2.1

Part 1: literature review

Based on the classification proposed in Fig. 1 , where dental devices were classified as implantable and non-implantable and sub-classified on the field of specialization and the contact with specific environments, searches were conducted using PubMed search engine for devices subdivided into 7 groups namely:

• –

  Implantable for oral surgery

• –

  Implantable for implantology

• –

  Implantable for periradicular surgery

• –

  Non-implantable for restorative dentistry-prosthodontics

• –

  Non-implantable for restorative dentistry-operative dentistry

• –

  Non-implantable for restorative dentistry-endodontics

• –

  Non-implantable for orthodontics

For all the groups the following keywords were included: antimicrobial, antibacterial, dental/oral. Following this preliminary search, other terms that were more specific to each group were inserted to reduce the number of articles retrieved. Articles within a 10-year range were included i.e. , from 2008 to 2018. Articles not written in English, all review articles and articles where antimicrobials were added to the materials but the testing of other properties was undertaken were excluded. The key words and outputs are shown in Table 1 .

For the oral surgery all the five searches indicated in Table 1 were undertaken. For the Implantology group, the output of 695 articles was further filtered to select the most relevant papers: articles with materials tested without specifying a dental application, use of bacterial strains not specific to oral applications, biomaterials not used to manufacture dental implants, or coatings for dental implants, materials/pieces not used for implantation in bone or to test/report medical treatment associated to dental implants or cases in which the antimicrobial testing was not significant, were excluded. These exclusion criteria led to the selection of 68 articles.

For the periradicular surgery the introduction of the term root-end reduced the search to 17 articles and six fulfilled the criteria. For the non-implantable materials, the introduction of specific key words also reduced the number of articles retrieved and all these were read and the exclusion criteria applied as indicated in Table 1 .

The details per output included the author’s first name, date of publication, full citation, the materials tested, the strains used, whether the testing was performed in vitro , ex vivo or in vivo and also details of any other testing performed.

2.2

Part 2: recommendations for antimicrobial testing

Based on the results of the literature review, recommendations for antimicrobial testing of dental devices are given. These recommendations will take into consideration pre-testing parameters such as ageing and the method of pre-sterilization of the device that needs to be noted in every test. Furthermore, the selection of the strain for both mono-species and multi-species testing and recommendations for the microbiological testing of the devices are made, and include in vitro , ex vivo and in vivo testing. Suggestions for integrated microbial and cellular testing and the need of material characterization including chemical characterization methods for bulk, surface, elution and degraded material and physical and mechanical characterization are also made.

3.1

Implantable devices

3.1.1

Periodontal and oral surgery

In the periodontal and oral surgery materials, after excluding implants, only 14 papers were selected and a further 2 were removed due to hits in more than one search term for the same paper. A total of 12 papers from the last 10 years, were therefore reviewed. Most papers investigated materials developed for use as guided tissue regeneration (GTR) or guided bone regeneration (GBR) membranes (10 papers) while only one of the reviewed papers investigated a scaffold and one investigated microspheres. None of the reviewed articles studied other types of grafts, gels or devices used in oral and periodontal surgery. In vitro methods are the most common method of antimicrobial testing, with variations of the agar diffusion test were the most commonly used antimicrobial test (6 publications) and most of the studies (8 publications) only tested for antimicrobial properties using one antimicrobial test. None of the reviewed literature used ex vivo methods and only one study used an additional in vivo test on Beagle dogs as an animal model. Nonetheless, this model was only used to assess histopathological tissue changes and not antimicrobial efficacy. Fusobacterium nucleatum and Streptococcus mutans were the most common microorganisms used for antimicrobial testing (4 publications each) and all testing was done using single species infections. Although 7 different publications tested antimicrobial properties against multiple organisms, only one publication used 2 strains of the same microorganism.

Eight out of the 12 reviewed studies included material characterization, as well as biological testing in addition to the antimicrobial testing. And out of all of the reviewed literature only 2 articles used or referred to the commercially available equivalent in their publication. Lack of standardization in antimicrobial testing of materials used in oral and periodontal surgery was observed with a large variation of organisms being tested and in one case the strains used were not even described. There is mostly disparity when testing for bone regenerative materials as some studies only test for typical osteomyelitis causing organisms such as Staphylococcus aureus , but do not include other typical oral microorganisms in their testing.

