Biocompatibility of Poly(Acrylonitrile-Butadiene-Styrene) Nanocomposites Modiﬁed with Silver Nanoparticles

Article Biocompatibility of Poly(acrylonitrile-butadiene-styrene) Nanocomposites Modiﬁed with Silver Nanoparticles

Magdalena Zi ˛abka 1,*, Michał Dziadek 2 and Elzbieta˙ Menaszek 3 1 AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Department of Ceramics and Refractories, al. Mickiewicza 30, 30-059 Krakow, Poland 2 AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Department of Glass Technology and Amorphous Coatings, al. Mickiewicza 30, 30-059 Krakow, Poland; [email protected] 3 Jagiellonian University, Collegium Medicum, Faculty of Pharmacy, Department of Cytobiology, ul. Medyczna 9, 30-688 Krakow, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-12-617-25-23

Received: 18 October 2018; Accepted: 11 November 2018; Published: 13 November 2018

Abstract: We evaluated the biological, mechanical, and surface properties of polymer nanocomposites manufactured via plastics processing, extrusion, and injection moulding. The aim of this study was to identify the interaction of fibroblasts and osteoblasts with materials intended for middle ear implants. We examined if silver nanoparticles (AgNPs) may change the mechanical parameters of the polymer nanocomposites. In our study, the biostable polymer of thermoplastic acrylonitrile-butadiene-styrene (ABS) copolymer was used. Silver nanoparticles were applied as a modifier. We discuss surface parameters of the materials, including wettability and roughness, and evaluated the microstructure. The mechanical parameters, such as the Young’s modulus and tensile strength, were measured. Cytotoxicity tests were conducted on two cell lines: Hs680.Tr human fibroblasts and Saos-2 human osteoblasts. Cell viability, proliferation, and morphology in direct contact with nanocomposites were tested. Based on the results, the incorporated modifier was found to affect neither the number of osteoblasts nor the fibroblast cells. However, the addition of AgNPs had a relatively small effect on the cytotoxicity of the materials. A slight increase in the cytotoxicity of the test materials was observed with respect to the control, with the cytotoxicity of the materials tending to decrease after seven days for osteoblast cells, whereas it remained steady for fibroblasts. Based on optical microscope observation, the shape and morphology of the adhered cells were evaluated. After seven days of culture, fibroblasts and osteoblasts were properly shaped and evenly settled on the surface of both the pure polymer and the silver nanoparticle-modified composite. Water droplet tests demonstrated increased hydrophilicity when adding the AgNPs to ABS matrices, whereas roughness tests did not show changes in the surface topography of the investigated samples. The 0.5% by weight incorporation of AgNPs into ABS matrices did not influence the mechanical properties.

Keywords: nanocomposites; biocompatibility; cell viability; ﬁbroblasts; osteoblasts

1. Introduction Nanoparticle-polymer composites have potential for application as the next generation of instructive biomaterials. They may display parameters comparable to casual composites, potentially allowing the development of modern devices for antibacterial treatment, medical imaging, tissue engineering, drug delivery, cancer therapy, and dental applications [1–3]. Obtaining optimal biological, mechanical, physical, and chemical properties is critical during the development of materials, especially for materials that will be implanted for more than 30 days. Medical devices, including implants, are

Polymers 2018, 10, 1257; doi:10.3390/polym10111257 www.mdpi.com/journal/polymers Polymers 2018, 10, 1257 2 of 13 classified with regard to the level of risk posed to the human body due to their implantation according to the definitions and rules of the 93/42/EEC Directive of the European Parliament and of the Council, also known as the Medical Devices Directive (MDD) [4]. This classification is simple: the higher the class, the higher the risk for the person after implantation. Classification may vary depending on the usage or dosage. All products that are supposed to be implanted for less than 30 days are classified in the second group. Implantation for more than 30 days classifies them into the third class. Additionally, even if a product will be implanted for a short period of less than 30 days but contains a drug that may affect healing results, it is assigned to the third class. Consequently, all materials developed to be integrated in the human body should be safe and biocompatible. Measurements of biological reactions and parameters assessing the possibility of using certain materials have been gathered in International Standard ISO 10993 [5]. Considering all of the above, during the development of a material, all properties cannot be worse at the end of an experiment than at the beginning—they should improve from the starting point. The addition of even a small amount of nanoparticles (NPs) to the polymer matrix and then adding the nanocomposite to the body will affect the interaction of nanoparticles with cells. Interaction with immune cells may result in molecular reactions, which may lead to higher sensitivity to disease and cancer growth [6]. Use of nanoparticles may not only affect various reactions within cells but also cause undesired allergic reactions [7–9]. Modification of composites with silver (Ag) nanoparticles may influence their antibacterial properties. Silver nanoparticles (AgNPs) are biocidal and their mechanism of toxicity is related to the damage of cell membranes and oxidative stress [10,11]. Silver ions can cause deformation of the protein structure by binding to them via disulphide bonds in the cytoplasm. Since the malformed proteins are incorporated into the plasma membrane, cell permeability is altered, leading to cell death [12]. Understanding how a nanocomposite reacts with cells is a challenge from a toxicological point of view, but is crucial for future solutions in biomedicine. Cell research including human cell lines may provide information on the biocompatibility of nanomaterials [13]. The ideal middle ear prosthesis for ossicles reconstruction should be biocompatible, readily available, technically easy to use, and have the best possible biomechanical properties [14]. Considering biological factors, the prosthesis material ought to be antibacterial and stable in the environment, especially in the case of chronic middle ear inflammation. As far as the mechanical properties are concerned, the middle ear implant should retain its shape and measurements for a prolonged period of time, along with tensile strength and rigidity. The materials used for middle ear prostheses should be resistant to live load and fatigue conditions. Additionally, middle ear implants have to be adaptable to the micro-movements between the tympanic membrane and the middle ear [15]. Many different materials have been used for ossiculoplasty. One of them is titanium due to its high biocompatibility, low weight, and ease of use [16,17]. Polymeric prostheses made of polytetrafluoroethylene (PTFE) [18] and ceramic prostheses made of aluminum oxide ceramic (Al2O3)[19] or bioactive hydroxyapatite (HA) [20] are also used. Composite prostheses, such as Plastipore, PTFE, Polycel (thermal-fused Plastipore), HAPEX (polyethylene composites reinforced with HA), Flex-HA (a mixture of silastic—silicone plastic—and HA) and those created by Bojrab et al. [21] and Hahn et al. [22] are available in the medical devices market. However, none of these currently available alloplastic materials fulfill the antibacterial requirement. The use of autogenic ossicles is not recommended in the case of chronic middle ear infection, otosclerosis, or cholesteatoma, since they contribute to the recurrence of the underlying disease [23]. As such, alloplastic materials, especially with bactericidal properties, seem to be a promising alternative to the aforementioned illnesses. Our previous results [24] showed that a middle ear prosthesis made of an ABS matrix and silver nanoparticles had antibacterial activity against Gram-positive and Gram-negative bacteria and was biocompatible in long-lasting tests on animals. Additionally, we proved that AgNPs accelerate the healing process of surrounded tissues, which is crucial to the length of convalescence after the reconstruction of ossicle chain. Polymers 2018, 10, 1257 3 of 13

