selb, Germany). The experiments have been conducted from ambient the 3D printed samples, while the droplet profile was recorded with a CCD video camera after 5 s in (25 C) up to 800 C in oxygen atmosphere with a heating rate of 10 K/min, while for the temperature all◦ measurements.◦ In order to form the water droplets onto the 3D printed samples (with variable calibration, Curie point standards were utilized. The sessile drop method was employed to investigate the wettability of 3D printed PLA/AgNP with different layer thickness (100 and 300 µm layer thickness) by H2O using the Dataphysics OCA 20 (Dataphysics, Filderstadt, Germany) contact angle analyser system. The environment experimental conditions for all measurements were 65% relative humidity at 25 1 C. All samples were kept ± ◦ for drying at 60 ◦C under vacuum overnight before performing the contact angle measurements. A droplet of deionized water with 2 µL volume was dispensed onto the surface of the 3D printed samples, while the droplet profile was recorded with a CCD video camera after 5 s in all measurements. In order to form the water droplets onto the 3D printed samples (with variable printed layer thickness), from which the respective contact angles have been determined using each single droplet profile, Biomimetics 2020, 5, 42 7 of 22 a microsyringe was employed. The reported values are representative of at least five different samples of which three measurements were performed at different positions, as a means to acquire statistically valid values. Optical microscopy (OM) has been performed with Keyence VHX-6000 optical microscope (Keyence, Itasca, IL, USA). Scanning electron microscopy (SEM) investigations were performed using a FEI NanoSem 200 (FEI, Eindhoven, The Netherlands) at an accelerating voltage of 2 kV. Prior to the SEM analysis, a thin layer (3 nm) of platinum was deposited by sputtering to avoid charging effects as reported elsewhere [51,52].

2.5. Antibacterial Activity The antibacterial activity of PLA/AgNP 3D printed samples was tested against Gram-positive Staphylococcus aureus (S. aureus, ATCC 25923), as well as Gram-negative Escherichia coli (E. coli, ATCC 25922) bacteria strains, according to the standard shake flask method (ASTM-E2149-01). The antibacterial tests have been carried out in a typical microbiology laboratory. All glassware, materials and relevant cell culture hardware have been sterilized before the experiments using an autoclave at 121 ◦C/1.5 atm for 20 min. Moreover, all cultures have been grown in petri dishes in a laminar flow hood (LFH). The laboratory temperature maintained between (23–25 ◦C) using an air conditioning facility for the microbiology laboratory. The growth of bacteria in liquid cultures was determined by measuring the optical density at 600 nm (OD600) with a Helios Epsilon Photometer (Thermo Scientific, Waltham, MA, USA). This method is known to provide quantitative data for measuring the reduction rate in a specific CFU number at a specific time. The CFU has been expressed further into average colony forming units per millilitre (CFU mL 1) of buffer solution in the flask. Specifically, S. aureus and E. coli cultures of · − bacteria were grown on nutrient agar overnight, while they have been transferred then into a nutrient broth (NB) with an initial optical density (OD) of 0.1 at 660 nm and allowed to grow at 37 ◦C and 110 rpm. Upon reaching an OD of 0.3 at 660 nm which is known as the beginning of the logarithmic phase, the cultures were centrifuged and washed twice with saline at pH 6.5 to yield a final bacterial concentration of approximately 108 CFU mL 1. Afterwards, a PLA/AgNP small piece (~0.5 gr) and · − 4.5 mL of a saline solution were inserted into a vial with an inner diameter of 2.5 cm, while 500 µL of the strain cells were poured into the vial using a micropipette. In these conditions, it has been achieved an initial bacterial concentration in the vial of appr. 107 CFU mL 1. The bacterial suspensions were · − incubated and shaken then at 37 ◦C and 230 rpm for up to 120 min (2 h). As a final step, samples of 100 µL each were taken at a specified time (30, 60, and 120 min), diluted tenfold in saline and then transferred onto nutrient agar plates. The plates were allowed to grow then at 37 ◦C for 24 h to determine the number of surviving bacteria. The antimicrobial activity is reported in terms of percentage of bacteria reduction calculated as the ratio between the number of surviving bacteria before and after the contact with the control (PLA) and PLA/AgNP 3D printed samples, making use of the following formula: Bacteria reduction (%) = ((A B)/A) 100 (1) − × where A and B are the average number of bacteria before and after the contact with the PLA/AgNP samples. For each bacteria strain, the experimental protocol was conducted three times (n = 3), while the antibacterial activity against S. aureus and E. coli after 30, 60, and 120 min of contact is reported as the mean standard deviation (SD). All the data were analysed also by one-way analysis of ± variance (ANOVA) and differences between the means were assessed with Neuman–Keuls’s multiple comparison tests to determine the significant variation of the PLA/AgNP bactericidal activity compared to the PLA control sample. Differences were considered significant at p < 0.05. Biomimetics 2020, 5, 42 8 of 22

2.6. Tensile Tests

BiomimeticsThe tensile 2020, 5, testsx FOR werePEER REVIEW performed according to the ASTM D638-02a standard, using an8 Imadaof 22 MX2 (Northbrook, IL, USA) tensile test apparatus, equipped with standardized grips. The chuck of thethe tensile tensile test test machine machine was was setset atat aa 1010 mmmm/min/min speedspeed for for testing. testing. All All specimens specimens were were tested tested for for the the determination of their tensile properties at room temperature (~23 °C). determination of their tensile properties at room temperature (~23 ◦C).

2.7.2.7. Micro-Hardness Micro-Hardness Tests Tests Micro-hardnessMicro-hardness tests tests were were performed performed according according to the to specificationsthe specifications of the of ASTM the ASTM E384-17 E384-17 standard. Thestandard. micro-Vickers The micro-Vickers method was method applied, was with applied, 0.2 kg forcewith scale0.2 kg (1.962 force N) scale and (1.962 10 s indentation N) and 10 time. s indentation time. A typical 136° apex angle Vickers diamond pyramid was used as indenter. A typical 136◦ apex angle Vickers diamond pyramid was used as indenter. Experiments were carried Experiments were carried out with an Innova Test 400-Vickers (Maastricht, The Netherlands) out with an Innova Test 400-Vickers (Maastricht, The Netherlands) apparatus. apparatus. 3. Results and Discussion 3. Results and Discussion 3.1. Raman Spectra of PLA/Ag/PEG and PLA/Ag/PVP 3D Printed Samples 3.1. Raman Spectra of PLA/Ag/PEG and PLA/Ag/PVP 3D Printed Samples Figure3a shows the Raman spectra of PLA /Ag/PEG (rec-01) and PLA/Ag/PVP (rec-03) 3D printed samples,Figure while 3a Figure shows3 bthe the Raman molecular spectra architectures of PLA/Ag/PEG of PLA, (rec-01) PEG, and and PVP. PLA/Ag/PVP It should be(rec-03) mentioned 3D printed samples, while Figure 3b the molecular architectures of PLA, PEG, and PVP. It should be that only the PLA/Ag/PEG (rec-01) and PLA/Ag/PVP (rec-03) spectra are depicted, since they have been mentioned that only the PLA/Ag/PEG (rec-01) and PLA/Ag/PVP (rec-03) spectra are depicted, since found to exhibit identical spectral features compared to that of PLA/Ag/PEG (rec-02) and PLA/Ag/PVP they have been found to exhibit identical spectral features compared to that of PLA/Ag/PEG (rec-02) (rec-04) materials, respectively. It is known from literature that silver presents a lattice vibrational mode and PLA/Ag/PVP (rec-04) materials, respectively. It is known from literature that silver presents a between 50 and 300 cm 1, which depends on the chemical compound silver is present, i.e., as , lattice vibrational mode− between 50 and 300 cm−1, which depends on the chemical compound silver nitrate, or as some other compound [55]. Specifically, the peak located at 244 cm 1 for the is present, i.e., as oxide, nitrate, chloride or as some other compound [55]. Specifically, the− peak PLA/Ag/PEG spectrum, as well as a “shoulder” in the case of PLA/Ag/PVP system, corresponds to located at 244 cm−1 for the PLA/Ag/PEG spectrum, as well as a “shoulder” in the case of thePLA/Ag/PVP metallic AgNPs system, formed, corresponds being in to good the metallic agreement AgNPs with formed, the Raman being spectrum in good agreement of 99.99% purewith silverthe wiresRaman reported spectrum elsewhere of 99.99% [56 pure]. silver wires reported elsewhere [56].

FigureFigure 3. 3. (a(a)) RamanRaman spectraspectra ofof PLAPLA/Ag/PEG/Ag/PEG and and PL PLAA/Ag/PVP/Ag/PVP 3D 3D printed printed samples, samples, and and (b () bthe) the molecularmolecular structures structures ofof PLA,PLA, PEG,PEG, andand PVPPVP compounds of of which which the the Raman Raman characteristic characteristic features features havehave been been assigned. assigned.

