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.
1
Introduction
Peri-implant disease is regarded as an infectious disease, whereby oral pathogens, usually gram-negative micro-organisms, initiate host inflammatory responses . 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 . 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 . 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 and often co-aggregates together with Pr. intermedia . 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 . 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 . 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 . Published data suggests that cobalt chromium metal surfaces demonstrate reduced bacterial adherence compared with titanium, although this is not significant for all studies . With regards to surface roughness, studies have suggested higher bacterial adherence due to irregularities on polymeric surfaces compared to ultrasmooth surfaces . However, for the majority of these studies the adherence of just one bacterial species was investigated, such as Staphylococcus epidermidis , Streptococcus mutans , or Staphylococcus aureus 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 .
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.
2
Materials and methods
2.1
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 Atmosphere Controlled System. A. actinomycetemcomitans was maintained on FAA plates containing 5% defibrinated horse blood at 37 °C in 5% CO 2 in air (Sanyo CO 2 incubator). Liquid cultures of P. gingivalis , F. nucleatum and Pr. intermedia were grown under anaerobic conditions at 37 °C in Fastidious Anaerobe Broth (FAB; Lab M), while A. actinomycetemcomitans was cultured in FAB at 37 °C in 5% CO 2 in air. Culture purity was determined by colony appearance on FAA, Gram staining and microscopic analysis.
2.2
Bacterial growth curves
A. actinomycetemcomitans , previously grown on FAA for 48 h in 5% CO 2 in air at 37 °C, was sub-cultured into 10 ml FAB and cultured in 5% CO 2 in air at 37 °C overnight without agitation. The broth culture was diluted to an initial inoculum of ∼1 × 10 7 colony forming units (CFUs)/ml and OD 600 readings were recorded every hour for 18 h using a FLUOstar Omega plate reader with an atmospheric control unit (O 2 /CO 2 Mpc GV; BMG Labtech GmbH, Ortenberg, Germany). Growth curves were plotted from three separate repeat experiments.
Pr. intermedia, F. nucleatum, P. gingivalis, all previously grown on FAA for 72 h in an anaerobic environment at 37 °C, were sub-cultured into 10 ml FAB prior to overnight culture in anaerobic conditions without agitation (exception P. gingivalis cultured for 40 h). Culture broths were diluted to an initial inoculum of 1 × 10 8 CFUs/ml ( Pr. intermedia ), 5 × 10 7 CFUs/ml ( F. nucleatum ) or 1 × 10 8 CFUs/ml ( P. gingivalis ) and OD 600 readings were recorded every hour for 20 h using an IMPLEN GmbH (Müchen, Germany) OD600 DiluPhotometer™. Bacterial numbers along the growth curves obtained were determined using the Miles and Misra technique on FAA plates. A. actinomycetemcomitans , Pr. intermedia , F. nucleatum and P. gingivalis were cultured in FAB broth, as described, for determination of the bacterial growth curves and the broth was diluted to a range of optical densities which correspond to points on the growth curves. These dilutions were further serially diluted 1 in 10 in sterile PBS to form a dilution series from 10 1 to 10 8 . Aliquots of these dilutions (10 μl) were inoculated onto FAA plates and cultured for 24 h ( P. gingivalis cultured for 40 h) at the appropriate atmospheric condition before counting bacterial colonies to determine plating numbers.
2.3
Metal surfaces and hydrophobicity assessment
Metal discs were supplied by Renishaw plc as either electropolished Cobalt Chromium (CoCr-polished) or machined Titanium 6-Aluminium 4-Vanadium (Ti64) (Ti-milled).
The Ti-milled discs were prepared from Ti64 (ASTM Grade 5) bar stock on a Schaublin 102 N manual lathe. The CoCr-polished discs were additively manufactured from Renishaw CoCr DG1™ metal powder on the Renishaw AM250 via the process of Selective Laser Melting (SLM). Build supports were manually removed and the disc surfaces dressed using a handheld rotary tool and tungsten carbide burr. The discs then underwent an electropolishing process in which they were connected to the positive side of a power supply (hence acting as the anode), submerged in an electrolyte along with a conformal cathode, and a voltage was applied in order to give a predetermined current density over the discs for a set time period. The resulting electrochemical reaction caused material to be preferentially removed from surface maxima on the disc, giving a polished appearance.
Surfaces of the discs were imaged using a Leica TCS SP2 AOBS spectral confocal microscope with a 20× objective lens. A reflectance signal was generated using a 488 nm laser, with the detector sampling reflected light from the surface of the discs. Optical sections were taken at 0.6 μm intervals over the entire surface of the disc and Z-stack sections were reconstructed into single maximal intensity projections. Surface roughness measurements of discs were taken using a Surftest SV-2000 profilometer (Mitutoyo, Hampshire, UK) across a distance of 25 μm at a speed of 0.1 mm/s (n = 6). Mean roughness values (Ra) and maximum heights (Ry) were calculated by Surfpak-SV software (Mitutoyo, Hampshire, UK) and surface profiles combined from 6 individual measurements. Hydrophobicity was measured for each surface (n = 6) using a Dynamic Contact Angle Analyser 312 (Cahn Instruments Inc., Thermo Electron Corporation), using deionised distilled water as the wetting medium. Prior to each measurement discs were placed in a drying cabinet at 95 °C overnight. Each disc was repeatedly assessed on 5 different days where environmental temperature recording were equivalent. Advancing and receding contact angles were calculated using the least squares analysis generated by Cahn Application Software. Average advancing contact angles and standard deviations were calculated.