In this manuscript, we present how AgNPs affect the physicochemical and biological properties of polymer composites. Given that the scientiﬁc literature describes the toxic action of silver nanoparticles, we wanted to determine if the proposed amount of the additive is safe for materials designed for otolaryngology.

2. Materials and Methods

2.1. Material Manufacturing Two commercially-available polymers—ABS poly(acrylonitrile-butadiene-styrene)-Novodur HD 15 (INEOS Styrolution, Frankfurt, Germany; ABS-N) and Elix M205FC (Elix Polymers, La Canonja, Spain; ABS-E)—as well as composite materials modiﬁed with 0.5 wt % AgNPs (NanoAmor, Katy, TX, USA) with a purity of 99.9%, 80-nm particle size, and density of 10.49 g cm−3 were shaped into 10-mm-diameter discs. Polymer and composite materials were manufactured using plastics processing methods, extrusion, and injection moulding. The procedure for obtaining specimens was as follows. First, the granulates were prepared and dried in the laboratory dryer at 80 ◦C for 6 h. Next, the AgNPs were incorporated and homogenized with polymer granules in the plasticizing chamber using a 0.8-m-long screw at a homogenization temperature of 240 ◦C. Subsequently, the material was injected into the steel moulding form, cooled, and extracted. The injection parameters were selected and adapted for the process according to data sheet of the polymer manufacture. Injection temperature in the three zones was 240 ◦C, injection pressure was 80 kg cm−2, and ﬂow was 80%.

2.2. Material Evaluation

2.2.1. Scanning Electron Microscopy A Nova NanoSEM 200 scanning electron microscope (SEM; FEI, Eindhoven, The Netherlands) coupled with a Genesis XM X-ray microanalysis system (EDAX, Tilburg, The Netherlands) featuring the Sapphire Si(Li) energy dispersive X-ray (EDX) detector was used to perform the detailed examination of the microstructure of the produced materials. The measurements and observations were conducted in high vacuum conditions, with back scatter electron detector (BSE) at an accelerated voltage of 10–18 kV. The samples were coated with a carbon layer.

2.2.2. Atomic Force Microscopy The topographical evaluation of the ABS-E, ABS-N, and their composites with AgNPs was performed via atomic force microscope (AFM, MultiMode 8 Bruker microscope, Karlsruhe, Germany), using antimony-doped silicon tips (spring constant = 40 N m−1), operating in tapping mode. Image analysis was performed using XEI 1.7.1 software (Park Systems, Suwon, Korea). Root mean square roughness (Rq) and the arithmetical mean roughness (Ra) values were calculated based on three AFM height images collected at three different places on the materials and are expressed as the mean ± standard deviation (SD).

2.2.3. Surface Wettability The surface wettability was evaluated by static water contact angle measurements. The contact angle was determined by the sessile drop method with an automatic drop shape analysis (DSA) system, DSA 10 Mk2 (Kruss GmbH, Hamburg, Germany). Ultrahigh quality (UHQ) water droplets of 0.25 µL were applied on each pure and dry sample. The experiments were carried out in constant temperature and humidity conditions. The apparent contact angle was calculated as an average of 10 measurements and is expressed as a mean ± standard deviation (SD). Polymers 2018, 10, 1257 4 of 13

2.2.4. Tensile Test

Tensile strength (σM) and Young’s modulus (Et) were determined using a universal testing machine, the Inspect Table Blue 5 kN with a 5-kN load cell (Hegewald&Peschke, Nossen, Germany). The pre-load force was 1 N, the test speed was 50 mm min−1. The samples for measurements were prepared according to EN ISO 527-1 [25]. Mechanical parameters were calculated by averaging 10 measurements and are expressed as mean ± SD.

