Streptococcus mutans is able to form a high-affinity biofilm on material surfaces. S mutans has also been detected around infected implants. Bioactive glasses (BAGs) have been shown to possess antibacterial effects against S mutans and other microorganisms. This in vitro study was performed to investigate the influence of BAG air abrasion on S mutans biofilm on sandblasted and acid-etched titanium surfaces. Sandblasted and acid-etched commercially pure titanium discs were used as substrates for bacteria (n = 107). The discs were immersed in an S mutans solution and incubated for 21 hours to form an S mutans biofilm. Twenty colonized discs were subjected to air abrasion with Bioglass 45S5 (45S5 BAG), experimental zinc oxide containing BAG (Zn4 BAG), and inert glass. After the abrasion, the discs were incubated for 5 hours in an anaerobic chamber followed by an assessment of viable S mutans cells. Surface morphology was evaluation using scanning electron microscopy (n = 12). The thrombogenicity of the glass particle–abraded discs (n = 75) was evaluated spectrophotometrically using whole-blood clotting measurement at predetermined time points. Air abrasion with 45S5 and Zn4 BAG eradicated S mutans biofilm. Significantly fewer viable S mutans cells were found on discs abraded with the 45S5 or Zn4 BAGs compared with the inert glass (P < .001). No significant differences were found in thrombogenicity since blood clotting was achieved for all substrates at 40 minutes. Air abrasion with BAG particles is effective in the eradication of S mutans biofilm from sandblasted and acid-etched titanium surfaces. Zn4 and 45S5 BAGs had similar biofilm-eradicating effects, but Zn4 BAG could be more tissue friendly. In addition, the steady release of zinc ions from Zn4 may enhance bone regeneration around the titanium implant and may thus have the potential to be used in the treatment of peri-implantitis. The use of either BAGs did not enhance the speed of blood coagulation.
Introduction
Dental implants have become an established treatment modality to replace missing teeth in different clinical situations. The establishment of bacteria on implant surfaces may induce inflammation of the peri-implant mucosa, and if left untreated, the inflammation can extend apically and result in bone resorption and subgingival infection, a condition that has been termed peri-implantitis.1 Therefore, one of the main objectives of peri-implantitis therapy is plaque removal.2 Studies have shown that bacterial adhesion to the implant surface is greatly influenced by the implant's surface roughness; hence, the amount of adhered bacterial cells is dramatically increased with increased surface roughness.3 Concurrently, other studies have shown that the biofilm formation is similar on all implant surfaces, irrespective of the roughness.4–6
Implant surface biofilm formation is initiated by the adhesion of gram-positive bacterial colonizers,7 which are assumed to facilitate the titanium surface colonization by the secondary bacterial colonizers, leading to the establishment of an anaerobic gram-negative microbial environment.8 Streptococcus mutans isolates have a substantial ability to form biofilm and survive at low pH values.9 Furthermore, S mutans has the ability to produce an insoluble polymer matrix with a high affinity to solid surfaces such as oral tissues or implants.10 Attachment of early colonizers of oral biofilm on titanium surfaces depends on both the surface topography and bacterial species involved.11 S mutans was chosen for this study because it is able to form a high-affinity biofilm and has been found to be at a higher level around infected rather than around healthy implants.12 Accordingly, S mutans might participate in the process that can lead to the development of peri-implantitis and eventually implant failure.13
Hench and colleagues in 1970 introduced the bioactive glass (BAG) 45S5 (composition in the system SiO2-Na2O-P2O5-CaO). Bioactive glasses are a group of biomaterials used in the fields of dentistry and orthopedics. In addition to being biocompatible, BAGs have the ability to stimulate repair and regeneration of hard tissues as they have demonstrated the capability of forming direct chemical bonds with both hard and soft tissues.14,15 Various compositions of BAGs have been developed for preparation of scaffolds16 and as a coating material for implants.17 The BAGs that have been used for air polishing yielded better results in terms of stain removal and patient comfort as compared with traditional sodium bicarbonate powder.18
The well-known BAGs 45S5 and S53P4 have previously been shown to have an antibacterial effect against S mutans along with several other oral microorganisms.19,20 The antibacterial activity of BAGs is predominantly due to high pH and osmotic effects caused by the nonphysiological concentrations of silica, sodium, and calcium ions dissolved from the glass.21,22 Stoor et al22 investigated the antibacterial effect of BAG against planktonic Aggregatibacter actinomycetemcomitans. In their experiment, BAG led to the bacteria losing their viability within 1 hour.22 Allan et al reported that the use of 45S5 BAG inhibited several oral bacteria (including Streptococcus sanguis, Streptococcus mutans, and Actinomyces viscosus).21 Zhang et al23 have also demonstrated that BAG without any special bactericidal components exhibited antibacterial activity against a large selection of bacteria in a concentration-dependent manner. In their study, the BAG S53P4 inhibited the proliferation of all tested bacteria including Escherichia coli, Pseudomonas aeruginosa, Moraxella catarrhalis, Enterococcus faecalis, and Staphylococcus epidermidis.23
Incorporation of zinc oxide (ZnO) to BAG has been reported to improve its biocompatibility and enhance the antimicrobial activity of the composition.24 ZnO has demonstrated antibacterial activity against S mutans as a result of the release of the Zn2+ ion, which causes interruption of protein synthesis and interference with DNA replication.25 In addition, Zn2+ may alter the function of the bacterial cell's membrane and the enzymatic activity within the cell that causes impairment in acid production during the glycolysis process.26,27 Furthermore, generation of H2O2 from the surface of ZnO powder has been reported to provide antibacterial activity.28
Favorable interactions between the implant surface and blood system is of absolute importance in determining the success of the implant's performance.29–31 The blood clot that facilitates cell migration and proliferation can be considered a natural scaffold on the tissue-implant interface.