2.4
Bacterial adherence studies
Bacterial adherence studies were performed for each surface at bacterial concentrations representative of the exponential mid-log growth phase and the stationary phase. Briefly, the respective bacterial cultures were prepared in FAB as described above and then diluted in further broth to give the following OD 600 ; P. gingivalis mid-log, 0.75, stationary 1.3; F. nucleatum mid-log 0.35, stationary 0.75; Pr. intermedia mid-log 0.75, stationary 1.6; A. actinomycetemcomitans mid-log 0.13, stationary 0.19. In addition, the adherence of co-cultures for F. nucleatum / P. gingivalis or F. nucleatum / P. gingivalis / Pr. intermedia were examined. Bacteria were prepared to OD equivalent to either their mid-log or stationary concentration and then combined.
Metal discs (CoCr-polished and Ti-milled) were sterilised by autoclaving at 121 °C for 15 min and dried prior to use. Discs were placed into wells of a 24 well plate and 2 ml bacterial suspension was pipetted onto each disc (FAB culture broth served as a negative control) and culture was continued for 2 h under the appropriate growth conditions described above. Discs were removed and washed with 1 ml PBS in a fresh well, with very gentle agitation for 1 min. Bacteria adherent to the disc was stained with 10 μl of LIVE/DEAD ® BacLight™ Bacterial Viability stain (Molecular Probes; as per manufacturer’s instructions) for 30 min prior to examination by confocal laser scanning fluorescence microscopy (Leica SP5 Confocal Microscope) at x40 magnification. Five random images were obtained for each sample using LAS-AF software, version 2.6.0.7266. All digital images were examined to determine the percentage area of fluorescence (representing the bacterial coverage) on the disc surface using ImageJ. Thresholds were applied to the images and converted into binary black and white images to allow for quantification of bacterial coverage. Discs were mechanically cleaned using a toothbrush and re-autoclaved. Removal of bacteria was confirmed by re-examination under confocal microscopy. The bacterial adherence assay was repeated on the same batch of discs on one further occasion.
2.5
Statistical analysis
For each bacterial species (grown to either mid-log or stationary phase) and each surface analysed, the mean and standard error of the mean (SEM) was determined. Statistical significance was assessed using an ANOVA test with Tukey-Kramer correction test for multiple comparisons (Instat, GraphPad Software, San Diego, USA). Growth curve raw data were graphically depicted using BMG Labtech Mars data analysis software.
2
Materials and methods
2.1
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 Atmosphere Controlled System. A. actinomycetemcomitans was maintained on FAA plates containing 5% defibrinated horse blood at 37 °C in 5% CO 2 in air (Sanyo CO 2 incubator). Liquid cultures of P. gingivalis , F. nucleatum and Pr. intermedia were grown under anaerobic conditions at 37 °C in Fastidious Anaerobe Broth (FAB; Lab M), while A. actinomycetemcomitans was cultured in FAB at 37 °C in 5% CO 2 in air. Culture purity was determined by colony appearance on FAA, Gram staining and microscopic analysis.
2.2
Bacterial growth curves
A. actinomycetemcomitans , previously grown on FAA for 48 h in 5% CO 2 in air at 37 °C, was sub-cultured into 10 ml FAB and cultured in 5% CO 2 in air at 37 °C overnight without agitation. The broth culture was diluted to an initial inoculum of ∼1 × 10 7 colony forming units (CFUs)/ml and OD 600 readings were recorded every hour for 18 h using a FLUOstar Omega plate reader with an atmospheric control unit (O 2 /CO 2 Mpc GV; BMG Labtech GmbH, Ortenberg, Germany). Growth curves were plotted from three separate repeat experiments.
Pr. intermedia, F. nucleatum, P. gingivalis, all previously grown on FAA for 72 h in an anaerobic environment at 37 °C, were sub-cultured into 10 ml FAB prior to overnight culture in anaerobic conditions without agitation (exception P. gingivalis cultured for 40 h). Culture broths were diluted to an initial inoculum of 1 × 10 8 CFUs/ml ( Pr. intermedia ), 5 × 10 7 CFUs/ml ( F. nucleatum ) or 1 × 10 8 CFUs/ml ( P. gingivalis ) and OD 600 readings were recorded every hour for 20 h using an IMPLEN GmbH (Müchen, Germany) OD600 DiluPhotometer™. Bacterial numbers along the growth curves obtained were determined using the Miles and Misra technique on FAA plates. A. actinomycetemcomitans , Pr. intermedia , F. nucleatum and P. gingivalis were cultured in FAB broth, as described, for determination of the bacterial growth curves and the broth was diluted to a range of optical densities which correspond to points on the growth curves. These dilutions were further serially diluted 1 in 10 in sterile PBS to form a dilution series from 10 1 to 10 8 . Aliquots of these dilutions (10 μl) were inoculated onto FAA plates and cultured for 24 h ( P. gingivalis cultured for 40 h) at the appropriate atmospheric condition before counting bacterial colonies to determine plating numbers.