2.2.5. In Vitro Tests In vitro biological evaluation of the produced materials was carried out using two cell lines: Hs680.Tr (human tracheal fibroblast; ATCC, Manassas, VA, USA) and Saos-2 (human osteosarcoma; ATCC, Manassas, VA, USA) from 3 passages. In order to obtain the cell suspension, the culture was washed twice with phosphate buffered saline (PBS), followed by a 5% trypsin solution with EDTA (HyClone, San Angelo, TX, USA). After washing and centrifugation, the cells were suspended in fresh medium. Next, 1 mL of the resulting cell suspension at a density of 104 cells/mL was added to the wells of 48-well culture plates (Nunc, Roskilde, Denmark) containing sterile discs of the materials. The bottom surfaces of tissue culture polystyrene (TCPS) wells served as a control. TCPS also served as a negative control for cytotoxicity assays. Cell culture of the discs in direct contact with the test materials was carried out for 3 and 7 days of culture. ToxiLight 100% Lysis Reagent set (Lonza, Walkersville, MD, USA) was used to disintegrate the cytomembrane of the cultured cells. Then a ToxiLight Bioassay Kit (Lonza, Walkersville, MD, USA) was used in order to establish the entire number of cells confirming their viability and proliferation. The amount of the released adenylate kinase was assessed with a PolarStar Omega reader (BMG Labtech, Ortenberg, Germany). Furthermore, the metabolic activity of the cells after 3 and 7 days of culture was evaluated using the PrestoBlue® Cell Viability Reagent (Invitrogen, Waltham, MA, USA). PrestoBlue® reagent was added to each well with cells spread on TCPS and then materials ◦ were incubated for 2 h at 37 C in a 5% CO2 moistened atmosphere. Fluorescence was measured at 560/590 nm (excitation/emission, respectively) using a POLARstar Omega microplate reader (BMG Labtech, Ortenberg, Germany). Results are expressed as the mean ± SD from 8 measurements for each group. Cytotoxicity of the materials was assessed via the bioluminescence method, using ToxiLight Bioassay Kit (Lonza, Walkersville, MD, USA), which measures the release of the enzyme adenylate kinase (AK) from damaged cells. AK is a robust protein present in all eukaryotic cells that is released into the culture medium when cells die. The luminescence was measured with a PolarStar Omega plate reader spectrophotometer (BMG Labtech, Ortenberg, Germany). The test was conducted on the supernatant from the cell culture and the results were compared to the entire enzyme concentration (proportional to the entire number of cells) released from all cells. Results are expressed as the mean ± SD from 8 measurements for each group. Two samples of each series were used for morphological observation under a fluorescence optical microscope. The cells adhering to the materials were dyed for 1 min with acridine orange (AO) solution (Sigma, Saint Louis, MO, USA). After washing with PBS (HyClone, San Angelo, TX, USA), the cells were examined using an Olympus CX-41 (Olympus, Tokyo, Japan) with a fluorescence device. A digital E-520 camera (Olympus, Tokyo, Japan) was used to take photographs of the cells. A Nova NanoSEM 200 scanning electron microscope (SEM; FEI, Eindhoven, The Netherlands) was used to perform a detailed morphological examination of the cells adhered to the investigated materials. The observations were conducted in high vacuum conditions, with a back scatter electron detector (BSE) at an accelerated voltage of 10–18 kV. After 7 days of cell culture, materials were rinsed with PBS and then cells were fixed with 3% glutaraldehyde solution in sodium cacodylate buffer at pH 7.4 (POCh, Gliwice, Poland) for 0.5 h. Subsequently, the cells were dehydrated in a graded series of ethanol solution (70%, 80%, 90%, 96%, and 100%) and dried in air. Cell morphologies were evaluated after coating with carbon. Polymers 2018, 10, 1257 5 of 13 Polymers 2018, 10, x FOR PEER REVIEW 5 of 13

2.2.6.2.2.6. StatisticalStatistical analysisanalysis TheThe resultsresults werewere analysedanalysed usingusing one-wayone-way analysisanalysis ofof variancevariance (ANOVA)(ANOVA) withwith DuncanDuncan postpost hochoc tests,tests, whichwhich werewere performedperformed withwith StatisticaStatistica 1010 (StatSoft(StatSoft®®,, Tulsa,Tulsa, OK,OK, USA)USA) software.software. TheThe resultsresults werewere consideredconsidered statisticallystatistically signiﬁcantsignificant whenwhenp p< < 0.05.0.05.

3.3. ResultsResults

3.1.3.1. SEMSEM andand AFMAFM MeasurementsMeasurements AFMAFM andand SEMSEM imagesimages (Figure(Figure1 1)) show show the the relevant relevant differences differences in in the the surface surface morphology morphology of of the the investigatedinvestigated materials.materials. TheThe bothboth surfacessurfaces ofof ABS-EABS-E andand ABS-NABS-N polymerspolymers andand compositecomposite materialsmaterials werewere ratherrather homogenous. homogenous. However, However, in in the the case case of of ABS-N ABS-N materials, materials, the the surface surface appeared appeared smoother smoother in comparisonin comparison to ABS-Eto ABS-E materials. materials. The The SEM SEM images images of of the the pure pure ABS-E ABS-E and and its its nanocomposite nanocomposite showed showed a grain-likea grain-like structure, structure, which which was was more more visible visible in in the the AFM AFM images. images.

FigureFigure 1. SEM 1. SEM and AFMand AFM tapping tapping mode imagesmode images of pure polymersof pure polymers and polymers and polymers containing containing silver nanoparticles. silver nanoparticles. The ABS-N exhibited a smooth surface, whereas its cross-section was rougher compared to the ABS-NThe cross-section. ABS-N exhibited We also a smooth observed surface, small regionswhereas with its cross-section silver aggregates. was rougher The aggregates compared were to upthe toABS-N 5 micrometres cross-section. in size. We also observed small regions with silver aggregates. The aggregates were up to 5 micrometres in size.