−11 OnOn the the other other hand, hand, the the sharp sharp bandband locatedlocated at 222 cm − forfor the the PLA/Ag/PVP PLA/Ag/PVP sample, sample, not not present present inin PLA PLA/Ag/PEG/Ag/PEG sample’s sample’s spectrum, spectrum, isis attributedattributed toto the stretching vibrations of of Ag–N Ag–N [57] [57 ,[58],58], duedue to theto formationthe formation of aof chemical a chemical bond bond between between the the AgNPs AgNPs andand thethe PVP nitrogen nitrogen atoms atoms [58], [58 ],or or Ag–O Ag–O bondsbonds [57 [57],], due due to to the the interaction interaction ofof AgNPsAgNPs with the carboxylate groups groups (n (n Ag–OCO-) Ag–OCO-) of of PLA PLA [59]. [59 ]. InIn both both spectra, spectra, all all peaks peaks that that are are attributed attributed to to PLA PLA are are depicted depicted with with continuous continuous lines, lines, while while the specificthe specific bands bands assigned assigned to the to chemistry the chemistry of the of blendedthe blended reducing reducing agent agent polymeric polymeric material material (PEG (PEG and and PVP, respectively) are illustrated with dashed lines. In specific, both spectra show the PVP, respectively) are illustrated with dashed lines. In specific, both spectra show the characteristic −1 −1 characteristic signature of PLA presenting peaks at 465, 479,1 and 589 cm (C-O-C vibration),1 655 cm signature of PLA presenting peaks at 465, 479, and 589 cm− (C-O-C vibration), 655 cm− (C=O stretching (C=O stretching vibration), 936 cm−1 (C-COO vibration), 1252 and 1320 cm−1 (CH deformation vibration), 1596 cm−1 (asymmetric C=O stretching vibrations of carboxylate groups of PLA), and 2947 cm−1 (CH3 symmetric and asymmetric stretching vibration). The PLA/Ag/PEG spectrum has

Biomimetics 2020, 5, x FOR PEER REVIEW 9 of 22

additionally some bands due to PEG blended additive located at 811 cm−1 (C-O-C vibration), 1038 cm−1 (C-C stretching vibration of the PEG backbone macromolecular chains), 1375 cm−1 (CH3 deformation vibration), 1396 cm−1 (CH3 symmetric deformation vibration), 1504 cm−1 (CH2 or O-CH2 Biomimeticsvibration)2020 and, 5, 42 2884 cm−1 (CH3 symmetric and asymmetric stretching vibration). The PLA/Ag/PVP9 of 22 spectrum has additionally some bands due to PVP blended additive located at 384 cm−1 (C-CH3 stretching vibration) 770 cm−1 (C-N vibration), 989 cm−1 (C-C stretching vibration), 1062 cm−1(C-CH3 1 1 1 vibration),stretching 936 vibration), cm− (C-COO 1361 cm vibration),−1 (CH2 band 1252 vibration and 1320 modes cm− of (CHthe pyrro deformationlidone ring vibration), in PVP) and 1596 1385 cm − 1 (asymmetriccm−1 (CH3 deformation C=O stretching vibration) vibrations [60].of carboxylate groups of PLA), and 2947 cm− (CH3 symmetric and asymmetric stretching vibration). The PLA/Ag/PEG spectrum has additionally some bands 1 1 due3.2. to Thermogravimetric PEG blended additive Analysis locatedof Neat PLA at 811and cmPLA/Ag− (C-O-C Nanocomposite vibration), Filaments 1038 cm− (C-C stretching 1 vibrationFigure of the 4 shows PEG the backbone TGA graphs macromolecular in the range of chains), 55 to 550 1375 °C for cm the− (CHdifferent3 deformation materials produced vibration), 1 1 1 1396in cmthis− study.(CH3 Namely,symmetric the deformation neat PLA, as vibration), well as the 1504 nanocomposite cm− (CH2 orPLA/Ag/PEG O-CH2 vibration) (rec-01 and and 2884 rec-02) cm − (CHand3 symmetric PLA/Ag/PVP and (rec-03 asymmetric and rec-04) stretching extruded vibration). filaments, The all of PLA which/Ag /havePVP been spectrum produced has additionallyunder the 1 1 somesame bands experimental due to PVP parameters, blended additivecould be located seen. Specifically, at 384 cm− (C-CHfour distinct3 stretching thermal vibration) decomposition 770 cm − 1 1 1 (C-Nwindows vibration), can be 989 observed cm− (C-C indicated stretching as I, vibration), II, III, and 1062 IV in cm the− (C-CHTGA figure.3 stretching In the vibration), first one (I), 1361 up cmto − 1 (CH~1802 band °C, none vibration of the modesmaterials of exhibits the pyrrolidone any weight ring loss in (%). PVP) Therefore, and 1385 it can cm −be (CHdeduced3 deformation that all vibration)materials [60 are]. stable up 180 °C. On the contrary, from 180 °C and up to ~266 °C, the II temperature window appears, where the 3.2.observed Thermogravimetric weight loss Analysis (%) is attributed of Neat PLA to andthe PLAdecomposition/Ag Nanocomposite of PEG Filaments(rec-01 and rec-02) and PVP (rec-03 and rec-04), respectively. It can be observed that PLA/Ag/PEG (rec-01) shows the same Figure4 shows the TGA graphs in the range of 55 to 550 ◦C for the different materials produced in thisdecomposition study. Namely, trend the neatwith PLA,the PLA/Ag/PVP as well as the (rec-0 nanocomposite3), while PLA/Ag/PEG PLA/Ag/PEG (rec-02) (rec-01 similar and rec-02) to the and PLA/Ag/PVP (rec-04). This is more precisely explained by the fact that in both cases an equal amount PLA/Ag/PVP (rec-03 and rec-04) extruded filaments, all of which have been produced under the same of PEG and PVP, respectively, was added as the macromolecular reactive melt mixing reducing experimental parameters, could be seen. Specifically, four distinct thermal decomposition windows agent for the creation of AgNPs in the PLA matrix. From approximately 266 °C and up to 374 °C, can be observed indicated as I, II, III, and IV in the TGA figure. In the first one (I), up to ~180 C, corresponding to the temperature window III, the onset of PLA decomposition could be observed◦ none of the materials exhibits any weight loss (%). Therefore, it can be deduced that all materials are starting from 266 °C, while at 374 °C all polymer based substance in the different materials/filament stableformulations up 180 ◦C. has been fully decomposed (both PLA and PEG or PVP in the different recipes).

FigureFigure 4. 4.Thermogravimetric Thermogravimetric analysisanalysis (TGA)(TGA) graphs of of neat neat PLA, PLA, PLA/Ag/PEG, PLA/Ag/PEG, and and PLA/Ag/PVP PLA/Ag/PVP nanocompositenanocomposite filaments filaments fabricated fabricated underunder the four different different recipes recipes developed developed in inthis this study study in inthe the temperature window 55 to 550 °C. (a) Full temperature window (55 to 550 °C) of the TGA temperature window 55 to 550 ◦C. (a) Full temperature window (55 to 550 ◦C) of the TGA experiment, experiment, as well as (b) magnified temperature window from 400 to 550 °C showing more as well as (b) magnified temperature window from 400 to 550 ◦C showing more specifically the material specifically the material weight loss at 550 °C, where all the organic polymeric substances have been weight loss at 550 ◦C, where all the organic polymeric substances have been decomposed and the decomposed and the weight loss observed is attributed to the AgNPs. weight loss observed is attributed to the AgNPs. The final temperature window (IV) of 374 to 550 °C is mainly included to demonstrate the On the contrary, from 180 ◦C and up to ~266 ◦C, the II temperature window appears, where the remaining material after all polymer has been decomposed, which is attributed to the generated observed weight loss (%) is attributed to the decomposition of PEG (rec-01 and rec-02) and PVP (rec-03 and rec-04), respectively. It can be observed that PLA/Ag/PEG (rec-01) shows the same decomposition trend with the PLA/Ag/PVP (rec-03), while PLA/Ag/PEG (rec-02) similar to the PLA/Ag/PVP (rec-04). This is more precisely explained by the fact that in both cases an equal amount of PEG and PVP, respectively, was added as the macromolecular reactive melt mixing reducing agent for the creation of AgNPs in the PLA matrix. From approximately 266 ◦C and up to 374 ◦C, corresponding to the temperature window III, the onset of PLA decomposition could be observed starting from 266 ◦C, Biomimetics 2020, 5, 42 10 of 22

while at 374 ◦C all polymer based substance in the different materials/filament formulations has been fully decomposed (both PLA and PEG or PVP in the different recipes). BiomimeticsThe final 2020, temperature5, x FOR PEER REVIEW window (IV) of 374 to 550 ◦C is mainly included to demonstrate10 of 22 the remaining material after all polymer has been decomposed, which is attributed to the generated AgNPs AgNPs within the PLA matrix as solid metallic particles that are stable up to 550 °C (even up to 1000 within the PLA matrix as solid metallic particles that are stable up to 550 ◦C (even up to 1000 ◦C °C that the TGA experiments left to run). The remaining material at 550 °C for the different PLA/Ag that the TGA experiments left to run). The remaining material at 550 ◦C for the different PLA/Ag nanocompositenanocomposite filament, filament, attributed attributed to theto AgNPsthe Ag grownNPs grown within thewithin PLA the matrix, PLA is 9.37%matrix, (PLA is /9.37%Ag/PEG rec-01),(PLA/Ag/PEG 4.61% (PLA rec-01),/Ag/ PEG4.61% rec-02), (PLA/Ag/PEG 9.45% (PLA rec-02),/Ag/PVP 9.45% rec-03), (PLA/Ag/PVP and 4.06% (PLArec-03),/Ag and/PVP 4.06% rec-04), (PLA/Ag/PVP rec-04), respectively. All the solid AgNP remaining material at above 400 °C for the respectively. All the solid AgNP remaining material at above 400 ◦C for the different samples could be different samples could be seen in the magnified temperature range of 360–580 °C in the right seen in the magnified temperature range of 360–580 ◦C in the right hand-side, while as mentioned hand-side, while as mentioned above the PLA, PEG, and PVP materials have been already above the PLA, PEG, and PVP materials have been already decomposed at lower temperatures. decomposed at lower temperatures. 3.3. Contact Angle–Hydrophobicity–Antiadhesive Properties-Wettability 3.3. Contact Angle–Hydrophobicity–Antiadhesive Properties-Wettability Figure5 shows the water contact angle onto the 3D printed di fferent samples. Namely, Figure5a,b Figure 5 shows the water contact angle onto the 3D printed different samples. Namely, Figure correspond to the neat 3D printed PLA with 100 and 300 µm printed layer thickness, respectively. 5a,b correspond to the neat 3D printed PLA with 100 and 300 µm printed layer thickness, Figure5c,d represent the H 2O contact angles of PLA/Ag/PEG (rec-01) with 100 and 300 µm printed respectively. Figure 5c,d represent the H2O contact angles of PLA/Ag/PEG (rec-01) with 100 and 300 layer thickness, respectively. Finally, Figure5e,f show the H 2O contact angle of PLA/Ag/PVP (rec-03) µm printed layer thickness, respectively. Finally, Figure 5e,f show the H2O contact angle of with 100 and 300 µm printed layer thickness, respectively. PLA/Ag/PVP (rec-03) with 100 and 300 µm printed layer thickness, respectively.

