{'fraction_non_alphanumeric': 0.027751792952915497, 'fraction_numerical': 0.011849080137199874, 'mean_word_length': 5.871451526513122, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Strategies to Reduce Biofilm Formation in PEEK Materials Applied to Implant Dentistry—A Comprehensive Review

Polyether-ether-ketone (PEEK) has emerged in Implant Dentistry with a series of short-time applications and as a promising material to substitute definitive dental implants. Several strategies have been investigated to diminish biofilm formation on the PEEK surface aiming to decrease the possibility of related infections. Therefore, a comprehensive review was carried out in order to compare PEEK with materials widely used nowadays in Implant Dentistry, such as titanium and zirconia, placing emphasis on studies investigating its ability to grant or prevent biofilm formation. Most studies failed to reveal significant antimicrobial activity in pure PEEK, while several studies described new strategies to reduce biofilm formation and bacterial colonization on this material. Those include the PEEK sulfonation process, incorporation of therapeutic and bioactive agents in PEEK matrix or on PEEK surface, PEEK coatings and incorporation of reinforcement agents, in order to produce nanocomposites or blends. The two most analyzed surface properties were contact angle and roughness, while the most studied bacteria were Escherichia coli and Staphylococcus aureus. Despite PEEK’s susceptibility to biofilm formation, a great number of strategies discussed in this study were able to improve its antibiofilm and antimicrobial properties.


Introduction
The diverse microbiome that harbors in the oral cavity plays an important role in health maintenance through the development of the immune response and inhibition of the pathogen colonization [1]. However, under certain circumstances, normal microbiota may be responsible for many oral diseases [2,3]. Oral dysbiosis triggers important changes, reducing the number of beneficial bacteria and favoring the growth of potential pathogens [4]. This is particularly worrying in susceptible individuals affected by periodontitis, a biofilm related disease characterized by alveolar bone resorption, which may lead to tooth mobility and tooth loss [5,6]. In fact, periodontal patients who were rehabilitated with dental implants are more predisposed to develop peri-implant diseases, for which poor plaque control also acts as a primary etiologic factor [7].
In a systematic review carried out in 2017, [8] patient-level data and implant-level data indicated that peri-implantitis was present in 9.25% and 19.83% of analyzed cases, respectively, while mucositis

Strategies to Reduce Biofilm Formation in PEEK Materials Applied to Implant Dentistry
A full strategy with inclusion and exclusion criteria, as well as the flow chart of selected studies, are available as Supplementary Data. From a total of 376 studies initially found during the literature search, 33 were chosen for full text reading based on titles. Thereafter, 31 studies fulfilled the inclusion criteria of this review. Tables 1 and 2 reveal comprehensive information on pure and modified PEEK, respectively.

Strategies to Reduce Biofilm Formation in PEEK Materials Applied to Implant Dentistry
A full strategy with inclusion and exclusion criteria, as well as the flow chart of selected studies, are available as Supplementary Data. From a total of 376 studies initially found during the literature search, 33 were chosen for full text reading based on titles. Thereafter, 31 studies fulfilled the inclusion criteria of this review. Tables 1 and 2 reveal comprehensive information on pure and modified PEEK, respectively.

Study Characteristics
Amongst the included studies, 5 involved in vitro associated to in vivo (animal) investigations, while 26 were restricted to in vitro studies. In vivo (human) studies did not fulfill inclusion criteria of this review. Regarding PEEK modification strategies, 6 studies analyzed pure PEEK compared to other materials [30][31][32][33][34][35] (e.g., titanium, silicon, gold, silver, zinc oxide, zirconia, silicon nitride) and none of them revealed special antibiofilm or antimicrobial properties of PEEK material. A total of 25 studies used strategies to reduce biofilm and bacterial colonization on PEEK, which were able to successfully confer either antibiofilm or antimicrobial properties, or both, to the material. Regarding applications aimed at the investigated materials, orthopedic, dental and the treatment of bone defects were the most commonly mentioned, followed by the development of biomaterials in general.

Microbiological Analysis
The most commonly investigated bacteria were Escherichia coli and Staphylococcus aureus, but other microorganisms such as Streptococcus sanguinis, Streptococcus oralis, Streptococcus faecalis, Streptococcus gordonni, Streptococcus epidermidis, Pseudomonas aeruginosa, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum, Enterococcus faecalis, Candida albicans, Actinomyces naeslundii, Streptococcus mutans and Staphylococcus epidermidis were also studied. Microbiological analysis was very heterogenic, and several methods were used, which are summarized in Tables 1 and 2. Among the included methods, it should be highlighted that the most recurrent ones were plate-counting, for the determination of average colony forming units (CFU/mm 2 ); Real-Time Polymerase Chain Reaction (RT-PCR) and Live/Dead cells analysis, followed by FE-SEM and confocal laser scanning microscopy; bacterial growth inhibition zone tests; crystal violet assays; longevity and stability of antibacterial activity and agar diffusion assay.

Microbiological Analysis
The most commonly investigated bacteria were Escherichia coli and Staphylococcus aureus, but other microorganisms such as Streptococcus sanguinis, Streptococcus oralis, Streptococcus faecalis, Streptococcus gordonni, Streptococcus epidermidis, Pseudomonas aeruginosa, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum, Enterococcus faecalis, Candida albicans, Actinomyces naeslundii, Streptococcus mutans and Staphylococcus epidermidis were also studied. Microbiological analysis was very heterogenic, and several methods were used, which are summarized in Tables 1 and 2. Among the included methods, it should be highlighted that the most recurrent ones were plate-counting, for the determination of average colony forming units (CFU/mm 2 ); Real-Time Polymerase Chain Reaction (RT-PCR) and Live/Dead cells analysis, followed by FE-SEM and confocal laser scanning microscopy; bacterial growth inhibition zone tests; crystal violet assays; longevity and stability of antibacterial activity and agar diffusion assay.

