Polymeric nanocomposite biodegradable based on

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materials for bone and dental reconstruction [12] because it has excellent .... The films were stored in desiccators to keep their stability and to avoid potential ... 96-well plates, and cultured at 37°C in a humidified atmosphere of 5% CO2 95% air for ... supplemented with 10% FBS at 37°C and pH 7.2 for 24 h under static ...
Polymeric nanocomposite biodegradable based on agar matrix modified with porous hydroxyapatite or titanium dioxide fillers from biotemplates route N.L.V.Carreño1*, V.C Duarte1, A.M Barbosa1, C.O Avellaneda1, F.F Demarco2, F.Nedel2, E. Piva2, R.G Lund2, R Rhodes3, F L M Sam3 and S R P Silva3 1

Material Engineering, Technology Development Center, Federal University of Pelotas, Pelotas, RS, CEP 96010-610, Brazil

2

Post-Graduate Program in Dentistry, Federal University of Pelotas, Pelotas, RS, Brazil

3

Nano-Electronics Centre, Advanced Technology Institute (ATI), University of Surrey, Guildford, GU2 7XH, UK *e-mail: [email protected]

Abstract

In this paper, biodegradable composites were developed from agar (a polysaccharide derived from seaweed) to which were added fillers containing different concentrations of titanium dioxide (TiO2) or hydroxyapatite (HA). The fillers were synthesized by adapting the template method; a resin containing either TiO2 or HA precursors was deposited on the surface of cellulose membranes, resulting in the formation of hollow structures with high porosity. These materials were subsequently silanized to improve the affinity between filler and matrix by the formation of hydrogen bridges, thereby reducing the interfacial tension. The fillers were dispersed within the agar matrix by the solution intercalation process at concentrations of 0.5%, 1% and 2%, resulting in materials possessing high transparencies and adhesion to the mold surface. The materials were characterized by scanning electron microscopy (SEM) and Raman spectroscopy, which confirmed the presence of TiO2 and HA filler structures within the polymer matrix as well as their dimensions and degree of dispersion. Cytotoxicity assays found that the addition of the fillers does not result in increased inhibition of cell growth. The antimicrobial activity of the films was evaluated by the diffusion test in liquid over time, in which the HA composite demonstrated antimicrobial activity. Mechanical testing demonstrated a significant increase in the modulus of elasticity of the composite material compared with matrix control film formed by the agar. Based on these promising results, it is felt that this material bears further investigation, particularly considering large number of potential applications for biodegradable, antimicrobial biopolymers reinforced with TiO2 or hydroxyapatite.

Keywords: Agar, biodegradable polymers, porous fillers, Hydroxyapatite, Titanium dioxide.

Introduction

Global trends point to scientific advances in the development of new materials and highlight the importance of using raw materials from renewable sources. Concern for the environment have increased in the last decades the interest in study biodegradable polymers, which can be used as renewable resources, sustainable systems adapting to and reducing volume of waste synthetics polymeric [1]. Biodegradable films and coatings can be synthesized from natural polymers of animal or vegetable origin, such as polysaccharides, lipids and proteins being perfectly biodegradable, that is, safe for the environment [2]. There are also many biopolymers extracted from marine algae which have found applications within biotechnology [3]. The composite developed during this study uses a matrix based on agar (a hydrocolloid extracted from marine algae) which is used widely in food and pharmaceutical industry. Despite this few works have been conducted or reported using agar in the preparation of films. These polymers usually exhibit poor mechanical properties (such as elastic modulus and mechanical resistance) when compared to other materials. One way to improve the properties of polymeric systems is incorporate highly rigid particles into the polymer matrix. Introduction of particles with micrometric dimensions to polymeric resin has been a practice used in the polymer industry. For changing mechanical, thermal and electrical properties of the material, the volumetric fraction of reinforcement required is high (about 40-60%). However, this may negatively affect some properties of the polymer matrix as processability and density [4, 5]. Recently research into nanomaterials research has made it possible to process composite materials with a significantly reduced filler concentration (usually below 5%) [6]; the interaction between polymeric chains and fillers results in a composite possessing superior properties compared to the pristine polymer, and (depending upon the choice of filler used) can also improve the functionality of the material, providing the possibility of its use in novel applications [7]. When considering a suitable filler, is important to select a material has the characteristics that are advantages for the intended purpose, for example heat resistance or chemical reactivity. In this study titanium dioxide is used because it is highly compatible biomaterials and functionals, used widely as an antioxidant in bone implants of titanium metallic [8-10]. TiO2 also releases free radicals when exposed to UV light,