Figure 5. Water contact angle profiles of neat PLA, PLA/Ag/PEG (rec-01), and PLA/Ag/PVP (rec-03) Figure 5. Water contact angle profiles of neat PLA, PLA/Ag/PEG (rec-01), and PLA/Ag/PVP (rec-03) 3D 3D printed samples at 300 and 100 µm printed layer thickness, respectively, showing the printed samples at 300 and 100 µm printed layer thickness, respectively, showing the hydrophobic hydrophobic and anti-adhesive properties of the different 3D printed objects in this work (CA2: and anti-adhesive properties of the different 3D printed objects in this work (CA2: contact angle 2; contact angle 2; CA1: contact angle 1; CAaverage: contact angle average value in “°” from CA1 and CA2). CA1: contact angle 1; CAaverage: contact angle average value in “◦” from CA1 and CA2). (a,b): H2O (a,b): H2O contact angle of neat 3D printed PLA with 100 and 300 µm printed layer thickness, contact angle of neat 3D printed PLA with 100 and 300 µm printed layer thickness, respectively. respectively. (c,d): H2O contact angles of PLA/Ag/PEG (rec-01) with 100 and 300 µm printed layer (c,d): H O contact angles of PLA/Ag/PEG (rec-01) with 100 and 300 µm printed layer thickness, thickness,2 respectively. (e,f): H2O contact angle of PLA/Ag/PVP (rec-03) with 100 and 300 µm printed µ respectively.layer thickness, (e,f respectively.): H2O contact angle of PLA/Ag/PVP (rec-03) with 100 and 300 m printed layer thickness, respectively. It can be seen that in the case of PLA and PLA/Ag/PVP 3D printed samples, the contact angle It can be seen that in the case of PLA and PLA/Ag/PVP 3D printed samples, the contact angle increases with increasing the 3D printed object micro-roughness, achieved by decreasing the printed increasesobject layer with thickness increasing from the 3D300 printedto 100 µm. object This micro-roughness, is more precisely achieved explained by decreasing by the well-known the printed Cassie–Baxter model that drives the wetting phenomena of surfaces by different liquids taking into account different ranges of surface roughness i.e., micro-roughness, nano-roughness, etc. [61]. Moreover, by decreasing the roughness of surfaces, i.e., creating nanostructured surfaces with

Biomimetics 2020, 5, 42 11 of 22 object layer thickness from 300 to 100 µm. This is more precisely explained by the well-known Cassie–Baxter model that drives the wetting phenomena of surfaces by different liquids taking into account different ranges of surface roughness i.e., micro-roughness, nano-roughness, etc. [61]. Moreover, by decreasing the roughness of surfaces, i.e., creating nanostructured surfaces with specific features, one can achieve superhydrophobic surfaces following biomimetic nanostructured surfaces as for instance this of the well-known lotus leaf (otherwise known as “lotus leaf effect” to achieve super-hydrophobicity, anti-adhesive and self-cleaning surfaces). On the other hand, the H2O contact angle of PLA/Ag/PEG (rec-01) decreases for the 100 µm printed layer thickness sample, due to the existence of PEG blended material within the PLA matrix that is hydrophilic water soluble material. Interestingly enough, all 3D printed samples exhibit a hydrophobic behavior, namely PLA, PLA/Ag/PEG, and PLA/Ag/PVP which is important for their use in biomedical applications with anti-adhesive properties, preventing from the microorganism attachment and colonization. Specifically, the PLA/Ag/PVP 3D printed specimen with the smallest printed layer thickness dimension (100 µm), exhibited the highest H2O contact angle (>100◦) which is important both as antiadhesive surface as well as easily to be cleaned since bacteria and biofilm cannot be created due to the required hydrophilic surface.

3.4. Optical Microscopy Investigations Figure6 summarizes the optical microscopy images of the 3D printed PLA /AgNP nanocomposite samples using PEG (recipe 01 and 02), as well as PVP (recipe 03 and 04) as reactive melt blending additive for the reduction of Ag+ to metallic Ag0 (AgNPs). All images were acquired from 3D printed samples fabricated with 100 µm printed layer thickness. It can be observed that all 3D printed samples exhibited micro-roughness ranging from few microns to Rmax of approximately 300 µm. Moreover, all samples showed homogeneous printed layer thickness with very few voids and discontinuities in the interlayers attributed both to the optimum printing parameters used in our study, as well as the high quality of the produced PLA/AgNP nanocomposite filaments, i.e., structural homogeneity and diameter uniformity.

3.5. Microstructure Investigation via SEM Analysis Figures7 and8 present the 3D printed samples microstructure investigated from the 3D printed side-surface (all samples shown with 300 µm printed layer thickness), at low magnification demonstrating the printing process features, as well as at high magnification showing the existence of AgNPs. Specifically, Figure7a presents the microstructure of neat PLA with a layer thickness of approximately 300 µm, being in a good agreement with the resolution of the Intamsys Funmat HT 3D printer manufacturer’s technical specifications in terms of printer’s resolution. Figure7b shows an amorphous PLA surface morphology, typical for polymeric materials. Figure8a,b show the 3D printed PLA /Ag/PEG nanocomposite surface morphology using filament of recipe 01, while Figure8c,d PLA /Ag/PEG nanocomposite filament of recipe 02, where PEG has been utilized as the reactive melt blending additive for the reduction of Ag+ to metallic Ag0 (AgNPs). On the other hand, Figure8e,f shows the 3D printed PLA /Ag/PVP nanocomposite surface morphology using filament of recipe 03, while Figure8g,h shows the PLA /Ag/PVP nanocomposite filament of recipe 04, where PVP was employed as the reducing agent. For all samples, it can be observed a homogeneous 3D printed layer thickness as well as high quality of bonding of the layers, indicating: (i) the optimum set of the selected printing parameters, as well as (ii) the high quality of the produced PLA/AgNP nanocomposite filaments, i.e., structural homogeneity and diameter uniformity produced in our study by reactive melt-blending process. Moreover, from the SEM images it can be further deduced that high quality 3D printed objects have been manufactured with good adhesion between the layers, which could most likely result further in high mechanical performance 3D printed components. Finally, at high magnification images for all Biomimetics 2020, 5, 42 12 of 22 cases of the 3D printed PLA/AgNP nanocomposites, the existence of AgNPs with sizes ranging from 50–100 nm blended and homogeneously distributed within the PLA matrix can be observed. Biomimetics 2020, 5, x FOR PEER REVIEW 12 of 22

Figure 6. Optical microscopy 2D (left hand-side) and 3D (right hand-side) images of 3D printed Figure 6. Optical microscopy 2D (left hand-side) and 3D (right hand-side) images of 3D printed PLA/AgNP nanocomposites (at 100 µm printed layer thickness), utilizing PLA/Ag/PEG PLA/AgNP nanocomposites (at 100 µm printed layer thickness), utilizing PLA/Ag/PEG (rec-01)—(a,b); (rec-01)—(a,b); PLA/Ag/PEG (rec-02)—(c,d); (PLA/Ag/PVP (rec-03)—(e,f); and PLA/Ag/PVP PLA/Ag/PEG (rec-02)—(c,d); (PLA/Ag/PVP (rec-03)—(e,f); and PLA/Ag/PVP (rec-04)—(g,h) extruded (rec-04)—(g,h) extruded nanocomposite filaments, respectively. The 500 µm scale bar is inserted and nanocomposite filaments, respectively. The 500 µm scale bar is inserted and shown in each figure in shown in each figure in the left hand-side images, while the respective 3D images are shown in the the left hand-side images, while the respective 3D images are shown in the right hand-side with the right hand-side with the corresponding color scale bar indicating the micro-roughness of each corresponding color scale bar indicating the micro-roughness of each sample. sample.