Discussion
Investigations have demonstrated that peri-implantitis is a heterogeneous infection, in which periodontopathogens and opportunistic microorganisms act simultaneously [62][63][64]. Moreover, the disease has been associated to specific immunological alterations on peri-implant crevicular fluid levels of proinflammatory, anti-inflammatory and osteoclastogenesis-related chemokines [65]. Several studies analyzed in this review [30][31][32][33][34][35] investigated biofilm and antimicrobial properties of pure PEEK, demonstrating that the polymer is susceptible to biofilm colonization. Within a context in which PEEK clinical applications in Implant Dentistry are increasing [28], strategies to modify its surface to enhance its antimicrobial/antibiofilm properties are crucial.
It becomes even more important to improve PEEK materials with the above-mentioned properties when considering that biofilms are organized polymicrobial communities that offer bacteria protection against environmental factors and antibiotic treatments [66,67]. In vitro analysis of submucosal biofilm samples of 120 peri-implantitis sites revealed that 71.7% exhibited bacterial pathogens resistance to one or more of tested antibiotics (clindamycin, amoxicillin, doxycycline or metronidazole) [68]. Therefore, the identification of compounds capable of inhibiting biofilm formation or disrupt biofilm organization emerges as an attractive alternative to avoid peri-implant related infections [69,70]. It is important to notice that this approach is not expected to completely eliminate biofilm formation, but it is a very effective way of modifying oral ecology instead, reducing the number of pathogenic bacteria and favoring the growth of mutualistic species. By doing so, the host organism is provided with just the necessary advantage to defeat the pathogens using its own resources.
Additionally, it is important to analyze PEEK surface properties and its influence on biologic systems. For example, the PEEK hydrophobic surface associated to its bio inertness is a major concern when prospecting for the expansion of its application in Implant Dentistry [28,71], as this type of surface typically reduces cellular adhesion and does not promote osseointegration [22]. Numerous modifications have been proposed to overcome those limitations, such as blending with bioactive particles such as titanium dioxide, hydroxyapatite and fluorapatite [72][73][74]. Interestingly, the present review exposed that some of those strategies showed the favorable additional effect of reducing biofilm formation [41,44,54]. For example, a very promising candidate to replace metallic implants is carbon fiber reinforced PEEK (CFRPEEK) [75], which has similar elastic modulus to the human cortical bone [22]. One of the studies included in this review [44] proposed a dual zinc and oxygen plasma immersion ion implantation to modify CFRPEEK. Despite the fact that this strategy made the surface far more hydrophobic (contact angle shifted from 66.6 • to 144.1 • after surface modification), it also improved both osteogenic and antibacterial activities, as evaluated through MC3T3-E1 and rat bone mesenchymal stem cell development and through Staphylococcus aureus, MRSA and Staphylococcus epidermidis inhibition [44]. Those findings provide positive perspectives of the development of PEEK surfaces enhanced with bioactive and antibiofilm properties, which is favorable for PEEK-based dental implant development.
Bone cell activity on the PEEK surface is very important to achieve proper osseointegration on dental implants, but considering that an imperative application for PEEK in Implant Dentistry is as implant abutments [76], the gingival sealing must be analyzed as well, since it provides protection to implants against infections by potential pathogens [10]. Among the studies included in this review describing strategies for PEEK modification through the incorporation of antibiofilm agents, the embedding of lactams through the PEEK sulfonation process is worth mentioning [45]. Lactams are compounds analogous to furanones, which were initially isolated from the algae Delisea pulchra, and had been proved to be effective against Streptococus mutans biofilms [77]. An in vitro study [26] demonstrated that PEEK sulfonation positively interferes with the ability of fibroblasts L929 to spread over the surface of the material [26]. This corroborates previous indications that PEEK sulfonation is a suitable process for the development of modified PEEK abutments with embedded antibiofilm compounds.
In addition to the mentioned in vitro studies indicating these strategies as promising approaches to develop clinical materials biofilm resistant, an in vivo (human) investigation also revealed that PEEK healing abutments did not affect important parameters of peri-implant health, such as marginal bone loss and soft tissue recession, during a three-month evaluation period [78]. Therefore, it seems plausible to associate PEEK inherent favorable properties with adequate strategies to maximize its biological properties and consequently achieve even better clinical outcomes in the near future.

Conclusions
Within the scope of the present review, it may be concluded that pure PEEK is susceptible to biofilm formation and that several strategies presented here are able to significantly improve its antibiofilm and antimicrobial properties. Those strategies include the PEEK sulfonation process, incorporation of therapeutic and/or bioactive agents in the PEEK matrix or on the PEEK surface, PEEK coatings and incorporation of reinforcement agents to produce nanocomposites and/or blends. Since the use of PEEK in Implant Dentistry is increasing, those modifications are necessary in order to enable patients to benefit from these new materials which present great potential to prevent infections. Therefore, it is expected that further in vivo studies, both in animals and humans, will make available PEEK-based dental implants and improved implant abutments for clinical practice applications. Funding: This project was supported by a grant from the ITI Foundation, Switzerland.