3.1.2

Dental implants

For the implantable devices, among the 68 papers related to the dental implants sub-group, 52 reported antimicrobial studies involving bacteria single species testing and 16 used bacteria multi-species together for the testing. Porphyromonas gingivalis (24 papers), Streptococcus mutans (19 papers), Streptococcus sanguinis (15 papers) and Streptococcus gordonii (10 papers) were the most used bacteria strains. All the 16 studies implementing more than one bacterial strain in the testing investigated biofilm formation. 55 papers reported in vitro microbiological testing, three of which also presented an in vivo study. One paper was dedicated only to in vivo assessment. The in vitro methods were dominated by colony forming unit (CFU) counting, followed by minimum inhibitory concentration (MIC) and MTT and live/dead. Twenty-seven of the papers included other biological assessment and 32 papers included characterization of the materials. Commercially pure titanium was by far the most common material tested (54 papers). Titanium alloy (Ti6Al4V, 4 papers), and hydroxyapatite, PEEK, zirconia, and PLGA were almost single instance materials (one or two papers).

3.1.3

Endodontic corrective surgical materials

The root-end filling materials were only investigated for antimicrobial characteristics in 6 papers in the last 10 years. The papers investigated specifically hydraulic calcium silicate cements including mineral trioxide aggregate (MTA) and Biodentine and some antimicrobial additives such as silver nanoparticles and hinokitiol. In 5 papers in vitro methods including agar diffusion and the microdilution broth methods were used and only 1 paper used dentine substrate and conducted intratubular infection method thus using an ex vivo method. Enterococcus faecalis was the most frequent tested microorganism (6 papers); either as the only microorganism employed (4 papers) or with other oral bacteria. Two of the papers used more than one bacterial strain but only 1 used a polymicrobial culture. Only 1 paper carried out material characterization and biological testing of the materials.

3.2

Non-implantable devices

3.2.1

Restorative dentistry — prosthodontics

In the group of prosthetic restorative materials, after search specifications were applied, only 4 articles were found that studied antimicrobial characteristics in the fixed prosthetic field — one in metal crown, and 3 in ceramics. The antimicrobial properties of surface treatments of each material, such as plasma, silver nanoparticles coating and polarization-induced treatments were studied. These in vitro studies used direct contact and CFU counting. Streptococcus mutans was used in all papers, either as the only tested microorganism (2 papers) or with other oral bacteria. Two papers that described new surface treatments also carried out a variety of structural and adhesion studies and biological testing.

On the contrary, in the removable prosthetic field, a considerable amount of work in the antimicrobial testing of acrylic dentures – 49 articles were found. So, this group was further subdivided into three sub-groups: drug loaded acrylic resins, drug exposure acrylic resins ( e.g. testing after disinfection) and drug coated acrylic resins ( e.g. denture adhesives).

Most of these studies tested for Candida species – mostly Candida albicans , which was referred to as the primary cause of denture-related stomatitis. Single Candida albicans was tested in 28 studies, 7 cultured from clinical samples. Streptococcus mutans strains were the more relevant bacteria for antimicrobial tests (10 studies). Two studies tested only for Staphylococcus aureus and Pseudomonas aeruginosa , both focusing on bacteria that can form denture biofilm and can cause other systemic diseases like respiratory pneumonia. Five studies tested total clinical biofilm and one was a randomized clinical study.

Most of the in vitro testing consisted of only one method, usually direct contact assays ( e.g. CFU counting by agar plate culture method) and biofilm evaluation for bioactivity (dead/live staining – XTT reduction), biomass (crystal violet assay) and scanning electron microscopy (SEM). Nevertheless, at least 19 papers used two or three combined methods.

A considerable amount of studies of drug loaded acrylic resins also included material characterization and biological studies. Two studies of drug exposure or drug coated acrylic resins also included biological or material characterization evaluation.