Biomimetics 2020, 5, 42 13 of 22 Biomimetics 2020, 5, x FOR PEER REVIEW 13 of 22

FigureFigure 7. 7.Scanning Scanningelectron electron microscopymicroscopy (SEM) images images of of the the side-surface side-surface microstructure microstructure and and morphology for the different 3D printed samples in this study at two different magnifications (all 3D morphology for the different 3D printed samples in this study at two different magnifications (all 3D printed samples with 300 µm printed layer thickness): Neat PLA SEM image at 250× magnification printed samples with 300 µm printed layer thickness): Neat PLA SEM image at 250 magnification (a) and 1250× magnification (b), respectively. × (a) and 1250 magnification (b), respectively. × 3.6.3.6. Bactericidal Bactericidal Tests Tests TheThe antibacterial antibacterial activity activity of of the the PLA PLA/AgNP/AgNP 3D printed nanocomposites nanocomposites with with 100 100 µmµ printedm printed layer thickness, utilized for the FFF 3D printing process filaments produced by recipe-01, recipe-02, layer thickness, utilized for the FFF 3D printing process filaments produced by recipe-01, recipe-02, recipe-03, and recipe-04, was examined by reduction in S. aureus and E. coli bacteria viability (%) at recipe-03, and recipe-04, was examined by reduction in S. aureus and E. coli bacteria viability (%) at 30, 60, and 120 min duration of contact. It should be mentioned that differences were considered 30,significant 60, and 120 at minp < 0.05. duration It can be of contact.seen that ItE. shouldcoli cultures be mentioned of the bacteria that were differences eradicated were completely considered significantafter 120 at minp < of0.05. treatment It can be(p seen< 0.05) that withE. PLA/Ag/PEG coli cultures of(rec-01) the bacteria and PLA/Ag/PVP were eradicated (rec-03), completely while afterbeing 120 mineradicated of treatment (>90%) (usingp < 0.05) PLA/Ag/PEG with PLA /(rec-02)Ag/PEG and (rec-01) reaching and >70% PLA /inAg the/PVP reduction (rec-03), of whilebacteria being eradicatedviability ( >by90%) PLA/Ag/PVP using PLA (rec-04)./Ag/PEG (rec-02) and reaching >70% in the reduction of bacteria viability by PLA/TheAg/ PVPobserved (rec-04). required time window of 120 min to reduce the initial S. aureus and E. coli amount ofThe CFU observed to zero requiredis governed time by window the velocity of 120 minof Ag to+ reduceions release, the initial whichS. aureus are responsibleand E. coli amountfor the of CFUantibacterial to zero is governed activity (p by < the0.05). velocity It is worth of Ag mentioning+ ions release, that which even in are 60 responsible min of S. aureus for the and antibacterial E. coli activitytreatment (p < 0.05).with PLA/Ag/PEG It is worth mentioning (rec-01) and thatPLA/Ag/PVP even in 60 (rec-03), min of moreS. aureus than 80%and ofE. the coli bacteriatreatment have with PLAbeen/Ag /eradicated.PEG (rec-01) At and the PLAsame/Ag time,/PVP PLA/Ag/PEG (rec-03), more (rec-02) than and 80% PLA/Ag/PVP of the bacteria (rec-04) have consisting been eradicated. 3D At theprinted same objects, time, exhibited PLA/Ag/ PEGremarkably (rec-02) less and antibact PLA/Agerial/PVP activity (rec-04) due consistingto possibly 3D less printed amount objects, of exhibitedAgNPs remarkably existent onto less the antibacterial outer surface activity of duethe toPLA/AgNP possibly less3D amountprinted ofob AgNPsjects, being existent in good onto the agreement with the TGA experiments showing the generated AgNP solid content as well as the outer surface of the PLA/AgNP 3D printed objects, being in good agreement with the TGA experiments utilized AgNO3 precursor used in the reactive melt-mixing recipe. showing the generated AgNP solid content as well as the utilized AgNO precursor used in the reactive Overall, the 3D printed manufactured PLA/AgNP samples exhibit3 excellent antibacterial melt-mixingproperties recipe. with the greatest bactericidal effect on E. coli (>98%) for both the PLA/Ag/PEG (rec-01) andOverall, PLA/Ag/PVP the 3D printed (rec-03), manufactured and the lowest PLA on /S.AgNP aureus samples with 74.9% exhibit for excellentthe PLA/Ag/PEG antibacterial (rec-01) properties and with68.9% the greatest for the bactericidal PLA/Ag/PVP eff ect(rec-03), on E. colirespectively,(>98%) for at both 120 the min PLA of/ Agcontact/PEG ( (rec-01)p < 0.05). and It PLAis worth/Ag/PVP (rec-03),mentioning and the that lowest the PLA/Ag/PEG on S. aureus showedwith 74.9%higher forantibacterial the PLA /performanceAg/PEG (rec-01) both against and 68.9% E. coli for as the PLAwell/Ag as/PVP S. aureus (rec-03), at the respectively, different durations at 120min of contact. of contact This (couldp < 0.05). be more It is precisely worth mentioning explained by that the the PLAfact/Ag that/PEG the showed PEG reducing higher agent antibacterial additive in performance the system is both more against hydrophilicE. coli (shownas well also as byS. aureusthe waterat the differentcontact durations angle experiments) of contact. Thisso that could the be bacteria more preciselystrains could explained be easier by the attached fact that onto the the PEG sample reducing agentsurface additive to start in thefurther system their is coloniza more hydrophiliction, while at (shown the same also time by the the Ag water+ release contact is more angle pronounced experiments) so thatin the the PLA/PEG bacteria blended strains system could killin be easierg the bacteria attached more onto effectively. the sample surface to start further their colonization,The results while obtained at the same in our time study the Ag regarding+ release th ise moredifferences pronounced in bacteria in the viability PLA/PEG between blended systemGram-positive killing the and bacteria Gram-negative more effectively. strains using AgNPs as the antimicrobial agent, are in good agreement with findings recently reported by Huq et al. [62]. Specifically, in this work the growth The results obtained in our study regarding the differences in bacteria viability between curves of Gram-positive S. aureus Gram-negative E. coli bacteria strains cultured in R2A broth with Gram-positive and Gram-negative strains using AgNPs as the antimicrobial agent, are in good various concentrations of AgNPs have been reported, showing a higher AgNP susceptibility of agreementGram-negative with findings bacteria recentlycompared reported to that of by Gram Huq-positive et al. [ 62bacteria.]. Specifically, This was infurther this workexplained the growthdue curvesto distinctions of Gram-positive in the compositionS. aureus Gram-negative of the bacteriaE. cell coli wall,bacteria which strains was similarly cultured reported in R2A brothalso in with variousanother concentrations study [63]. PLA of surfaces AgNPs of have bare beenPLA retractor reported, exhibited showing no abactericidal higher AgNP effect. susceptibility The detailed of Gram-negative bacteria compared to that of Gram-positive bacteria. This was further explained due to distinctions in the composition of the bacteria cell wall, which was similarly reported also in another Biomimetics 2020, 5, 42 14 of 22 studyBiomimetics [63]. PLA2020, 5 surfaces, x FOR PEER of bareREVIEW PLA retractor exhibited no bactericidal effect. The detailed results14 of 22 are presented in Table1 (values shown represent the means SD of triplicate measurements; n = 3) and results are presented in Table 1 (values shown represent ±the means ± SD of triplicate measurements; Figures9 and 10. n = 3) and Figures 9 and 10.

FigureFigure 8. SEM8. SEM images images of theof side-surfacethe side-surface microstructure microstructure and and morphology morphology for thefor dithefferent different 3D printed 3D printed samples in this study at two different magnifications (all 3D printed samples with 300 µm samples in this study at two different magnifications (all 3D printed samples with 300 µm printed printed layer thickness): (a,b) PLA/Ag/PEG using rec-01 filament, (c,d) PLA/Ag/PEG using rec-02 layer thickness): (a,b) PLA/Ag/PEG using rec-01 filament, (c,d) PLA/Ag/PEG using rec-02 filament; filament; (e,f) PLA/Ag/PVP using rec-03 filament; and (g,h) PLA/Ag/PVP using rec-04 filament. (e,f) PLA/Ag/PVP using rec-03 filament; and (g,h) PLA/Ag/PVP using rec-04 filament.

Biomimetics 2020, 5, x FOR PEER REVIEW 15 of 22

Table 1. Antibacterial activity of 3D printed PLA/AgNP nanocomposites (@100 µm printed layer thickness) against S. aureus and E. coli after Different Incubation Times (n = 3, p < 0.05).

Reduction in Viability (%) Sample Formulation 30 min 60 min 120 min S. aureus 36.42 ± 2.02 57.20 ± 0.88 74.91 ± 3.78 PLA/Ag/PEG (rec-01) E. coli 79.17 ± 0.55 90.01 ± 0.95 94.86 ± 1.22 S. aureus 28.23 ± 3.08 47.45 ± 3.88 62.96 ± 5.78 PLA/Ag/PEG (rec-02) E. coli 49.17 ± 0.52 76.68 ± 0.95 88.97 ± 1.45 S. aureus 33.42 ± 3.02 52.20 ± 0.81 68.93 ± 3.32 PLA/Ag/PVP (rec-03) E. coli 60.17 ± 0.85 83.85 ± 0.95 92.97 ± 1.12 Biomimetics 2020, 5, 42 S. aureus 22.35 ± 2.68 40.21 ± 3.88 50.95 ± 5.78 15 of 22 PLA/Ag/PVP (rec-04) E. coli 46.17 ± 0.88 68.08 ± 0.99 73.28 ± 1.95

FigureFigure 9. Antibacterial9. Antibacterial activity activity of (a )of PLA (a/)Ag PLA/Ag/PEG/PEG (rec-01), (rec-01), (b) PLA /Ag(b)/PEG PLA/Ag/PEG (rec-02), (c )(rec-02), PLA/Ag /PVP(c) (rec-03)PLA/Ag/PVP and (d) (rec-03) PLA/Ag and/PVP (d (rec-04)) PLA/Ag/PVP 3D printed (rec-04) PLA 3D/AgNP printed nanocomposites PLA/AgNP nanocomposites with 100 µm 3D with printed 100 layerµm thickness,3D printed againstlayer thickness,S. aureus againstand E. S. coli aureusafter and 30, 60,E. coli and after 120 30, min 60, of and contact. 120 min Mean of contact. values Mean do not values do not differ significantly (p < 0.05). Biomimeticsdiffer significantly2020, 5, x FOR (PEERp < 0.05). REVIEW 16 of 22

3.7. Tensile Properties and Fracture Surface Analysis Tensile test experiments were performed on 3D printed dog bone-shaped samples manufactured from neat PLA extruded filament, as well as nanocomposite PLA/AgNP filament from the PLA/Ag/PEG (rec-01), PLA/Ag/PEG (rec-02), PLA/Ag/PVP (rec-03), and PLA/Ag/PVP (rec-04). Figure 11g shows typical stress–strain curves derived and calculated from the tensile testing of the nanocomposites. Both tensile strength and moduli results with the corresponding standard deviations are plotted in Figure 12a,b respectively for the different sample formulations.