resulting in the decomposition of organic material and bacteria on the surface. Finally, it has a number of other attractive properties, being low cost, nontoxic, and highly stable in aggressive environments, including within the human body [11]. In another study, the authors report that TiO2 and apatite dispersed in an acrylic resin demonstrated that both materials possess strong antifungal activities [9]. A bioceramic HA is used in this work to be one of is one of the most frequently used materials for bone and dental reconstruction [12] because it has excellent compatibility with bone and high osteoconductivity [13]. In comparison with the bulk material, the characteristics of nanoscale-HA (such as grain size, wettability and pore size) may control the interaction with proteins (adsorption configuration and bioactivity) in addition to modulating an improved adhesion of osteoblasts and long term functionality [14]. Several authors recommend use of particulate hydroxyapatite with an array of materials that constitute bonding agents, biocompatible and resorbable, responsible for preserving to migration of ceramic particles until incorporation into the tissue, minimizes the displacement of particles and facilitates handling and adaptation of material [15-19]. Hydroxyapatite is also used in the present work because it is a catalyst in decomposing of pollutants [20]; presents high capacity to remove heavy metals from waters and soils contaminated [21] which increases the possibilities of application of research material developed. So developing composites based on materials such as TiO2 and HA represents a creative alternative to researching new materials, and enables the development of innovative industrial applications. The aim of this study was to develop and investigate the composite polymeric consisting of a biodegradable matrix based on agar reinforced with different concentrations of either titanium dioxide or hydroxyapatite, defining compositions that present mechanical strength, low cytotoxicity and antimicrobial activity which allow exploiting these materials in biotechnological researches. These materials may also represent an alternative to replace of petroleum derivatives in production of polymeric compounds. Moreover, this is an area that is in constant development and provides an alternative for research of new materials allowing the development new industrial applications.

Experimental

Chemicals and reagents Agar and Tetraethyl orthosilicate (TEOS) 98% were obtained from Sigma Aldrich, Sorbitol 70% and Ethanol Absolute 99.5% were obtained from Synth, Glutaraldehyde 50% was obtained from Vetec. TiO2 and hydroxyapatite particles were synthesized by the template method, at temperatures of 600°C and 800°C respectively [22]. The immortalized mouse fibroblast cell line (NIH/3T3) cells were obtained from the Rio de Janeiro Cell Bank (PABCAM Federal University of Rio de Janeiro, RJ, Brazil); Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and WST-1 were purchased from Cultilab (Campinas, Brazil), Gibco (Grand Island, NY, USA) and Roche (Mannheim, Germany), respectively.

Fillers and Polymer composite preparation The fillers were obtained by an adaptation of the template method [22]. Calcium nitrate (Synth) and ammonium phosphate dibasic (Synth) were mixed at 80°C to complete homogenization and stirred with presence of CA (Aldrich), to posterior addition of Ethylene glycol (Aldrich). A Citric acid/metal ratio of 4:1 (mol) was used. The viscosity of our resin was keep at slightly less than water. Resin precursor obtained were subsequently deposited onto organic microporous membranes cellulose nitrate (pore diameter 0.2 µm) by means of a spin coating technique, selected due to uniform deposition of the resin onto substrate. Then, the material was subjected to heat treatment for two hours at 800°C in air atmosphere. In order to obtain the TiO2 fillers, the resin of TiO2 was obtained by addition of citric acid to titanium isopropoxide (Across) in proportion 3:1, distilled water added to the mixture and dissolution of the reagents by heating (150°C) and stirring, the residual resin was annealing at 600°C in air atmosphere.