3.2.2

Restorative dentistry — operative

In the group of restorative dentistry and operative materials/devices the initial search revealed also a great number of articles. In the sub-group for amalgam 64 were identified, for composite resin 59, for glass ionomer 329, while for luting cements 132. After the application of inclusion and exclusion criteria, the secondary outcomes limited the articles to a total number of 135. More specifically, 7 articles for amalgam, 24 for composite resin, 93 for glass ionomer and 11 for luting cements. From the total 135 studies, 15 were in vivo , 6 ex vivo while 126 were in vitro or in combination with the rest. In 104 of the 135 papers Streptococcus mutans was the strain tested, primarily as single and in a significant % of papers exclusively (52/135). Characteristically, in the 93 papers for glass ionomer cements 4 papers did not include S. mutans but instead other species ( L. casei (n 2), A. viscosus, C. albicans, S. milleri and E. faecalis ). In one study 11 species were involved ( S. mutans , S. salivarius , S. sorbinus , A. viscosus , A. naeslundii , A. odontolyticus , L. acidophilus , L. caesi , P. gingivalis , Prevotella intermedia , A. actinomycetemcomitans ), one study used 6 strains, three studies 5 while four studies 4 strains. Similarly, in the reviewed 24 papers for composite resins S. mutans was used in 16 (in 10/16 exclusively), in one study 6 different strains ( P. gingivalis, P. intermedia, P. nigrescens, A. actinomycetemcomitans, F. nucleatum and E. faecalis ), in one study 4 ( P. gingivalis, P. intermedia, A. actinomycetemcomitans and F. nucleatum ), while in 4 studies a dental plaque microcosm biofilm model was used.

For the in vitro testing, the methods usually applied were direct contact assays ( e.g. CFU counting), ADT, BAT, ABCT and biofilm evaluation for bioactivity (dead/live staining – XTT reduction), biomass (cristal violet assay), adhesion assays, MTT (in 10 studies) or MTS, fluorescence or confocal imaging and SEM. NMR was used in one study and very limited ELISA and rtPCR (in 4 studies). Ageing procedures were applied in 7 studies and thermocycling in only 1.

3.2.3

Restorative dentistry — endodontics

For the non-implantable devices, sub-groups restorative dentistry and endodontics after the inclusion and exclusion criteria were applied, the quantity of papers reduced significantly. Only 9 papers tested antimicrobial characteristics of pulp capping materials. From the 9 papers, 5 used the agar diffusion test to assess the antimicrobial activity; one evaluated the leachate rather than the materials themselves. The rest of the in vitro studies used the MIC, direct contact test and CFU counting. In general, the materials were tested using a variety of oral bacteria and bacteria related to dental caries. One paper used E. faecalis which is more linked to failed root canal therapy. Two of the papers included biological testing. None of the papers characterized the materials used. One of the papers was a clinical study isolating bacteria in samples of carious dentine in vivo . This was the only study that used a multispecies biofilm.

Inside the root canal, searches for endodontic filling materials yielded 40 articles that fell within the search specifications. Further searches for sealers and gutta-percha, resulted in finding more related papers. There were 13 papers published on antimicrobial activity of gutta-percha. Nine papers out of the total 13 that met the criteria used single species of E. faecalis for testing, 2 used more than 1 species but tested separately and only 2 used multispecies biofilm. Dentine substrate was used in 8 of the 13 papers. Although gutta-percha was tested in these studies, it was used either as control to test the efficacy of sealers used or else modified versions were investigated rather than the gutta-percha per se. Thus, although this material is used extensively in clinical endodontics, it is not tested very rigorously. For Resilon, another core filling material, only 2 papers met the criteria. Both papers used a single species of E. faecalis and dentine as a substrate was used in the antimicrobial testing. All the papers reviewed did not include any material characterization or any biological testing.

A considerable amount of work has been published in the antimicrobial testing of root canal sealers. Several prototype sealers have also been tried. So, this group was further subdivided into two, clinically available sealers and experimental sealers. This was done to be able to differentiate the testing undertaken for the clinically available materials and the experimental ones. For the testing of the clinically available sealers, 53 out of 55 used a single species culture. Thirty four of the papers tested only one strain and 21 included other microorganisms associated with the endodontic infections and also Candida albicans. Most of the in vitro testing consisted of only one method. Three papers used both in vitro and ex vivo methods and 11 used only ex vivo methods that included dentine substrate. There were no in vivo studies. Three papers included material characterization and 3 biological testing.