Polymers 2018, 10, x FOR PEER REVIEW 6 of 13 Polymers 2018, 10, 1257 6 of 13 Polymers 2018, 10, x FOR PEER REVIEW 6 of 13 The arithmetic average values of the roughness profile (Ra) and root mean-square roughness (Rq), collected from AFM images, did not show significant changes upon modification of the polymer The arithmetic average values of the roughness profileprofile (Ra) and root mean-square roughness matrices with silver nano-additive (Figure 2). However, we observed slight differences in Rq (Rq), collected from AFM images, did not show significant significant change changess upon modification modification of the polymer measured for the ABS-E pure polymer and its composite, which might be related to the grain matrices withwith silver silver nano-additive nano-additive (Figure (Figure2). However, 2). However, we observed we observed slight differences slight differences in Rq measured in Rq structuremeasured of for the the pure ABS-E polymer, pure whereaspolymer the and additi its coonmposite, of AgNPs which could might influence be related this difference. to the grain The for the ABS-E pure polymer and its composite, which might be related to the grain structure of the AFMstructure measurements of the pure revealed polymer, that whereas tested surfacesthe additi wereon of characterized AgNPs could by influence Ra and Rq this parameters difference. below The pure polymer, whereas the addition of AgNPs could influence this difference. The AFM measurements 65AFM nm measurements in the case of ABS-Nrevealed pure that polymer tested surfaces and its werecomposites, characterized and below by Ra 45 and nm Rq for parameters ABS-E materials. below revealed that tested surfaces were characterized by Ra and Rq parameters below 65 nm in the case of These65 nm resultsin the caseindicate of ABS-N the low pure surface polymer roughness and its of composites, the tested materials.and below 45 nm for ABS-E materials. ABS-N pure polymer and its composites, and below 45 nm for ABS-E materials. These results indicate These results indicate the low surface roughness of the tested materials. the low surface roughness of the tested materials.

Figure 2. The root mean-square roughness (Rq) and average roughness (Ra) of pure polymers and polymersFigure 2. ThecontainingThe root root mean-square mean-square silver nanoparticles roughness roughness (Rq)collected (Rq) and and averfrom averageage AFM roughness roughness images. (Ra) Statistically (Ra) of pure of pure polymers significant polymers and differencesandpolymers polymers containing (p < containing0.05) between silver silver thenanoparticles nanoparticlestested materials collected collected are mark from fromed AFMa-c AFM for images.Rq images. and A-BStatistically Statistically for Ra, respectively. signiﬁcantsignificant differences (p < 0.05) between the tested materials are mark markeded a-c for Rq and A-B for Ra, respectively. respectively. 3.2. Surface Wettability 3.2. Surface Wettability The contact angle measurements allowed us to evaluate the surface wettability of the tested samples.The contactThe The results results angle showed showedmeasurements that that the the allowed surfaces surfaces us of ofto all allevaluate polymers polymers the surfaceand composite wettability materials of the testedwere hydrophilic.samples. The For results both pure showed polymers that the(ABS-E surfaces and ABS-N),of all polymers the wettability and compositeangle was estimated materials at were the samehydrophilic. level, below For both 8585°◦ (Figurepure polymers 33).). (ABS-E and ABS-N), the wettability angle was estimated at the same level, below 85° (Figure 3).

Figure 3.3.The The static static water water contact contact angle ofangle pure of polymers pure po andlymers polymers and containingpolymers silvercontaining nanoparticles. silver Statistically signiﬁcant differences (p < 0.05) between the tested materials are marked a–c. nanoparticles.Figure 3. The Statistically static water significant contact differencesangle of pure(p < 0.05) polymers between and the polymerstested materials containing are marked silver a–c.Thenanoparticles. incorporation Statistically of silver significant nanoparticles differences in the (p < amount 0.05) between of 0.5 bythe weight tested materials into the polymerare marked matrix slightlya–c. inﬂuenced the wettability of the composites. In comparison to polymers, composite materials were more hydrophilic and the wettability angle was below 80◦.

Polymers 2018, 10, x FOR PEER REVIEW 7 of 13 Polymers 2018, 10, x FOR PEER REVIEW 7 of 13 The incorporation of silver nanoparticles in the amount of 0.5 by weight into the polymer matrix slightlyThe influencedincorporation the ofwettability silver nanoparticles of the composit in thees. amount In comparison of 0.5 by to weight polymers, into thecomposite polymer materials matrix slightlywere more influenced hydrophilic the wettability and the we ofttability the composit anglees. was In belowcomparison 80°. to polymers, composite materials Polymers 2018, 10, 1257 7 of 13 were more hydrophilic and the wettability angle was below 80°. 3.3. Mechanical Properties 3.3. Mechanical Properties Figure 4 shows the Young’s modulus (Et) and the tensile strength of materials (σM). The material Figure 4 shows the Young’s modulus (Et) and the tensile strength of materials (σM). The material basedFigure on the4 shows Elix M205FC the Young’s polymer modulus (ABS-E) (E t showed) and the significantly tensile strength higher of materialsvalues of (Eσt Mand). The σM compared material based on the Elix M205FC polymer (ABS-E) showed significantly higher values of Et and σM compared basedto materials on the Elix based M205FC on the polymer Novodur (ABS-E) HD 15 showed polymer significantly (ABS-N). The higher presence values of of silver Et and nanoparticlesσM compared in totothe materialsmaterials ABS-N basedbasedmaterial onon matrix thethe NovodurNovodur caused HDHD a statistically 1515 polymerpolymer si (ABS-N).gnificant(ABS-N). Theincrease presence in strength ofof silversilver parameters. nanoparticlesnanoparticles On inthein thetheother ABS-NABS-N hand, materialmaterial in the case matrixmatrix of ABS-E_0.5Ag causedcaused aa statisticallystatistically material, significantsinognificant significant increaseincrease changes inin strengthstrengthwere observed parameters.parameters. in comparison On thethe otherto the hand, unmodified in the case material. of ABS-E_0.5Ag material, no significant significant changes were observed in comparison toto thethe unmodifiedunmodified material.material.