The polymer composite was prepared as follows: In the first stage: TiO2 or HA, with corresponding concentrations of 0.5, 1 and 2 wt% of agar was added to 10 ml of ethanol absolute, followed by 10% this volume of TEOS (surface treatment by silanization) [23-25]. Any excess material was evaporated in the oven at 70°C. The silanization treatment aims to improve the chemical affinity between the particles and the polymer matrix through the formation of hydrogen bridges, resulting in the reduction of interfacial tension and thereby improving adhesion (Figure 1a). The methodology for obtaining the composite is referred to as a "solution intercalation", where in both particles and polymer are dispersed (or, in the case of the polymer, dissolve) and mixed strongly to promote association. The solvent is subsequently evaporated, to form the polymeric composite [26]. In order to achieve a suitable dispersion of nanoparticles, the material was sonicated in 180 mL of DI H2O using an ultrasonic probe (Sonics Vibra Cells) at a frequency of 40 kHz. Samples containing concentrations of 0.5 and 1wt% were sonicated for 30 minutes, while concentrations of 2% were sonicated for an hour. At this point, 3 g of agar was measured out and added to the dispersion, and the mixture was held at 100oC under stirring until the agar had completely dissolved. At this point, 1.5% sorbitol and 0.001% gluteraldehyde, plasticizers, and crosslinking agents were added to the vessel. The solution was poured warm (100°C) into a refractory container with internal dimensions of 15 cm x 27 cm. The material was held under ambient conditions (temperature 22°C) on a flat surface until complete composite polymerization (15 days). All of the composites obtained were transparent and continuous with a thickness of 0.1 mm. The films were stored in desiccators to keep their stability and to avoid potential contamination [27-29].

FIGURE 1: Scheme of porous and hollow fillers synthesis of TiO2 and HA from template with silane coupling agent (a); SEM image of the TiO2 (b) and HA (c) fillers. Images Scanning electron microscope (SEM) were obtained on a FEI Quanta 200 (FEI).

Filler and Polymer composite characterization The structural analysis of the hollow fiber ceramic membranes was carried out by X-ray diffraction (XRD; Shimadzu, model 6000), with CuKα radiation (λ = 1.5406 Ǻ), operating at room temperature. Scanning electron microscope (SEM) images were obtained on a FEI Quanta 200 (FEI). The surface of the film was covered with 30 nm of gold to prevent charging during analysis. Raman spectra were obtained on a Renishaw System 2000 micro Raman spectrometer (Renishaw) with a laser excitation line at 782 nm.

Cytotoxicity determination of particles (fillers) and composites

Cell Culture NIH/3T3 cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cells were cultured at 37°C in a humidified mixture of 95% air and 5% CO2 as previously described [30-32]. Experiments were carried out with cells in logarithmic growth phase.

Cytotoxicity determination of particles (fillers) Cells were seeded at a density of 2 x 104 cells per well in a volume of 100 µL in 96-well plates, and cultured at 37°C in a humidified atmosphere of 5% CO2 95% air for 24 h before evaluating via a WST-1 assay. Silanized TiO2 and HA were mixed in DMEM culture medium supplemented with 10% FBS. This was adjusted to a desired concentration of 4% solution before an equal volume of the dispersed TiO2 and HA fillers was added, bringing the total final concentration to 2%. The cells were incubated with particles of both materials for 24 h and 48 h. After this time, 20 µL WST-1 (Roche, Mannheim, Germany) was added to the medium of each well and the plates were incubated for a further 2h. At this point, 100 µL aliquots were removed from each well and the optical density at 450 nm was determined in a microplate reader (Thermo TPplate reader, Thermo Fisher Scientific) as previously described [30]. All observations were validated by at least two independent experiments for each experiment and analyzes were performed in triplicate.