FigureFigure 10. 10.Comparative Comparative spider spider graphs graphs for thefor antibacterial the antibact behaviorerial behavior for the fourfor the 3D printedfour 3D PLA printed/AgNP nanocompositesPLA/AgNP nanocomposites produced in produced this work in (with this 100workµm (with 3D printed100 µm layer3D printed thickness) layer againstthickness) (a) againstS. aureus and(a) S. (b )aureusE. coli andafter (b) 30,E. coli 60, after and 30, 120 60, min and of 120 contact. min of Valuescontact. areValues mean. are mean. Mean Mean values values do not do dinotffer significantlydiffer significantly (p < 0.05). (p < 0.05).

Figure 11. (a) Tensile test of a specimen, (b) Failed PLA/Ag/PEG (rec 1) specimens after the tensile tests, (c) Failed PLA/Ag/PEG (rec 2) specimens after the tensile tests, (d) Failed pure PLA specimens after the tensile tests, (e) Failed PLA/Ag/PVP (rec 3) specimens after the tensile tests, (f) Failed PLA/Ag/PVP (rec 4) specimens after the tensile tests, (g) Tensile stress vs. strain graphs for the neat PLA and the four recipes prepared in this work. All graphs are from the no 1 specimen of each case studied.

As it can be observed, there is a knock-down effect on the tensile strength of the 3D printed PLA/Ag/PEG (rec-01), PLA/Ag/PEG (rec-02), PLA/Ag/PVP (rec-03), and PLA/Ag/PVP (rec-04) as compared to the neat PLA 3D printed specimens. This is more precisely attributed to the low molecular weight PEG (Mn = 4600 g/mol) and PVP (Mn = 10,000 g/mol) additives in PLA/Ag/PEG (rec-01 and rec-02) as well as PLA/Ag/PVP (rec-03 and rec-04), respectively; utilized as the macromolecular reducing agents for the creation of AgNPs in the four different blend recipes. Moreover, it can be seen also that the effect of strength reduction is more prominent for the PLA/Ag/PEG (rec-03) as compared to the reference PLA. In general, the tensile strength knock-down is not significant as compared to the neat PLA reference material for all recipes tested, except the PLA/Ag/PVP (rec-03). Due to the reasons explained above, a minor decrease in the mechanical properties was expected. The main aim of this work was not to increase the mechanical properties of the produced nanocomposites, but to produce nanocomposites with improved antibacterial and

Biomimetics 2020, 5, 42 16 of 22

Table 1. Antibacterial activity of 3D printed PLA/AgNP nanocomposites (@100 µm printed layer thickness) against S. aureus and E. coli after Different Incubation Times (n = 3, p < 0.05).

Reduction in Viability (%) Sample Formulation 30 min 60 min 120 min S. aureus 36.42 2.02 57.20 0.88 74.91 3.78 PLA/Ag/PEG (rec-01) ± ± ± E. coli 79.17 0.55 90.01 0.95 94.86 1.22 ± ± ± S. aureus 28.23 3.08 47.45 3.88 62.96 5.78 PLA/Ag/PEG (rec-02) ± ± ± E. coli 49.17 0.52 76.68 0.95 88.97 1.45 ± ± ± S. aureus 33.42 3.02 52.20 0.81 68.93 3.32 PLA/Ag/PVP (rec-03) ± ± ± E. coli 60.17 0.85 83.85 0.95 92.97 1.12 ± ± ± S. aureus 22.35 2.68 40.21 3.88 50.95 5.78 PLA/Ag/PVP (rec-04) ± ± ± E. coli 46.17 0.88 68.08 0.99 73.28 1.95 ± ± ± Biomimetics 2020, 5, x FOR PEER REVIEW 16 of 22 3.7. Tensile Properties and Fracture Surface Analysis Tensile test experiments were performed on 3D printed dog bone-shaped samples manufactured from neat PLA extruded filament, as well as nanocomposite PLA/AgNP filament from the PLA/Ag/PEG (rec-01), PLA/Ag/PEG (rec-02), PLA/Ag/PVP (rec-03), and PLA/Ag/PVP (rec-04). Figure 11 shows typical stress–strain curves derived and calculated from the tensile testing of the nanocomposites. Both tensile strength and moduli results with the corresponding standard deviations are plotted in Figure 12a,b respectively for the different sample formulations. As it can be observed, there is a knock-down effect on the tensile strength of the 3D printed PLA/Ag/PEG (rec-01), PLA/Ag/PEG (rec-02), PLA/Ag/PVP (rec-03), and PLA/Ag/PVP (rec-04) as compared to the neat PLA 3D printed specimens. This is more precisely attributed to the low molecular weight PEG (Mn = 4600 g/mol) and PVP (Mn = 10,000 g/mol) additives in PLA/Ag/PEG (rec-01 and rec-02) as well as PLA/Ag/PVP (rec-03 and rec-04), respectively; utilized as the macromolecular reducing agents for the creation of AgNPs in the four different blend recipes. Moreover, it can be seen also that the effect of strength reduction is more prominent for the PLA/Ag/PEG (rec-03) as compared to the reference PLA. In general, the tensile strength knock-down is not significant as compared to the neat PLA reference material for all recipes tested, except the PLA/Ag/PVP (rec-03). Due to the reasons explained above, a minor decrease in the mechanical properties was expected. The main aim of this work was not to increase the mechanical properties of the produced nanocomposites, but to produce nanocompositesFigure 10. with Comparative improved spider antibacterial graphs for and the other antibact propertieserial behavior and testfor the mechanicalfour 3D printed behavior of PLA/AgNP nanocomposites produced in this work (with 100 µm 3D printed layer thickness) against the parts manufactured with these nanocomposites, which were expected to be similar to the polymer (a) S. aureus and (b) E. coli after 30, 60, and 120 min of contact. Values are mean. Mean values do not matrix material.differ significantly This is achieved(p < 0.05). with the recipes implemented and tested in this work.

FigureFigure 11. 11.(a) ( Tensilea) Tensile test test of aof specimen, a specimen, (b )(b Failed) Failed PLA PLA/Ag/PEG/Ag/PEG (rec (rec 1) 1) specimens specimens after after the the tensile tensile tests, tests, (c) Failed PLA/Ag/PEG (rec 2) specimens after the tensile tests, (d) Failed pure PLA specimens (c) Failed PLA/Ag/PEG (rec 2) specimens after the tensile tests, (d) Failed pure PLA specimens after the after the tensile tests, (e) Failed PLA/Ag/PVP (rec 3) specimens after the tensile tests, (f) Failed tensile tests, (e) Failed PLA/Ag/PVP (rec 3) specimens after the tensile tests, (f) Failed PLA/Ag/PVP PLA/Ag/PVP (rec 4) specimens after the tensile tests, (g) Tensile stress vs. strain graphs for the neat (rec 4) specimens after the tensile tests, (g) Tensile stress vs. strain graphs for the neat PLA and the four PLA and the four recipes prepared in this work. All graphs are from the no 1 specimen of each case recipes prepared in this work. All graphs are from the no 1 specimen of each case studied. studied.

As it can be observed, there is a knock-down effect on the tensile strength of the 3D printed PLA/Ag/PEG (rec-01), PLA/Ag/PEG (rec-02), PLA/Ag/PVP (rec-03), and PLA/Ag/PVP (rec-04) as compared to the neat PLA 3D printed specimens. This is more precisely attributed to the low molecular weight PEG (Mn = 4600 g/mol) and PVP (Mn = 10,000 g/mol) additives in PLA/Ag/PEG (rec-01 and rec-02) as well as PLA/Ag/PVP (rec-03 and rec-04), respectively; utilized as the macromolecular reducing agents for the creation of AgNPs in the four different blend recipes. Moreover, it can be seen also that the effect of strength reduction is more prominent for the PLA/Ag/PEG (rec-03) as compared to the reference PLA. In general, the tensile strength knock-down is not significant as compared to the neat PLA reference material for all recipes tested, except the PLA/Ag/PVP (rec-03). Due to the reasons explained above, a minor decrease in the mechanical properties was expected. The main aim of this work was not to increase the mechanical properties of the produced nanocomposites, but to produce nanocomposites with improved antibacterial and

Biomimetics 2020, 5, x FOR PEER REVIEW 17 of 22

otherBiomimetics properties 2020, 5, x FORand PEER test REVIEW the mechanical behavior of the parts manufactured with17 theseof 22 nanocomposites, which were expected to be similar to the polymer matrix material. This is achieved other properties and test the mechanical behavior of the parts manufactured with these with the recipes implemented and tested in this work. Biomimeticsnanocomposites,2020, 5, 42 which were expected to be similar to the polymer matrix material. This is achieved17 of 22 with the recipes implemented and tested in this work.