2.3
Metal surfaces and hydrophobicity assessment
Metal discs were supplied by Renishaw plc as either electropolished Cobalt Chromium (CoCr-polished) or machined Titanium 6-Aluminium 4-Vanadium (Ti64) (Ti-milled).
The Ti-milled discs were prepared from Ti64 (ASTM Grade 5) bar stock on a Schaublin 102 N manual lathe. The CoCr-polished discs were additively manufactured from Renishaw CoCr DG1™ metal powder on the Renishaw AM250 via the process of Selective Laser Melting (SLM). Build supports were manually removed and the disc surfaces dressed using a handheld rotary tool and tungsten carbide burr. The discs then underwent an electropolishing process in which they were connected to the positive side of a power supply (hence acting as the anode), submerged in an electrolyte along with a conformal cathode, and a voltage was applied in order to give a predetermined current density over the discs for a set time period. The resulting electrochemical reaction caused material to be preferentially removed from surface maxima on the disc, giving a polished appearance.
Surfaces of the discs were imaged using a Leica TCS SP2 AOBS spectral confocal microscope with a 20× objective lens. A reflectance signal was generated using a 488 nm laser, with the detector sampling reflected light from the surface of the discs. Optical sections were taken at 0.6 μm intervals over the entire surface of the disc and Z-stack sections were reconstructed into single maximal intensity projections. Surface roughness measurements of discs were taken using a Surftest SV-2000 profilometer (Mitutoyo, Hampshire, UK) across a distance of 25 μm at a speed of 0.1 mm/s (n = 6). Mean roughness values (Ra) and maximum heights (Ry) were calculated by Surfpak-SV software (Mitutoyo, Hampshire, UK) and surface profiles combined from 6 individual measurements. Hydrophobicity was measured for each surface (n = 6) using a Dynamic Contact Angle Analyser 312 (Cahn Instruments Inc., Thermo Electron Corporation), using deionised distilled water as the wetting medium. Prior to each measurement discs were placed in a drying cabinet at 95 °C overnight. Each disc was repeatedly assessed on 5 different days where environmental temperature recording were equivalent. Advancing and receding contact angles were calculated using the least squares analysis generated by Cahn Application Software. Average advancing contact angles and standard deviations were calculated.
2.4
Bacterial adherence studies
Bacterial adherence studies were performed for each surface at bacterial concentrations representative of the exponential mid-log growth phase and the stationary phase. Briefly, the respective bacterial cultures were prepared in FAB as described above and then diluted in further broth to give the following OD 600 ; P. gingivalis mid-log, 0.75, stationary 1.3; F. nucleatum mid-log 0.35, stationary 0.75; Pr. intermedia mid-log 0.75, stationary 1.6; A. actinomycetemcomitans mid-log 0.13, stationary 0.19. In addition, the adherence of co-cultures for F. nucleatum / P. gingivalis or F. nucleatum / P. gingivalis / Pr. intermedia were examined. Bacteria were prepared to OD equivalent to either their mid-log or stationary concentration and then combined.
Metal discs (CoCr-polished and Ti-milled) were sterilised by autoclaving at 121 °C for 15 min and dried prior to use. Discs were placed into wells of a 24 well plate and 2 ml bacterial suspension was pipetted onto each disc (FAB culture broth served as a negative control) and culture was continued for 2 h under the appropriate growth conditions described above. Discs were removed and washed with 1 ml PBS in a fresh well, with very gentle agitation for 1 min. Bacteria adherent to the disc was stained with 10 μl of LIVE/DEAD ® BacLight™ Bacterial Viability stain (Molecular Probes; as per manufacturer’s instructions) for 30 min prior to examination by confocal laser scanning fluorescence microscopy (Leica SP5 Confocal Microscope) at x40 magnification. Five random images were obtained for each sample using LAS-AF software, version 2.6.0.7266. All digital images were examined to determine the percentage area of fluorescence (representing the bacterial coverage) on the disc surface using ImageJ. Thresholds were applied to the images and converted into binary black and white images to allow for quantification of bacterial coverage. Discs were mechanically cleaned using a toothbrush and re-autoclaved. Removal of bacteria was confirmed by re-examination under confocal microscopy. The bacterial adherence assay was repeated on the same batch of discs on one further occasion.
2.5
Statistical analysis
For each bacterial species (grown to either mid-log or stationary phase) and each surface analysed, the mean and standard error of the mean (SEM) was determined. Statistical significance was assessed using an ANOVA test with Tukey-Kramer correction test for multiple comparisons (Instat, GraphPad Software, San Diego, USA). Growth curve raw data were graphically depicted using BMG Labtech Mars data analysis software.
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