Elsevier

Journal of Dentistry

Volume 53, October 2016, Pages 64-72
Journal of Dentistry

An assessment of early colonisation of implant-abutment metal surfaces by single species and co-cultured bacterial periodontal pathogens

https://doi.org/10.1016/j.jdent.2016.07.013Get rights and content

Abstract

Objective

Numerous studies have proposed that smooth metal surfaces reduce initial bacterial attachment in the establishment of an early biofilm formation. However, these studies have largely examined single bacterial species, which are not always relevant as pathogens identified as initiators of inflammatory peri-implantitis. This study investigated the adherence of four periodontally-relevant bacterial species to implant and abutment surfaces in current clinical use.

Methods

Discs of polished cobalt chromium (CoCr-polished) and milled titanium (Ti-milled), representing two clinically relevant surfaces, were prepared and surfaces were characterised. Bacterial species Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia and Aggregatibacter actinomycetemcomitans were cultured to mid-log or stationary growth phase. Co-cultures of P. gingivalis, F. nucleatum and P. gingivalis, F. nucleatum, Pr. intermedia were similarly prepared. Bacteria were inoculated onto discs for 2 h, stained with a live/dead fluorescent stain and percentage bacterial coverage was calculated by confocal microscopy and image analysis.

Results

CoCr-polished discs had smooth surfaces with gentle valley structures, whilst Ti-milled discs had sharp edged peaks. Both discs demonstrated a partial wetting ability capable of initiating bacterial adhesion. P. gingivalis, F. nucleatum and co-cultures, at both mid-log and stationary concentrations, demonstrated equally high coverage of both the smooth CoCr-polished and the rougher Ti-milled metal surfaces. Pr. intermedia and A. actinomycetemcomitans demonstrated lower surface coverage which was slightly higher for Ti-milled.

Conclusion

Variability was noted in the adherence potential for the respective periodontal pathogens examined. Particularly high adherence was noted for P. gingivalis and F. nucleatum, despite the manufacture of a smooth surface.

Clinical significance

Both surfaces studied may be used at implant-abutment junctions and both possess an ability to establish a bacterial biofilm containing a periodontally-relevant species. These surfaces are thus able to facilitate the apical migration of bacteria associated with peri-implantitis.

Introduction

Peri-implant disease is regarded as an infectious disease, whereby oral pathogens, usually gram-negative micro-organisms, initiate host inflammatory responses [1], [2]. Peri-implant mucositis describes the inflammation of the soft tissues surrounding the implant that can progress to peri-implantitis and involves apical migration of the bacteria, effecting inflammatory destruction of the supporting bone and potential loss of the implant [3]. Approximately 1000 bacterial species have been identified in the oral cavity, of which approximately 10 species have been implicated as periodonto-pathogens, that have a strong association as initiators of the host response leading to peri-implant disease [4], [5]. Of these periodonto-pathogens significant research literature is available to support pathogenic roles for opportunistic bacteria Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas gingivalis [reviewed by 4]. Despite P. gingivalis being identified as a low abundance biofilm species, evidence suggests that it can orchestrate inflammatory destruction [6] and often co-aggregates together with Pr. intermedia [7]. The adherence of periodonto-pathogens to dental implants or abutments exposed to the oral cavity can lead to the transfer of bacteria to the trans-gingival region around the implant/abutment leading to inflammation of the soft tissues. The elimination of biofilms from implant surfaces is thus a significant objective in preventing peri-implant disease.

Virulence factors, such as lipopolysaccharides and other bacterial surface proteins released from the bacteria, are now well regarded as initiators of inflammatory destruction via activation of the innate and acquired immune response [8], [9], [10]. Continued burden of bacterial virulence factors can trigger an excessive immune response and an imbalanced homeostatic response of the resident connective tissue cells, leading to net activation of tissue degradation pathways, such as osteoclast activation and destruction of the supporting bone. In addition, peri-implantitis progression is determined by the nature of the host immune response to these specific microorganisms within the biofilm. In susceptible individuals, inflammatory destruction is exasperated by genetic and/or systemic factors such as neutrophil dysfunction, osteoporosis, diabetes and smoking. Environmental factors, such as macro/micro occlusal stresses placed on the supporting bone can also lead to inappropriate immune responses, particularly if long-term incorrect loading of the implant occurs, which may increase susceptibility to inflammatory bone loss. These conditions can only be resolved via eradication of the bacterial pathogens as initiators of the host response [3]. If implants have been used as replacements for teeth lost as a consequence of periodontal disease, then similar compromising factors would be expected to be present in allowing the progression to peri-implantitis.

