Targeting biofilm superhydrophobic coating to reduce microbial accumulation on titanium surface
João Gabriel Silva Souza1, Jairo Matozinho Cordeiro1, Martinna Bertolini2, Amanda B. de Almeida1, Raphael Cavalcante Costa1, Francisco H. Nociti Junior1, Elidiane
Cipriano Rangel, Valentim Adelino Ricardo Barão
1 Piracicaba Dental School, University of Campinas (UNICAMP), Piracicaba, São Paulo, Brazil.
2 University of Connecticut School of Dental Medicine, Farmington, CT, USA. 3 São Paulo State University (UNESP), Engineering College, Sorocaba, Brazil.
ABSTRACT
Polymicrobial infections are one of the most common reasons for implanted biomaterials failure. Thus, studies have been proposing the use of superhydrophobic coatings due to its non-fouling properties in order to reduce microbial adhesion on biomaterials. Therefore, the aim of the current study was to develop, for the first time, a one-step technique using low pressure plasma technology to create a biocompatible superhydrophobic coating on titanium (Ti) surface to reduce polymicrobial biofilms formation. Based on this, we developed a superhydrophobic coating on Ti by glow discharge plasma technology using Ar, O2 and HMDSO gases,
and physical chemical and biological characterization were performed. Data analysis showed that newly developed coating presented: i) increased significantly roughness and hydrophobicity (contact angle over 150°) (p<0.05), ii) enhanced corrosion resistance (p<0.05), iii) slightly altered protein adsorption composition at proteomic level and iv) reduced microbial (bacterial and fungal) adhesion and biofilm formation (p<0.05). Furthermore, coated surfaces did not affect fibroblast viability and proliferation. Thus, this new superhydrophobic coating developed by one-step glow discharge plasma technique is a promising biocompatible strategy to reduce microbial adhesion and biofilm formation on Ti surface.
INTRODUCTION
Titanium (Ti) is the main material used for the manufacture of orthopedic and dental implant devices due to its excellent biocompatibility and biomechanical properties (Elias et al., 2008; Özcan and Hämmerle, 2012). However, abiotic surfaces may serve as a substrate for microorganism adhesion and biofilm colonization, triggering local infections in the surrounding tissues (Arciola et al., 2018). In the oral environment, implant surfaces are immediately coated by proteins adsorbed from saliva, which are responsible to mediate different biological processes (Kalasin et al., 2009; Rabe et al., 2011). In addition, microorganisms from the oral microbiome will colonize pellicle-coated surfaces via adhesion-receptor leading to biofilm formation and accumulation (Nobbs et al. 2009). Microorganism growing in biofilms present higher antimicrobial resistance and increased metabolism (Costerton et al., 1995; Flemming and Wingender, 2010; Bowen et al., 2018), being a critical virulence factor in the pathogenesis of bacterial infections (Bowen et al., 2018). Polymicrobial infections due to biofilms are the main reason for dental implant failure (Berglundh et all., 2018), and it has been associated with an exacerbated inflammatory response and tissue destruction (Nguyen Vo et al., 2017). Although host epithelium and innate immunity will activate inflammatory mediators in response to oral pathogens, this pathway can even enhance a transition process for a more aggressive and multidrug- resistent biofilm (Marsh and Devine, 2011), which is challenging to be treated due to wide microbiological composition and structure (Flemming and Wingender, 2010; Arciola et al., 2018). In order to improve potential antibacterial properties of such Ti- made implantable devices, biocompatible surface coatings have been proposed.
Surface coatings on Ti has gained attention for dental implant applications and are promising approaches to control biofilm formation. Different technologies have been applied, such as glow discharge and electrolytic plasma, and photocatalytic materials (Ferraris and Spriano, 2016), showing promising results. Several antimicrobials, molecules, compounds and ions were already functionalized on Ti surface as biofilm-targeting approaches to reduce viable microorganisms in biofilms (Ferraris and Spriano, 2009). However, in general, such strategies have been unsuccessful as they may affect tissue health (Patil et al., 2019), only present short- term efficiency (Kaufman et al., 2008; Smeets et al., 2017). Additionally, bactericidal and/or bacteriostatic effect does not mean the complete eradication of biofilms and
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these approaches can leave remaining biofilm on the surface, which even containing dead cells, can still promote colonization by new pathogenic organisms (Koo et al., 2017).
Because microorganism adhesion is the first step for biofilm formation, previous studies have proposed physical-chemical modifications of Ti surface to reduce microbial attachment (Song et al., 2015). Among these, wettability is an important mediator of microbial adhesion, as molecules and microbial cells can adhere to surface through charge or hydrophobic interactions (Busscher et al., 2008; Nobbs et al., 2009), and therefore, superhydrophobic surfaces (contact angle greater than 150°) (Falde et al., 2016) have been investigated in regarding to its non-fouling property (Falde et al., 2016; Bartlet et al., 2018). Chemical vapor deposition (Tombesi et al., 2019), fluorine based polymer (Huang et al., 2015) and laser system (Truong et al., 2012) have been used to achieve superhydrophobic profile. However, these approaches are two-step techniques, since they are typically fabricated in a roughened surface, which is prebuilt before hydrophobic treatment (Lai et al., 2013). Superhydrophobic surfaces have been shown to affect human-cell adhesion and proliferation (Falde et al., 2016), to promote corrosion resistance, mechanical and thermal-stress resistance (Huang et al., 2015; Yoo et al., 2018), and to present the capacity to reduce microbial adhesion (Zhang et al., 2013; Hwang et al., 2017). Interestingly, the microbiological effect of superhydrophobic films on Ti surface have been demonstrated for specific bacterial species (such as Staphylococcus aureus and Escherichia coli) and, in special, only during the process of initial adhesion (Fadeva et al., 2011; Hwang et al., 2017; Chang et al., 2018), and one may assume that by affecting only specific microorganisms at the initial stages of biofilm formation superhydrophobic films may not represent a promising strategy to deal with mature polymicrobial biofilms that naturally occur in oral infections. Moreover, some cell cytotoxicity has been described for superhydrophobic films on Ti using laser oxidation technique (Chang et al., 2018) due the chemical compostion of film used. Therefore, to the best our knowledge no previous study has developed a biocompatible superhydrophobic coating on Ti aiming at successfully reducing biofilm formation for dental implant application at transmucosal abutment connections level.
In the current investigation, it was hypothesized that a one-step and non- thermal technique created by glow discharge plasma technology using gases at low pressure to modify Ti surfaces would reduce microbial adhesion and biofilm
formation. In addition to the potential ability of superhydrophobic films to affect biofilm formation on Ti surfaces, it was also investigated whether such films would affect fibroblast cell cultures and materials electrochemical stability.
2 MATERIALS AND METHODS