Figure 12. (a) Comparative tensile strength graph and (b) tensile mod. of elasticity for all the materials studied. FigureFigure 12. 12.( a()a) Comparative Comparative tensiletensile strengthstrength graph and and ( (bb)) tensile tensile mod. mod. of ofelasticity elasticity for for all allthe the materials studied. materials studied. The generation of AgNPs by reactive melt mixing extrusion resulted in a slight decrease of the modulusTheThe generation generationof elasticity of of AgNPs AgNPsfor all by by the reactivereactive 3D meltmeltprinte mixingd PLA/Ag/PEG extrusion extrusion resulted resulted(rec-01), in in aPLA/Ag/PEG aslight slight decrease decrease (rec-02), of ofthe the PLA/Ag/PVP (rec-03) and PLA/Ag/PVP (rec-04) specimens when compared to the neat PLA 3D modulusmodulus of elasticityof elasticity for allfor the all 3D the printed 3D PLAprinte/Agd /PEGPLA/Ag/PEG (rec-01), PLA(rec-01),/Ag/PEG PLA/Ag/PEG (rec-02), PLA (rec-02),/Ag/PVP printedPLA/Ag/PVP sample. (rec-03) and PLA/Ag/PVP (rec-04) specimens when compared to the neat PLA 3D (rec-03) and PLA/Ag/PVP (rec-04) specimens when compared to the neat PLA 3D printed sample. printedFigure sample. 13 shows SEM images of the tensile test fractured areas of the neat PLA specimens and Figure 13 shows SEM images of the tensile test fractured areas of the neat PLA specimens and the specimensFigure 13 manufactured shows SEM images with the of thefour tensile different test recipesfractured of thisareas study. of the Images neat PLA show specimens a more brittle and the specimens manufactured with the four different recipes of this study. Images show a more brittle behaviorthe specimens of the manufactured neat PLA, when with compared the four different to the four recipes nanocomposites, of this study. Imageswhich is show in agreement a more brittle with behavior of the neat PLA, when compared to the four nanocomposites, which is in agreement with the thebehavior stress- ofstrain the neatgraphs PLA, produced when compared during the to experi the fourments nanocomposites, of this work. Thewhich more is in ductile agreement behavior with of stress- strain graphs produced during the experiments of this work. The more ductile behavior of the thethe recipestress- strain1 nanocomposite graphs produced is also during verified the experi by thements images of this takenwork. Thein the more fracture ductile areasbehavior of ofthe recipe 1 nanocomposite is also verified by the images taken in the fracture areas of the specimens. Also, specimens.the recipe 1Also, nanocomposite it was evident is also that verified extrusio by nthe produced images takennanocomposites in the fracture with areas no internalof the itstructuring wasspecimens. evident faults,Also, that extrusionsinceit was a uniform evident produced structure that nanocomposites extrusio can ben observedproduced with in nonanocomposites all internal different structuring materials with developedno faults, internal since in a uniformthisstructuring work. structure faults, can since be observeda uniform in structure all different can be materials observed developed in all different in this materials work. developed in this work.

Figure 13. SEM images of the tensile test fractured areas (a,b) Neat PLA, (c) PLA/Ag/PEG rec-01 FigureFigure 13. 13.SEM SEM images images ofof thethe tensiletensile test fractured areas areas (a (a,b,b) )Neat Neat PLA, PLA, (c ()c PLA/Ag/PEG) PLA/Ag/PEG rec-01 rec-01 specimen, (d) PLA/Ag/PEG rec-02 specimen, (e) PLA/Ag/PVP rec-03 specimen, and (f) PLA/Ag/PVP specimen,specimen, (d ()d PLA) PLA/Ag/PEG/Ag/PEG rec-02 rec-02specimen, specimen, (e) PLAPLA/Ag/PVP/Ag/PVP rec-03 rec-03 specimen, specimen, and and (f) ( fPLA/Ag/PVP) PLA/Ag/PVP rec-04 specimen. rec-04rec-04 specimen. specimen.

3.8. Micro-Hardness Properties of 3D Printed PLA and PLA/AgNP Nanocomposites

The calculated microhardness of the 3D printed manufactured samples consisting of neat PLA extruded filament, as well as nanocomposite PLA/AgNP filament from the PLA/Ag/PEG (rec-01), PLA/Ag/PEG (rec-02), PLA/Ag/PVP (rec-03), and PLA/Ag/PVP (rec-04) are depicted in Figure 14g. As it can be seen, the microhardness of the neat PLA is marginally higher than the microhardness of the four Biomimetics 2020, 5, x FOR PEER REVIEW 18 of 22

3.8. Micro-Hardness Properties of 3D Printed PLA and PLA/AgNP Nanocomposites The calculated microhardness of the 3D printed manufactured samples consisting of neat PLA extruded filament, as well as nanocomposite PLA/AgNP filament from the PLA/Ag/PEG (rec-01), BiomimeticsPLA/Ag/PEG2020, 5(rec-02),, 42 PLA/Ag/PVP (rec-03), and PLA/Ag/PVP (rec-04) are depicted in Figure18 14g. of 22 As it can be seen, the microhardness of the neat PLA is marginally higher than the microhardness of the four nanocomposites, showing that these specific fillers effect on the microhardness of the PLA nanocomposites, showing that these specific fillers effect on the microhardness of the PLA polymer polymer matrix is negligible. Some microhardness variation and decrease for the PLA/AgNP matrix is negligible. Some microhardness variation and decrease for the PLA/AgNP nanocomposite nanocomposite samples could be attributed to some plasticization effect that has been induced to the samples could be attributed to some plasticization effect that has been induced to the PLA due to the PLA due to the blended macromolecular chains of the PEG (rec-01 and rec-02) and PVP (rec-03 and blended macromolecular chains of the PEG (rec-01 and rec-02) and PVP (rec-03 and rec-04) that have rec-04) that have been utilized as reducing agents in the melt state for the in-situ growth of the been utilized as reducing agents in the melt state for the in-situ growth of the AgNPs. AgNPs.

Figure 14. (a(a) )Measuring Measuring the the Vickers Vickers Micro-Hardness Micro-Hardness of of a a specimen, specimen, ( (bb)) PLA/Ag/PEG PLA/Ag/PEG (rec (rec 1) specimen, (c(c)) PLA PLA/Ag/PEG/Ag/PEG (rec (rec 2) specimen,2) specimen, (d) Pure(d) PLAPure specimen,PLA specimen, (e) PLA /(Age) /PLA/Ag/PVPPVP (rec 3) specimen, (rec 3) (specimen,f) PLA/Ag (/fPVP) PLA/Ag/PVP (rec 4) specimen, (rec 4) specimen, and (g) Micro-Hardness and (g) Micro-Hardness Vickers results Vickers of results neat PLA of neat and PLA the fourand therecipes four prepared recipes prepared in this work. in this work.

4. Conclusions Conclusions Since PLA has been proven to be safe for biomedicalbiomedical applications, cost effective, effective, safe, and an environmentally suitablesuitable material material for for printing, printing, 3D 3D printed printed antimicrobial antimicrobial and and anti-adhesive anti-adhesive objects objects have havebeen producedbeen produced herein herein via the via 3D th printinge 3D printing FFF 3D printingFFF 3D printing process. process. PLA/AgNP PLA/AgNP antimicrobial antimicrobial filaments filamentshave been have produced been produced by reactive bymelt reactive mixing, melt viamixing, blending via blendi of (i) PLA,ng of AgNO(i) PLA,3, AgNO and PEG,3, and as PEG, well as (ii)well PLA, as (ii) AgNO PLA,3 ,AgNO and PVP.3, and PEG PVP. and PEG PVP and have PVP been have utilized been asutilized alternative as alternative macromolecular macromolecular reducing reducingagent polymeric agent polymeric materials materials in both cases. in both The cases. PLA The as thePLA polymer as the polymer matrix andmatrix AgNO and3 AgNOas the3 AgNP as the precursorAgNP precursor salt. AgNPs salt. AgNPs have been have generated been generated in the PLA in the polymer PLA polymer matrix, yieldingmatrix, yielding extremely extremely efficient efficientantimicrobial antimicrobial (AM) objects (AM) as obje determinedcts as determined by the reduction by the inreduction Staphylococcus in Staphylococcus aureus (S. aureusaureus) and (S. aureusEscherichia) and Escherichia coli (E. coli) bacteriacoli (E. coli viability) bacteria (%) viability after being (%) exposed after being for exposed 30, 60, and for 120 30, min,60, and respectively. 120 min, respectively.Such behavior Such is in behavior the direction is in of the mimicking direction nature of mimicking mechanism nature to kill mechanism these specific to kill types these of bacteria,specific whiletypes of the bacteria, same e ffwhileect is the possible same toeffect be expectedis possible in to other be expected types of in bacteria, other types too, andof bacteria, can expand too, and the cancurrent expand study the in current the future study in thisin the direction. future in this direction. The advantagesadvantages of of the the current current study’s study’s protocol protocol lie in thelie factin the that: fact (i) molecularlythat: (i) molecularly the Ag+ stemming the Ag+ stemmingfrom the AgNO from 3thedissociated AgNO3 dissociated in the polymer in the melt polymer are distributed melt are indistributed volume of in the volume PLAmelt of the getting PLA meltreduced getting further reduced in-situ further during in-situ the melt-mixing during the compounding melt-mixing process,compounding (ii) it is process, a clean process (ii) it is avoiding a clean processmixing ofavoiding e.g., Ag nanopowders,mixing of e.g., (iii) Ag ready-made nanopowders, AgNPs (iii) are knownready-made to be expensive,AgNPs are (iv) known AgNPs to could be expensive,agglomerate (iv) during AgNPs processing could agglomerate and affect during the rheology processing of the and system affect increasing the rheology the meltof the viscosity, system increasingand (v) there the is melt no guaranteeviscosity, orand full (v) characterization there is no guarantee about theor full NP qualitycharacterization (size, monodispersity), about the NP whilequality in (size, many monodispersity), cases of commercially while available in many AgNPs cases of the commercially suppliers do available not disclose AgNPs the particle the suppliers surface dochemistry, not disclose affecting the particle both the surface nanodispersion chemistry, process affecting as both well the as thenanodispersion nanocomposites’ process antimicrobial as well as properties.the nanocomposites’ It could beantimicrobial envisaged thatproperties. the proposed It could 3D be printed envisaged AM that objects the proposed demonstrated 3D printed in this workAM objects with tunabledemonstrated anti-adhesive in this work properties with tunabl varyinge anti-adhesive the 3D printed properties object printed varying layer the 3D thickness, printed could possibly substitute a great number of metallic objects, i.e., titanium consisting nosocomial equipment, etc. with a much lower cost. Biomimetics 2020, 5, 42 19 of 22

Author Contributions: M.P., L.T., and E.V. contributed to the preparation of the 3D printed samples and the fabrication of the PLA/AgNP nanocomposite filaments produced by reactive melt-mixing. M.P., L.T., E.V., and M.L. decided the optimum protocol and reaction parameters for creating the silver nanoparticles into the PLA matrix. M.P., L.T., and E.V. designed the 3D model for the dog-bone shaped samples utilised for the mechanical tests. L.T. and M.L. performed the optical and electron microscopy investigations and the related image analysis as well as the TGA experiments and data analyses. L.T. carried out the antibacterial activity tests of 3D printed PLA/AgNP nanocomposites. M.P., L.T., E.V., and N.V. designed the experimental protocol in this work, and all contributed to the analysis and discussion of the data. M.P. and L.T. wrote the first draft of the paper. M.P., L.T., E.V., M.L., and N.V. coordinated the mentioned activities. The manuscript was written through contributions of all authors. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: L.T. gratefully acknowledges the Bodossaki Foundation for financial support in the framework of institutional scholarships. Conflicts of Interest: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