The design and production of implants and abutments follow BS EN ISO standards. Whilst genotoxicity studies affecting bacterial mutations are performed, bacterial adherence is not currently a requirement and thus no standard GLP protocol has been established. Adherence of bacteria is dependent upon the physiochemical nature of the cell wall, fimbriae attached to the bacteria and the surface characteristics of the metal surface. Previous studies have indicated that bacterial adherence can be affected by crystallinity, depth of the oxide layer and surface roughness [11]. Published data suggests that cobalt chromium metal surfaces demonstrate reduced bacterial adherence compared with titanium, although this is not significant for all studies [12], [13], [14]. With regards to surface roughness, studies have suggested higher bacterial adherence due to irregularities on polymeric surfaces compared to ultrasmooth surfaces [15], [16]. However, for the majority of these studies the adherence of just one bacterial species was investigated, such as Staphylococcus epidermidis [13], [17], Streptococcus mutans [18], or Staphylococcus aureus [19] all of which are species associated with plaque formation and the development of a caries lesion rather than peri-implantitis. No studies have investigated the adherence of the different periodontally relevant bacterial species which can guide bacteria to sites for the initiation of peri-implantitis. Further, these studies fail to take into account that peri-implantitis is initiated following the colonisation by a polymicrobial biofilm [4], [5].

The aim of this study was to investigate the adherence of populations of periodontally relevant bacterial species to an electropolished smooth cobalt chromium surface and milled titanium surface prepared as discs, representing surfaces of dental abutments and implants. Of note, bacteria adhering to the abutment surfaces may derive from free-floating rapidly growing bacteria in saliva or slow dividing bacteria living in a biofilm. The same species can have significantly different properties, recognising that bacteria existing in densely protected environments cooperate and interact in different ways to planktonic bacterial suspensions, facilitated by altered quorum sensing. To investigate whether this affected adherence properties of bacteria to metal surfaces, this study also looked to compare bacterial properties in log-growth stage and stationary phase of culture. The study lays the foundation for development of research protocols which is a relevant consideration in the functioning of implants and abutments within the oral cavity.

Section snippets

Growth conditions of anaerobic bacteria

The strains used in this study were Porphyromonas gingivalis NCTC11834, Fusobacterium nucleatum ATCC49256, Prevotella intermedia NCTC13070 and Aggregatibacter actinomycetemcomitans DSM8324.

P. gingivalis, F. nucleatum and Pr. intermedia were maintained on Fastidious Anaerobe Agar (FAA; Lab M Ltd., Lancashire, UK) plates containing 5% defibrinated horse blood (TCS Biosciences Ltd., Buckingham, UK) at 37 °C in an anaerobic environment (80% nitrogen, 10% carbon dioxide, 10% hydrogen) in a Modular

Surface characteristics of disc surfaces

All surfaces had a similar partial wetting ability. The average advancing contact angles, for n = 6, were calculated as 78.39° ± 1.735° for the CoCr-polished surfaces and 83.176° ± 2.531° for Ti-milled surfaces. Topographical images and surface roughness profiles for the CoCr-polished and Ti-milled surfaces are shown in Fig. 1. The Ti-milled surfaces demonstrated a sharp edged concentric circular pattern consistent with the milling procedure used in their preparation. The CoCr-polished surfaces had a

Discussion

Within this study two very contrasting metal surfaces were examined for their ability to form a bacterial biofilm consisting of periodonto-pathogens with high clinical relevance leading to the induction of peri-implantitis and thus the potential loss of an endosseous prosthesis. The highly polished CoCr (CoCr-polished) represents a surface commercially used for dental abutments with a purported reduced bacterial adherence and is compared with a contrasting milled titanium (Ti-milled surface),

Acknowledgements

This work was supported through a research collaboration funded by Renishaw plc. All authors participated in study design. In addition RPCJ performed all experiments; LM performed confocal; RJW supervised the co-ordination of the study.

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