Cytotoxicity determination of composites Samples of polymer composites with various concentrations of fillers HA or TiO2 (0.5%, 1% and 2%), respectively, were prepared in a ratio of 91.6 mm2 of surface area per milliliter cell culture medium following the recommendations of the standard ISO 10993-12 [33]. The samples were then sterilized by exposure to UV (ultraviolet) for 40 min on each side of the film, and pre-incubated in DMEM culture medium

supplemented with 10% FBS at 37°C and pH 7.2 for 24 h under static conditions as described previously [34, 36]. Cells were seeded under standard conditions at 37°C for 24 h. The sample of each polymeric material maintained in the medium for 24 h was removed and 200 µL aliquots was incubated for a further 24 h. After this period 20 µL of WST-1 (Roche, Mannheim, Germany) was added to each well and the plates were incubated for 2 h. 100 µL aliquots were removed from each well and the optical density at 450 nm was determined in a microplate reader (Thermo TP-plate reader, Thermo Fisher Scientific, Waltham, MA, USA). Percent inhibition of cell growth was determined as follows: the inhibition rate = (1 - Abs492treatedcells/Abs492control cells) × 100 [34]. All observations were validated by at least two further independent experiments and analyzes were performed in triplicate.

Antimicrobial activity determination of composites Inoculum Standardization We used the Gram-negative bacterial strain E. Coli (Escherichia coli). Bacterial cells were grown in TSA (Trypticase Soy Agar) for a period of 24 h under aerobic atmosphere at 37°C. Upon reaching the stationary phase of growth, the bacterial inoculum was placed in TSB (Tryptone Soy Broth) and subsequently standardized spectrophotometer in exponential phase 107 CFU / mL.

Antimicrobial activity of the composite over time The rate of diffusion of materials solids and hydrophobic is reduced in solid media, in addition, the surface tension offered by these materials can mask the results of tests involving the formation of inhibition zones. Therefore, this experiment was conducted in order to determine the effect of immersion time of the composite and consequently the distribution of these materials in the growth medium on the microbial activity thereof [35]. An experiment was designed to determine the effect of the immersion time of the film in the growth medium on its antimicrobial activity. In brief, samples with 0.01 g and 0.5 cm2 of film of each material, previously sterilized by UV, was immersed in 4

mL of TSB for two time periods (48 h and 120 h). After the corresponding time, the films were removed from the solution and 40 µL of standard bacterial inoculum of E. coli in exponential phase (10 7 CFU/mL) was inoculated into the tubes and incubated at 37 °C for 24 h. Serial dilutions to 107 with peptone water were made and 20 µL of each dilution plated in Petri dishes with 15 mL of TSA culture medium. Colonies were counted after incubation at 37°C for 24 h. Counts were performed in triplicate. Separately, tubes with 4 mL of TSB and 0.01 g of film were stored for 120 h at 37°C, after which the films were removed and 40 µL standard bacterial inoculum of E. coli in exponential phase (107 CFU/mL) was inoculated into the tubes and incubated at 37°C for 24 h. Serial dilutions to 107 with peptone water were made and 20 µL of each dilution plated in Petri dishes with 15 mL of TSA culture medium. Colonies were counted after incubation at 37°C for 24 h. Counts were performed in triplicate.

Mechanical characteristics The test for determining the tensile properties of the flexible structures (polymeric materials) involved the separation, at a constant speed, of the claws that fasten the ends of the bodies of the test piece (Figure 2a), registering throughout the trials the force or resistance to deformation of material (elongation). Tensile testing was performed on an Instron Universal Testing Machine, model E3000 (Instron, Massachusetts, USA), with traction velocity of 10mm/min and load of 50 N, according to ASTM D 638 (1989) and ISO 527-1 (1993), under standard temperature and pressure. The specimens for tensile tests were made according to ASTM D 638 (Figure 2b), and the tests performed after complete drying of materials.

FIGURE: a) Illustration of the specimen for tensile test with dimensions as set out in ASTM D 638 and ISO 527-1 b) Sample tensile test in a universal testing machine Instron.

Data Analysis For cytotoxicity assays data sets were analyzed using a two-way ANOVA followed by Tukey test for multiple comparisons. The microbiological data as analyzed using ANOVA and Student Newman Keuls complement to comparisons multiple. For mechanical assays data sets were analyzed using a one-way ANOVA followed by Tukey test for multiple comparisons. The significance level adopted was p