References

1. Tappa, K.; Jammalamadaka, U.; Weisman, J.A.; Ballard, D.H.; Wolford, D.D.; Pascual-Garrido, C.; Wolford, L.M.; Woodard, P.; Mills, D.K. 3D Printing Custom Bioactive and Absorbable Surgical Screws, Pins, and Bone Plates for Localized Drug Delivery. J. Funct. Biomater. 2019, 10, 17. [CrossRef][PubMed] 2. Tzounis, L.; Bangeas, P.; Exadaktylos, A.K.; Petousis, M.; Vidakis, N. Three-Dimensional Printed Polylactic Acid (PLA) Surgical Retractors with Sonochemically Immobilized Silver Nanoparticles: The Next Generation of Low-Cost Antimicrobial Surgery Equipment. Nanomaterials 2020, 10, 985. [CrossRef][PubMed] 3. Tyler, B.; Gullotti, D.; Mangraviti, A.; Utsuki, T.; Brem, H. Polylactic acid (PLA) controlled delivery carriers for biomedical applications. Adv. Drug Deliv. Rev. 2016, 107, 163–175. [CrossRef][PubMed] 4. Mohammadi, M.S.; Bureau, M.; Nazhat, S. Polylactic acid (PLA) biomedical foams for tissue engineering. In Biomedical Foams for Tissue Engineering Applications; Netti, P.A., Ed.; Woodhead Publishing: Cambridge, UK, 2014; pp. 313–334. 5. Nasab, S.M.; Tabari, M. Antimicrobial properties and permeability of Poly lactic Acid nanocomposite films containing Zinc Oxide. Nanomed. Res. J. 2018, 3, 125–132. 6. Lewicki, J.P.; Rodriguez, J.N.; Zhu, C.; Worsley, M.A.; Wu, A.S.; Kanarska, Y.; Horn, J.D.; Duoss, E.B.; Ortega, J.M.; Elmer, W.; et al. 3D-Printing of Meso-structurally Ordered Carbon Fiber/Polymer Composites with Unprecedented Orthotropic Physical Properties. Sci. Rep. 2017, 7, 43401. [CrossRef][PubMed] 7. Christ, J.F.; Aliheidari, N.; Pötschke, P.; Ameli, A. Bidirectional and Stretchable Piezoresistive Sensors Enabled by Multimaterial 3D Printing of Carbon Nanotube/Thermoplastic Polyurethane Nanocomposites. Polymers 2018, 11, 11. [CrossRef][PubMed] 8. Rankin, T.M.; Giovinco, N.A.; Cucher, D.J.; Watts, G.; Hurwitz, B.; Armstrong, D.G. Three-dimensional printing surgical instruments: Are we there yet? J. Surg. Res. 2014, 189, 193–197. [CrossRef] 9. Chen, J.V.; Dang, A.B.C.; Lee, C.S.; Dang, A.B.C. 3D printed PLA Army-Navy retractors when used as linear retractors yield clinically acceptable tolerances. 3D Print. Med. 2019, 5, 16–19. [CrossRef] 10. Yue, J.; Zhao, P.; Gerasimov, J.Y.; Van De Lagemaat, M.; Grotenhuis, A.; Rustema-Abbing, M.; Van Der Mei, H.C.; Busscher, H.J.; Herrmann, A.; Ren, Y. 3D-Printable Antimicrobial Composite Resins. Adv. Funct. Mater. 2015, 25, 6756–6767. [CrossRef] 11. An, J.; Teoh, J.E.M.; Suntornnond, R.; Chua, C.K. Design and 3D Printing of Scaffolds and Tissues. Engineering 2015, 1, 261–268. [CrossRef] 12. Pitsalidis, C.; Ferro, M.; Iandolo, D.; Tzounis, L.; Inal, S.; Owens, R.M. Transistor in a tube: A route to three-dimensional bioelectronics. Sci. Adv. 2018, 4, eaat4253. [CrossRef][PubMed] 13. Sodian, R.; Schmauss, D.; Schmitz, C.; Bigdeli, A.; Haeberle, S.; Schmoeckel, M.; Markert, M.; Lueth, T.; Freudenthal, F.; Reichart, B.; et al. 3-Dimensional Printing of Models to Create Custom-Made Devices for Coil Embolization of an Anastomotic Leak After Aortic Arch Replacement. Ann. Thorac. Surg. 2009, 88, 974–978. [CrossRef][PubMed] Biomimetics 2020, 5, 42 20 of 22

14. Kurenov, S.N.; Ionita, C.N.; Sammons, D.; Demmy, T.L. Three-dimensional printing to facilitate anatomic study, device development, simulation, and planning in thoracic surgery. J. Thorac. Cardiovasc. Surg. 2015, 149, 973–979.e1. [CrossRef][PubMed] 15. Bangeas, P.; Tzounis, L.; Karolos, I.; Stavrides, E.; Paramythiotis, D.; Michalopoulos, A.; Tsioukas, V.; Tsoulfas, G.; Papadopoulos, V.; Exadaktylos, A. Evaluation of three-dimensional printed models in surgical education: A paradigm of a new educational method for the simulation of surgery environment. HPB 2018, 20, S779. [CrossRef] 16. Sodian, R.; Schmauss, D.; Markert, M.; Weber, S.; Nikolaou, K.; Haeberle, S.; Vogt, F.; Vicol, C.; Lueth, T.; Reichart, B.; et al. Three-Dimensional Printing Creates Models for Surgical Planning of Aortic Valve Replacement After Previous Coronary Bypass Grafting. Ann. Thorac. Surg. 2008, 85, 2105–2108. [CrossRef] 17. Kolyva, C.; Biglino, G.; Pepper, J.R.; Khir, A.W. A Mock Circulatory System with Physiological Distribution of Terminal Resistance and Compliance: Application for Testing the Intra-Aortic Balloon Pump. Artif. Organs 2010, 36, E62–E70. [CrossRef] 18. Lioufas, P.A.; Quayle, M.R.; Leong, J.C.; McMenamin, P.G. 3D Printed Models of Cleft Palate Pathology for Surgical Education. Plast. Reconstr. Surg. Glob. Open 2016, 4, e1029. [CrossRef] 19. Bangeas, P. Three-dimensional printing as an educational tool in colorectal surgery. Front. Biosci. 2019, 11, 29–37. [CrossRef] 20. Vidakis, N.; Petousis, M.; Maniadi, A.; Koudoumas, E.; Liebscher, M.; Tzounis, L. Mechanical Properties

of 3D-Printed Acrylonitrile–Butadiene–Styrene TiO2 and ATO Nanocomposites. Polymers 2020, 12, 1589. [CrossRef] 21. Tzounis, L.; Petousis, M.; Grammatikos, S.A.; Vidakis, N. 3D Printed Thermoelectric Polyurethane/Multiwalled Carbon Nanotube Nanocomposites: A Novel Approach towards the Fabrication of Flexible and Stretchable Organic Thermoelectrics. Materials 2020, 13, 2879. [CrossRef] 22. Tzounis, L.; Petousis, M.; Liebscher, M.; Grammatikos, S.A.; Vidakis, N. Three-Dimensional (3D) Conductive Network of CNT-Modified Short Jute Fiber-Reinforced Natural Rubber: Hierarchical CNT-Enabled Thermoelectric and Electrically Conductive Composite Interfaces. Materials 2020, 13, 2668. [CrossRef] [PubMed] 23. Bekas, D.; Hou, Y.; Liu, Y.; Panesar, A. 3D printing to enable multifunctionality in polymer-based composites: A review. Compos. Part B Eng. 2019, 179, 107540. [CrossRef] 24. Venkatesan, J.; Kim, S.-K.; Shim, M.S. Antimicrobial, Antioxidant, and Anticancer Activities of Biosynthesized Silver Nanoparticles Using Marine Algae Ecklonia cava. Nanomaterials 2016, 6, 235. [CrossRef][PubMed] 25. Jeong, S.H.; Hwang, Y.H.; Yi, S.C. Antibacterial properties of padded PP/PE nonwovens incorporating nano-sized silver colloids. J. Mater. Sci. 2005, 40, 5413–5418. [CrossRef] 26. Ilana, P.; Guy, A.; Nina, P.; Geoffrey, G.; Serguei, M.; Aharon, G. Sonochemical coating of silver nanoparticles on textile fabrics (nylon, polyester and cotton) and their antibacterial activity. Nanotechnology 2008, 19, 245705. 27. Nina, P.; Galina, A.; Guy, A.; Eldar, E.; Yuri, K.; Anil Vithal, G.; Bo-Jung, C.; Yong-Chien, L.; Aharon, G. Depositing silver nanoparticles on/in a glass slide by the sonochemical method. Nanotechnology 2008, 19, 435604. 28. Chen, Y.-H.; Hsu, C.-C.; He, J.-L. Antibacterial silver coating on poly(ethylene terephthalate) fabric by using high power impulse magnetron sputtering. Surf. Coat. Technol. 2013, 232, 868–875. [CrossRef] 29. Jiang, S.; Qin, W.; Guo, R.; Zhang, L. Surface functionalization of nanostructured silver-coated polyester fabric by magnetron sputtering. Surf. Coat. Technol. 2010, 204, 3662–3667. [CrossRef] 30. Gorenšek, M.; Gorjanc, M.; Bukošek, V.; Kovaˇc,J.; Petrovic, Z.L.; Puaˇc,N. Functionalization of Polyester Fabric by Ar/N2Plasma and Silver. Text. Res. J. 2010, 80, 1633–1642. [CrossRef] 31. Islam, S.-U.; Shahid, M.; Mohammad, F. Green Chemistry Approaches to Develop Antimicrobial Textiles Based on Sustainable Biopolymers—A Review. Ind. Eng. Chem. Res. 2013, 52, 5245–5260. [CrossRef] 32. Cheng, X.; Ma, K.; Li, R.; Ren, X.; Huang, T. Antimicrobial coating of modified chitosan onto cotton fabrics. Appl. Surf. Sci. 2014, 309, 138–143. [CrossRef] 33. Deng, X.; Nikiforov, A.; Coenye, T.; Cools, P.; Aziz, G.; Morent, R.; De Geyter, N.; Leys, C. Antimicrobial nano-silver non-woven polyethylene terephthalate fabric via an atmospheric pressure plasma deposition process. Sci. Rep. 2015, 5, 10138. [CrossRef][PubMed] Biomimetics 2020, 5, 42 21 of 22