Figure 4. Young’s modulus (Et) and tensile strength (σM) of pure polymers and polymers containing Figure 4. Young’s modulus (E ) and tensile strength (σ ) of pure polymers and polymers containing Figuresilver 4.nanoparticles. Young’s modulus Statistically (Ett) and significant tensile strength differences (σMM) of(p pure< 0.05) polymers between an thed polymers tested materials containing are silver nanoparticles. Statistically signiﬁcant differences (p < 0.05) between the tested materials are silvermarked nanoparticles. a-c for Et and Statistically A-B for σM significant, respectively. differences (p < 0.05) between the tested materials are marked a-c for E and A-B for σ , respectively. marked a-c for Ett and A-B for σM, respectively. 3.4.3.4. Cell Cell Viability/Proliferation Viability/Proliferation Test Test 3.4. Cell Viability/Proliferation Test FigureFigure5A,B 5A,B shows shows metabolic metabolic activity activity of of Saos-2 Saos-2 (osteoblast) (osteoblast) and and Hs-680.Tr Hs-680.Tr (ﬁbroblasts) (fibroblasts) cells cells after after 3 Figure 5A,B shows metabolic activity of Saos-2 (osteoblast) and Hs-680.Tr (fibroblasts) cells after and3 and 7 days 7 days of culture,of culture, as evaluatedas evaluated by theby the PrestoBlue PrestoBlue test. test. 3 and 7 days of culture, as evaluated by the PrestoBlue test.

Figure 5. Metabolic activity of Saos-2 (A) and Hs680.Tr (B) cells after 3 and 7 days of culture in direct contact with the materials tested in the Presto-Blue test. The results are presented as mean Figure 5. Metabolic activity of Saos-2 (A) and Hs680.Tr (B) cells after 3 and 7 days of culture in direct values ± standard deviation. Statistically significant differences (p < 0.05) between the tested materials Figurecontact 5. withMetabolic the materials activity oftested Saos-2 in (theA) Presto-Bluand Hs680.Tre test. (B )The cells results after 3 are and presented 7 days of asculture mean in values direct ± are marked a-d for 3-day culture and A-D for 7-day culture. contactstandard with deviation. the materials Statistica testedlly insignificant the Presto-Blu differencese test. The(p < results 0.05) between are presented the tested as mean materials values are ± standard deviation. Statistically significant differences (p < 0.05) between the tested materials are Formarked all the a-d materials, for 3-day culture a significant and A-D increase for 7-day in culture. metabolic activity of osteoblast and fibroblast cells marked a-d for 3-day culture and A-D for 7-day culture. was observed after 7 days of culture. This increase indicates high levels of viability and proliferation of both types of cells. After 7 days of experiment, the osteoblast and fibroblast cells cultured on all of the tested materials showed slightly reduced metabolic activity, as compared to the control material (TCPS). In the case of osteoblasts, metabolic activity of cells was on a similar level in both groups of materials Polymers 2018, 10, x FOR PEER REVIEW 8 of 13

For all the materials, a significant increase in metabolic activity of osteoblast and fibroblast cells was observed after 7 days of culture. This increase indicates high levels of viability and proliferation of both types of cells. After 7 days of experiment, the osteoblast and fibroblast cells cultured on all of the tested materials showed slightly reduced metabolic activity, as compared to the control material Polymers(TCPS).2018 In, 10the, 1257 case of osteoblasts, metabolic activity of cells was on a similar level in both groups8 of 13 of materials (ABS-E and ABS-N) after both culture periods. While in tests conducted with the use of (ABS-Efibroblasts, and ABS-N)cells on afterABS-N both and culture ABS-N_0.5Ag periods. While after in7-day tests culture conducted exhibited with thethe use lowest of ﬁbroblasts, metabolic cellsactivity. on ABS-N and ABS-N_0.5Ag after 7-day culture exhibited the lowest metabolic activity. FigureFigure6 A,B6A,B shows shows the the relative relative number number of of cells cells of of both both cell cell lines lines after after 3 3 and and 7 7 days days of of culture culture in in directdirect contact contact with with the the test test materials materials determined determined in in the the ToxiLight ToxiLight test. test.