For the experimental sealers, seven out of the eight papers used E. faecalis single microorganism for testing. All the methods employed were in vitro without using dentine substrate. Two studies included material characterization and 3 biological assessment of the materials. No literature on the antimicrobial properties of post materials has been found.

3.2.4

Orthodontics

A significant amount of work has been published in the antimicrobial testing of materials for orthodontic application, divided in two major fields: drug-loaded orthodontic cement systems ( e.g. resin-based or glass ionomer cements) and nanoparticles coated brackets/wires ( e.g. silver). For the evaluation, 54 of the papers used a single species with all of them except one opting for the strain Streptococcus mutans . The remaining papers included other microorganisms associated with the oral cavity ( e.g.Streptcoccus sanguinis , Porphyromonas gingivalis ) and also Candida albicans . Multi-species testing was performed in 6 papers which used human saliva as the inoculum resembling the microcosm oral biofilm environment. Additionally, in 1 paper, the presence of 16 g-negative periodontal-related microorganisms were tested on metallic brackets. The in vitro testing consisted mainly of direct contact assays ( e.g. agar diffusion assay) and biofilm evaluation ( e.g. dead/live staining, SEM). Two papers evaluating orthodontic brackets used ex vivo methods that included extracted maxillary premolars. There were 14 in vivo studies with all of them with humans except one using rats. Thirty-six papers included material characterization and 8 cytotoxicity studies.

3.3

Recommendations for standardization of antimicrobial testing

Based on the results of the literature review, it is clear that there is a lack of standardization of testing at all levels. Furthermore, data such as ageing and pre-sterilization if any is not always included. Most of the studies in each field of research did not include material characterization nor cell biological assessment to evaluate the interplay between the antimicrobial efficacy and damage to the host. The following recommendations are thus being proposed to alleviate this lack of standardization.

3.3.1

Pre-testing parameters

The following parameters need to be noted when undertaking antimicrobial testing of dental devices. These include the ageing of the specimens as well as the pre-sterilization method used, if any.

3.3.1.1

Ageing

Ageing of materials and devices refers to the gradual process in which the properties of a material, structure, or system, change (for better or worse), over time or with use, due to biological, chemical, mechanical or physical agents. Ageing affects the shelf life of a device and changes the device profile thus altering its characteristics. Artificial ageing may be necessary for a number of tests. If the material or device is artificially aged this must be done following standard methods. The accelerated ageing methods and the specific duration of the test should also be noted. For all devices, cycles of acidic/neutral/basic pH for a given time are necessary. In addition:

• –

  For implantable devices incubation in simulated body fluid at 37 ± 1 °C is recommended;

• –

  For non-implantable devices the dynamic ageing dependent on the specific application is recommended. This should follow specifications listed in ISO/TS 11405:2015 [ ].

3.3.1.2

Pre-sterilization

Pre-sterilization of the device is generally necessary prior to antimicrobial testing. Pre-sterilization should be performed following established standard protocols outlined in ISO standards and should be in line with the device use in the clinical setting. Importantly, the methods employed neither shall affect the material properties nor the antimicrobial properties. The ISO standards outline the use of ethylene oxide in ISO 11135-1:2007 [ ], radiation in ISO 11137-1:2015 [ ], moist heat in ISO 17665-1:2006 [ ], dry heat in ISO 20857:2010 [ ], low temperature sterilization and formaldehyde in ISO 25424:2009 [ ] and liquid chemicals in ISO 14160:2011 [ ]. It is important that all microorganisms are eliminated and tests should be conducted to ensure a microorganism free device prior to the start of testing as indicated in the relevant ISO standards [ , ].

3.3.2

Recommendations for antimicrobial testing

3.3.2.1

Description of the relevant strains

The testing can be performed using single or multiple microbial strains. The most relevant strains to be used for testing, categorized according to the material classification in Fig. 1 are listed in Table 3 . In each case there is only one strain suggested for use as first line material testing. The strains are chosen as they are native to the location where the material is being implanted. Since most of the oral infections are polymicrobial, it is also important to test the antimicrobial activity of the materials against multi-microbial cultures ( Table 4 ) as second line testing procedure. Non-implantable devices in contact with the oral mucosa should also be tested for the relevant strains of fungi, like Candida albicans using ATCC 10231 strain.

Table 3

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