34. D’Agostino, A.; Taglietti, A.; Desando, R.; Bini, M.; Patrini, M.; Dacarro, G.; Cucca, L.; Pallavicini, P.; Grisoli, P. Bulk Surfaces Coated with Triangular Silver Nanoplates: Antibacterial Action Based on Silver Release and Photo-Thermal Effect. Nanomaterials 2017, 7, 7. [CrossRef][PubMed] 35. Shrivastava, S.; Bera, T.; Singh, S.K.; Singh, G.; Ramachandrarao, P.; Dash, D. Characterization of Antiplatelet Properties of Silver Nanoparticles. ACS Nano 2009, 3, 1357–1364. [CrossRef][PubMed] 36. Rai, M.; Yadav, A.; Gade, A. Silver nanoparticles as a new generation of antimicrobials. Biotechnol. Adv. 2009, 27, 76–83. [CrossRef][PubMed] 37. Feng, Q.L.; Wu, J.; Chen, G.Q.; Cui, F.Z.; Kim, T.N.; Kim, J.O. A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J. Biomed. Mater. Res. 2000, 52, 662–668. [CrossRef] 38. Asharani, P.V.; Mun, G.L.K.; Hande, M.P.; Valiyaveettil, S. Cytotoxicity and Genotoxicity of Silver Nanoparticles in Human Cells. ACS Nano 2008, 3, 279–290. [CrossRef] 39. Sawant, S.N.; Selvaraj, V.; Prabhawathi, V.; Doble, M. Antibiofilm Properties of Silver and Incorporated PU, PCLm, PC and PMMA Nanocomposites under Two Shear Conditions. PLoS ONE 2013, 8, e63311. [CrossRef] 40. Tzounis, L.; Hegde, M.; Liebscher, M.; Dingemans, T.; Pötschke, P.; Paipetis, A.S.; Zafeiropoulos, N.E.; Stamm, M. All-aromatic SWCNT-Polyetherimide nanocomposites for thermal energy harvesting applications. Compos. Sci. Technol. 2018, 156, 158–165. [CrossRef] 41. Tzounis, L.; Gärtner, T.; Liebscher, M.; Pötschke, P.; Stamm, M.; Voit, B.; Heinrich, G. Influence of a cyclic butylene terephthalate oligomer on the processability and thermoelectric properties of polycarbonate/MWCNT nanocomposites. Polymers 2014, 55, 5381–5388. [CrossRef] 42. Liebscher, M.; Gärtner, T.; Tzounis, L.; Miˇcušík, M.; Pötschke, P.; Stamm, M.; Heinrich, G.; Voit, B. Influence of the MWCNT surface functionalization on the thermoelectric properties of melt-mixed polycarbonate composites. Compos. Sci. Technol. 2014, 101, 133–138. [CrossRef] 43. Wode, F.; Tzounis, L.; Kirsten, M.; Constantinou, M.; Georgopanos, P.; Rangou, S.; Zafeiropoulos, N.E.; Avgeropoulos, A.; Stamm, M. Selective localization of multi-wall carbon nanotubes in homopolymer blends and a diblock copolymer. Rheological orientation studies of the final nanocomposites. Polymers 2012, 53, 4438–4447. [CrossRef] 44. Triebel, C.; Vasylyev, S.; Damm, C.; Stara, H.; Özpınar, C.; Hausmann, S.; Peukert, W.; Münstedt, H. Polyurethane/silver-nanocomposites with enhanced silver ion release using multifunctional invertible polyesters. J. Mater. Chem. 2011, 21, 4377–4383. [CrossRef] 45. Damm, C. Silver Ion Release from Polymethyl Methacrylate Silver Nanocomposites. Polym. Polym. Compos. 2005, 13, 649–656. [CrossRef] 46. Damm, C.; Münstedt, H.; Rösch, A. The antimicrobial efficacy of polyamide 6/silver-nano- and microcomposites. Mater. Chem. Phys. 2008, 108, 61–66. [CrossRef] 47. Parida, D.; Simonetti, P.; Frison, R.; Bülbül, E.; Altenried, S.; Arroyo, Y.; Balogh, Z.; Caseri, W.; Ren, Q.; Hufenus, R.; et al. Polymer-assisted in-situ thermal reduction of silver precursors: A solventless route for silver nanoparticles-polymer composites. Chem. Eng. J. 2020, 389, 123983. [CrossRef] 48. Jaggessar, A.; Shahali, H.; Mathew, A.; Yarlagadda, P.K. Bio-mimicking nano and micro-structured surface fabrication for antibacterial properties in medical implants. J. Nanobiotechnol. 2017, 15, 64. [CrossRef] 49. Hasan, J.; Chatterjee, K. Recent advances in engineering topography mediated antibacterial surfaces. Nanoscale 2015, 7, 15568–15575. [CrossRef] 50. Zhang, C.; McAdams, D.A.; Grunlan, J.C. Nano/Micro-Manufacturing of Bioinspired Materials: A Review of Methods to Mimic Natural Structures. Adv. Mater. 2016, 28, 6292–6321. [CrossRef] 51. Liz-Marzán, L.M.; Lado-Touriño, I. Reduction and Stabilization of Silver Nanoparticles in by Nonionic Surfactants. Langmuir 1996, 12, 3585–3589. [CrossRef] 52. Andersson, M.; Alfredsson, V.; Kjellin, P.; Palmqvist, A.E.C. Macroscopic Alignment of Silver Nanoparticles in Reverse Hexagonal Liquid Crystalline Templates. Nano Lett. 2002, 2, 1403–1407. [CrossRef] 53. Koczkur, K.M.; Mourdikoudis, S.; Skrabalak, S.E.; Polavarapu, L. Polyvinylpyrrolidone (PVP) in nanoparticle synthesis. Dalton Trans. 2015, 44, 17883–17905. [CrossRef][PubMed] 54. Nam, S.; Parikh, D.V.; Condon, B.D.; Zhao, Q.; Yoshioka-Tarver, M. Importance of poly(ethylene glycol) conformation for the synthesis of silver nanoparticles in aqueous solution. J. Nanoparticle Res. 2011, 13, 3755–3764. [CrossRef] Biomimetics 2020, 5, 42 22 of 22

55. Carrasco, P.M.; Tzounis, L.; Mompean, F.J.; Strati, K.; Georgopanos, P.; Garcia-Hernandez, M.; Stamm, M.; Cabañero, G.; Odriozola, I.; Avgeropoulos, A.; et al. Thermoset Magnetic Materials Based on Poly(ionic liquid)s Block Copolymers. Macromolecules 2013, 46, 1860–1867. [CrossRef] 56. Tzounis, L.; Debnath, S.; Rooj, S.; Fischer, D.; Mäder, E.; Das, A.; Stamm, M.; Heinrich, G. High performance natural rubber composites with a hierarchical reinforcement structure of carbon nanotube modified natural fibers. Mater. Des. 2014, 58, 1–11. [CrossRef] 57. Harada, M.; Inada, Y.; Nomura, M. In situ time-resolved XAFS analysis of silver particle formation by photoreduction in polymer solutions. J. Colloid Interface Sci. 2009, 337, 427–438. [CrossRef] 58. Ramirez, D.; Jaramillo, F. Facile one-pot synthesis of uniform silver nanoparticles and growth mechanism. DYNA 2016, 83, 165–170. [CrossRef] 59. Chowdhury,J.; Ghosh, M. Concentration-dependent surface-enhanced Raman scattering of 2-benzoylpyridine adsorbed on colloidal silver particles. J. Colloid Interface Sci. 2004, 277, 121–127. [CrossRef] 60. Mukherjee, P.K.; Roy, M.; Mandal, B.P.; Dey, G.K.; Ghatak, J.; Tyagi, A.K.; Kale, S.P. Green synthesis of highly stabilized nanocrystalline silver particles by a non-pathogenic and agriculturally important fungusT. asperellum. Nanotechnology 2008, 19, 075103. [CrossRef] 61. Maiti, N.; Kapoor, S.; Mahal, H.S.; Mukherjee, T. Adsorption of CGA on colloidal silver particles: DFT and SERS study. Chem. Phys. Lett. 2007, 444, 338–345. [CrossRef] 62. Huq, A. Green Synthesis of Silver Nanoparticles Using Pseudoduganella eburnea MAHUQ-39 and Their Antimicrobial Mechanisms Investigation against Drug Resistant Human Pathogens. Int. J. Mol. Sci. 2020, 21, 1510. [CrossRef][PubMed] 63. Chatterjee, T.; Chatterjee, B.K.; Majumdar, D.; Chakrabarti, P. Antibacterial effect of silver nanoparticles and the modeling of bacterial growth kinetics using a modified Gompertz model. Biochim. Biophys. Acta (BBA) Gen. Subj. 2015, 1850, 299–306. [CrossRef][PubMed]

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