Figure 6. The relative number of Saos-2 (A) and Hs680.Tr (B) cells after 3 and 7 days of culture in direct contact with the tested materials in the ToxiLight test. The results are presented as mean values Figure 6. The relative number of Saos-2 (A) and Hs680.Tr (B) cells after 3 and 7 days of culture in ± standard deviation. Statistically significant differences (p < 0.05) between the tested materials are direct contact with the tested materials in the ToxiLight test. The results are presented as mean values marked a-b for 3-day culture and A-B for 7-day culture. ± standard deviation. Statistically significant differences (p < 0.05) between the tested materials are Themarked results a-b offor the 3-day study culture indicate and A-B a significant for 7-day culture. increase in the relative number of both fibroblasts and osteoblasts after 7 days of the experiment. The results again confirm that the materials promote cells proliferation.The results of Similarthe study results indicate were a significant obtained forincr bothease polymerin the relative materials. number For of both both cellsfibroblasts lines, theand number osteoblasts of cells after grown 7 days on of all the of experiment. the materials Th fore results 3 days again was significantly confirm that lower, the materials as compared promote to TCPS.cells proliferation. In turn, after Similar 7-day cultureresults therewere wereobtained no significantfor both polymer differences materials. between For theboth tested cells sampleslines, the number of cells grown on all of the materials for 3 days was significantly lower, as compared to TCPS. and control material (besides Saos-2 cells cultured on ABS-E sample). In the case of Saos-2 cells, for In turn, after 7-day culture there were no significant differences between the tested samples and material containing silver nanoparticles (ABS-E_0.5Ag and ABS-N_0.5Ag) an increase in the number of control material (besides Saos-2 cells cultured on ABS-E sample). In the case of Saos-2 cells, for cells at 7 days of culture was observed in comparison to pure polymers (ABS-E and ABS-N), however material containing silver nanoparticles (ABS-E_0.5Ag and ABS-N_0.5Ag) an increase in the number the changes were not statistically significant. Furthermore, the number of osteoblasts grown on both of cells at 7 days of culture was observed in comparison to pure polymers (ABS-E and ABS-N), composite materials for 7 days was on similar level, as compared to TCPS. In addition, due to the size however the changes were not statistically significant. Furthermore, the number of osteoblasts grown of nanomaterials, their additive can modulate cell-material interaction, improving biocompatibility. on both composite materials for 7 days was on similar level, as compared to TCPS. In addition, due 3.5.to Cytotoxicitythe size of Testnanomaterials, their additive can modulate cell-material interaction, improving biocompatibility. Figure7A,B shows the cytotoxicity of materials after 3 and 7 days of Saos-2 and Hs-680.Tr cell culture,3.5. Cytotoxicity evaluated Test on the basis of the level of AK adenylate kinase released into the culture medium during cell culture. InFigure the case7A,B ofshows the Elixthe cytotoxicity polymer for of osteoblastmaterials after cells, 3 and the cytotoxicity7 days of Saos-2 level and for Hs-680.Tr ABS-E and cell ABS-E_Ag0.5culture, evaluated decreased on the after basis seven of the days level of of culture. AK adenylate The measured kinase released values forinto both the culture materials medium were slightlyduring highercell culture. for TCPS but did not exceed 16% after three days of experiment and 9% after seven days. In the case of fibroblast lines cultured on the ABS-E group of materials, a slight increase in cytotoxicity was observed with respect to the control material; however, these values did not exceed 9% after both culture periods. In the case of the Novodur polymer for osteoblast cells, the cytotoxicity level for ABS-N and ABS-N_Ag0.5 decreased after seven days of culture. The measured values for both materials were slightly higher than for TCPS and the ABS-E group of materials but did not exceed 16% after three days of experiment and 10% after seven days. In the case of fibroblast lines cultured on the ABS-N group of materials, a slight increase in cytotoxicity was observed with respect to the control material after three days of cell culture. However, these values did not exceed 9%. For the seven-day period, the cytotoxicity increased up to 14%. This behaviour was observed for both pure Polymers 2018, 10, x FOR PEER REVIEW 9 of 13

Polymers 2018, 10, 1257 9 of 13

Figure 7. Cytotoxicity of materials evaluated in contact with Saos-2 (A) and Hs680.Tr (B) cells. The polymerresults ABS-N are presented and composite as mean material values ± ABS-N_0.5Ag.standard deviation. The Statistically results showed significant that the differences materials (p do< not Polymers 2018, 10, x FOR PEER REVIEW 9 of 13 exhibit0.05) cytotoxic between effects,the tested while materials encouraging are marked the a-d proliferation for 3-day culture of ﬁbroblasts and A-C for and 7-day osteoblasts. culture.

In the case of the Elix polymer for osteoblast cells, the cytotoxicity level for ABS-E and ABS- E_Ag0.5 decreased after seven days of culture. The measured values for both materials were slightly higher for TCPS but did not exceed 16% after three days of experiment and 9% after seven days. In the case of fibroblast lines cultured on the ABS-E group of materials, a slight increase in cytotoxicity was observed with respect to the control material; however, these values did not exceed 9% after both culture periods. In the case of the Novodur polymer for osteoblast cells, the cytotoxicity level for ABS- N and ABS-N_Ag0.5 decreased after seven days of culture. The measured values for both materials were slightly higher than for TCPS and the ABS-E group of materials but did not exceed 16% after three days of experiment and 10% after seven days. In the case of fibroblast lines cultured on the ABS- N group of materials, a slight increase in cytotoxicity was observed with respect to the control material after three days of cell culture. However, these values did not exceed 9%. For the seven-day period, the cytotoxicity increased up to 14%. This behaviour was observed for both pure polymer Figure 7. Cytotoxicity of materials evaluated in contact with Saos-2 (A) and Hs680.Tr (B) cells. The ABS-NFigure and 7.composite Cytotoxicity material of materials ABS-N_0.5Ag. evaluated inThe contact results with showed Saos-2 that(A) and the Hs680.Tr materials (B do) cells. not Theexhibit results are presented as mean values ± standard deviation. Statistically significant differences (p < 0.05) cytotoxicresults effects, are presented while encouraging as mean values the prol± standardiferation deviation. of fibroblasts Statistically and osteoblasts.significant differences (p < between the tested materials are marked a-d for 3-day culture and A-C for 7-day culture. 0.05) between the tested materials are marked a-d for 3-day culture and A-C for 7-day culture. 3.6. Cell Cultures Observation In the case of the Elix polymer for osteoblast cells, the cytotoxicity level for ABS-E and ABS- Based on the images obtained from the fluorescence microscope shown in Figures 8 and 9, both E_Ag0.5Based decreased on the images after seven obtained days from of culture. the fluorescence The measured microscope values shownfor both in materials Figures8 wereand9 slightly, both osteoblast and fibroblast cells were observed to have proper morphology and were evenly spread osteoblasthigher for andTCPS fibroblast but did not cells exceed were observed16% after tothree have days proper of experiment morphology and and 9% were after evenlyseven days. spread In homogeneously on ABS-E, ABS-N, and their composites modified with silver nanoparticles surfaces. homogeneouslythe case of fibroblast on ABS-E, lines cultured ABS-N, and on the their ABS-E composites group modifiedof materials, with a silverslight nanoparticlesincrease in cytotoxicity surfaces. was observed with respect to the control material; however, these values did not exceed 9% after both culture periods. In the case of the Novodur polymer for osteoblast cells, the cytotoxicity level for ABS- N and ABS-N_Ag0.5 decreased after seven days of culture. The measured values for both materials were slightly higher than for TCPS and the ABS-E group of materials but did not exceed 16% after three days of experiment and 10% after seven days. In the case of fibroblast lines cultured on the ABS- N group of materials, a slight increase in cytotoxicity was observed with respect to the control material after three days of cell culture. However, these values did not exceed 9%. For the seven-day period, the cytotoxicity increased up to 14%. This behaviour was observed for both pure polymer ABS-N and composite material ABS-N_0.5Ag. The results showed that the materials do not exhibit cytotoxic effects, while encouraging the proliferation of fibroblasts and osteoblasts.

3.6. Cell Cultures Observation Figure 8. Fluorescence microscope images of of Saos-2 cells cells in in direct contact with with the the tested materials. materials. Based on the images obtained from the fluorescence microscope shown in Figures 8 and 9, both PolymersMagnificationMagniﬁcation 2018, 10, x FOR 20× PEER.. REVIEW 10 of 13 osteoblast and fibroblast cells were observed to have proper morphology and were evenly spread homogeneously on ABS-E, ABS-N, and their composites modified with silver nanoparticles surfaces.

FigureFigure 9.9. FluorescenceFluorescence microscopemicroscope imagesimages ofof Hs680.TrHs680.Tr cellscells inin directdirect contactcontact withwith the tested materials. MagniﬁcationMagnification 2020××.

ForFigure all 8.materials, Fluorescence a significant microscope increase images in of theSaos-2 number cells in of direct Saos-2 contact and Hs with 680.Tr the tested cells wasmaterials. observed after Magnificationseven days of 20 culture.×. This increase confirms that the high level of proliferation of both types of cells. The number of cells adhered to both the examined materials, evaluated using microscopic photographs, was similar to that observed for the control material (TCPS). This result is correlated with the dependencies shown in the PrestoBlue and ToxiLight tests. Based on the images obtained from the scanning electron microscope shown in Figures 10 and 11, both osteoblast and fibroblast cells were present all over the observed regions.

Figure 10. SEM images of Saos-2 cells in direct contact with the tested materials. Magnification 1000×.

Figure 11. SEM images of Hs680.Tr cells in direct contact with the tested materials. Magnification 1000×.

However, more cells were observed after seven days of cells culture, which correlates with the cell viability presented in Figures 5 and 6 and cell morphology in Figures 8 and 9. Osteoblast and fibroblast cells spread over the material surfaces and showed flattened and proper morphology. The cellular network was formed by well-established, multiple cytoplasmic extensions, providing cell– cell and cell–surface interactions. Silver nanoparticles did not influence the number of cells adhering to the polymers. All the observed regions appeared similar for pure polymers and composites.

4. Discussion According to the work of Ziąbka and co-workers [26,27], the nanomodifier changes the surface properties of the polymer matrix, primarily causing an increase in roughness and surface area. The surface topography has a direct effect on cell adhesion, proliferation, and differentiation, as well as the expressions of the extracellular matrix proteins in direct contact with the material. Materials with

Polymers 2018, 10, x FOR PEER REVIEW 10 of 13

PolymersFigure2018, 9.10 Fluorescence, 1257 microscope images of Hs680.Tr cells in direct contact with the tested materials.10 of 13 Magnification 20×.

For all materials, a significant significant increase in the number of Saos-2 and Hs 680.Tr cells was observed after seven days days of of culture. culture. This This increase increase confirms confirms that that the the high high level level of ofproliferation proliferation of ofboth both types types of ofcells. cells. The The number number of ofcells cells adhered adhered to to both both the the examined examined materials, materials, evaluated using microscopicmicroscopic photographs, was similar to that observed for the controlcontrol material (TCPS). This result is correlated with the dependenciesdependencies shownshown inin thethe PrestoBluePrestoBlue andand ToxiLightToxiLight tests.tests. Based onon thethe imagesimages obtainedobtained from from the the scanning scanning electron electron microscope microscope shown shown in Figuresin Figures 10 and10 and 11, both11, both osteoblast osteoblast and and fibroblast fibroblast cells cells were were present present all over all over the observedthe observed regions. regions.

Figure 10. SEM images of Saos-2 cells in direct contactcontact withwith thethe testedtested materials.materials. MagniﬁcationMagnification 1000 1000×.×.

FigureFigure 11. 11.SEM SEM images images of Hs680.Trof Hs680.Tr cells cells in direct in direct contact contac witht with the tested the tested materials. materials. Magnification Magnification 1000× . 1000×. However, more cells were observed after seven days of cells culture, which correlates with the cell viabilityHowever, presented more cells in were Figures observed5 and6 andafter cellseven morphology days of cells in Figuresculture,8 which and9. correlates Osteoblast with and the fibroblastcell viability cells presented spread over in Figures the material 5 and surfaces 6 and cell and morphology showed flattened in Figures and proper8 and 9. morphology. Osteoblast Theand cellularfibroblast network cells spread was formed over the by material well-established, surfaces and multiple showed cytoplasmic flattened extensions,and proper providing morphology. cell–cell The andcellular cell–surface network interactions. was formed Silver by well-established, nanoparticles did multiple not influence cytoplasmic the number extensions, of cells providing adhering to cell– the polymers.cell and cell–surface All the observed interactions. regions Silver appeared nanoparticles similar for did pure not polymersinfluence the and number composites. of cells adhering to the polymers. All the observed regions appeared similar for pure polymers and composites. 4. Discussion 4. DiscussionAccording to the work of Zi ˛abkaand co-workers [26,27], the nanomodifier changes the surface propertiesAccording of the to polymer the work matrix, of Ziąbka primarily and co-workers causing an [26,27], increase the in nanomodifier roughness and changes surface the area. surface The surfaceproperties topography of the polymer has a directmatrix, effect primarily on cell causing adhesion, an increase proliferation, in roughness and differentiation, and surface asarea. well The as thesurface expressions topography of the has extracellular a direct effect matrix on cell proteins adhesion, in direct proliferation, contact with and the differentiation, material. Materials as well with as highthe expressions surface roughness of the extracellular have been shown matrix to proteins promote in contact direct contact osteogenesis with the [28 ].material. Osteoblast-biomaterial Materials with interactions directly affect the development of the bone–implant interface. Zareidoost et al. [29] showed that cells cultured for 3, 7, and 14 days on samples with rough surfaces exhibited a higher cell proliferation rate compared to polished and chemically modified material surfaces. Webster et al. [30] noticed that nanostructured surfaces are capable of adsorbing more vitronectin. They observed that surfaces with nanoscale topography were more favorable to osteoblast adhesion compared to the other cell types. Puckett et al. [31] established that the lower the width of the nano rough regions of the patterned substrates (from 80 to 22 µm), the lower the number of adhered osteoblasts. Furthermore, osteoblast adhesion was improved on the deposited nano rough Ti region compared to the non-treated region with microscale topography, regardless of width. This suggests that osteoblasts are able to recognize surface roughness. However, surfaces exhibiting more nano characteristic dimensions favor osteoblast adhesion. Polymers 2018, 10, 1257 11 of 13

In our work, we did not notice a correlation between roughness and number of adhered cells because the addition of silver nanoparticles did not change the Ra and Rq parameters. Therefore, the cell numbers for pure polymers and composites were comparable. Furthermore, the human cell diameter is 10–15 microns for fibroblasts and 20–30 microns for osteoblasts, which may suggest that nano roughness becomes too insignificant for fibroblasts and osteoblasts to affect cell behavior, such as attachment and proliferation rate. Some research has been conducted on fibroblast behavior in contact with surfaces exhibiting nanotopographical features. Some of these studies revealed the positive impact of surface topography on cell adhesion, whereas another showed the adverse effects of nanostructures [32]. Khang et al. observed that increased cellular function can be related to modified surface wettability resulting from the presence of nanofeatures. In turn, this can affect surface energy, therefore promoting adsorption of proteins, such as fibronectin and vitronectin, containing cell adhesion motif (RGD). Modified surface wettability also stimulates subsequent cellular functions (e.g., extracellular matrix deposition) [33]. Maschhoff et al. showed that the density was the highest for fibroblasts cultured for 18 h on ultrasonicated ZnO/PVC nanocomposites modified with the smallest ZnO nanoparticles [34]. Nanocomposites with smaller nanoparticles (10 nm in diameter) promoted fibroblast proliferation and simultaneously reduced bacterial adhesion and propagation. In our research, we observed wettability changes occurring with the addition of AgNPs, but the nanoparticles we incorporated into the ABS polymers were 80 nm in diameter, which might not affect cell number. There is no precise strength requirements for the materials used for middle ear prostheses. The implant material should have mechanical properties that are as close as possible to those of the host tissues. Since the chain of auditory ossicles is a complex structure with a wide range of Young’s modulus for particular elements (ligaments, muscles, joint, and bones), ranging from 0.049 MPa for ligaments to 14 GPa for bones, it is difficult to design a perfect material for its reconstruction [35]. For this reason, all the materials tested in this study perform biomechanical functions and may be used for middle ear implants.

5. Conclusions In conclusion, the cytotoxicity, viability, and proliferation tests conducted using fibroblast and osteoblastic cells showed that the tested poly(styrene-acrylonitrile-butadiene) polymers from ELIX Polymers and INEOS Styrolution are highly biocompatible. Furthermore, the results suggest that these materials possess sufficient mechanical properties for ossicular chain reconstruction. The addition of 0.5% by weight of silver nanoparticles did not significantly affect their biological and mechanical properties. The surface roughness of composite materials was not significantly affected, while wettability was slightly improved. Relative numbers of osteoblast and fibroblast cells did not change upon polymer modification with a nanomodifier. This study demonstrates that both pure polymers and composites are biocompatible, suggesting that the nanocomposites may successfully be used as a medical implant material, such as for middle ear prosthesis.

Author Contributions: Conceptualization, M.Z.; Investigation, M.Z., M.D. and E.M.; Methodology, M.Z.; Project administration, M.Z.; Writing—original draft, M.Z. Acknowledgments: This work was financially supported by the National Centre for Research and Development grant no: LIDER/154/L-6/14/NCBR/2015. The authors thank Elix-Polymers, in particular Aurelie Mannell and Ignacio Buezas Sierra for polymer delivery. Conflicts of Interest: The authors declare no conflict of interest.

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