This study set out to investigate the influence of air abrasion with BAG particles on S mutans biofilm on sandblasted and acid-etched (SA) titanium surfaces. Furthermore, the study aimed to determine whether air abrasion with BAG influences the blood coagulation on infected SA surfaces. This study was based on the working hypothesis that the BAG particle air abrasion of the SA titanium surface with S mutans biofilm decreases the counts of S mutans cells and enhances early blood clot formation.
Materials and Methods
SA of titanium samples
The SA procedures were performed in house. Grade 5 commercially pure titanium discs were cut into squares of 10 mm and a thickness of 1 mm. The discs were subjected to sandblasting with large-grit aluminum oxide particles (250–500 μm) using an air pressure of 5 bar followed by acid etching in an HCl (60%) and H2SO4 (70%) acid mixture for 1 hour at 60°C. Then, the discs were thoroughly rinsed with deionized water in an ultrasonic bath for 20 minutes to eliminate acid residues. Discs were allowed to dry in a hot air oven for 30 minutes at 50°C.
Glass particle preparation for air-abrasion
Zn4 and 45S5 BAGs were melted in house. Batches giving 300 g of glass were mixed with analytical grade chemical Na2CO3 (Sigma-Aldrich), CaHPO4·2H2O (Sigma-Aldrich), ZnO (Sigma-Aldrich), CaCO3 (Fluka), H3BO3 (Merck), and Belgian glass quality quartz sand for SiO2. The oxide composition of 45S5 BAG in mol % is 46.1 SiO2, 24.3 Na2O, 2.6 P2O5, and 26.9 CaO, whereas for Zn4 BAG is 44.1 SiO2, 24.3 Na2O, 2.6 P2O5, 24.9 CaO, and 4.0 ZnO. The glasses were melted in an uncovered platinum crucible at 1360°C for 3 hours in air. After casting, the obtained glass blocks were annealed at 520°C for 1 hour and then slowly cooled in the oven. The blocks were then crushed and remelted to ensure homogeneity. The annealed blocks were crushed and sieved to provide particles of the size range fraction 25–120 μm. The particle morphology was examined with a scanning electron microscope (SEM; Leo Gemini 1530, Hamburg, Germany).
In vitro simulated body fluid testing of the glasses
The in vitro bioactivity of the BAGs was tested in simulated body fluid (SBF). The SBF was prepared following the protocol by Kokubo et al.33 In the static ion dissolution tests, BAG particles (300–500 μm) were immersed in the SBF using the concentration 100 mg mL−1. The samples were placed in containers and then in a shaking incubator (rotation speed 120 rpm) at 37°C for 48 hours. Then, the particles were separated from the fluid, and the reaction was stopped by washing with acetone and drying in an oven at 40°C. The ion concentrations dissolved from the BAGs were measured using an inductively coupled plasma-atomic emission spectrometer (ICP-OES; Optima 5300 DV, Perkin Elmer, Downers Grove, Ill). The pH measurements were performed using the concentration of 10-mg glass particles (300–500 μm) to 4 mL SBF. The pH of the SBF was measured in triplicate at 37°C at 2, 10, 30, 60, and 120 minutes.
The dynamic dissolution tests were performed using the method described in detail by Fagerlund et al.33 Fresh SBF at 37°C was continuously fed at 0.2 mL min−1 through a bed of glass particles (285 mg, 300–500 μm). The solution was collected at selected time points for 15 min followed by measurement of the ion concentrations released from the glass particles using ICP-OES. The measurements were continued up to 50 hours.
Biofilm formation
The biofilm experiments were performed with a method used in our laboratory earlier.34 Briefly, the SA discs were first coated with 1 mL pasteurized whole saliva diluted (1:3) with phosphate-buffered saline (PBS) in 24-well cell culture plates for 30 minutes at 37°C. Afterward, the discs were rinsed with 1.5 mL PBS. The processing of the saliva has been described in detail previously.34
To form the biofilm on the SA discs, the reference strain S mutans Ingbritt was grown in brain heart infusion medium (BHI; Becton-Dickinson and Company, Sparks, Md) overnight at 37°C. The cells were collected with centrifugation (5000g, 10 minutes) and then suspended in fresh BHI containing 0.03% sucrose, A550 = 0.05. Sucrose was added to the medium to promote polysaccharide production. The SA discs were immersed in 1.8 mL of the above S mutans suspension, and 200 μL of the pasteurized saliva was added. The incubation was performed in a Whitley A35 anaerobic chamber (Don Whitley Scientific Ltd, Shipley, UK) at +37°C under 80% N2, 10% CO2, and 10% H2 atmosphere for 21 hours.
Air abrasion with BAG particles and culturing the samples
A total of 107 SA discs with the biofilms were rinsed once in PBS and then immersed in 1 mL PBS. The entire surface of the SA discs was subjected to air abrasion with the BAGs or inert glass (n = 99). Eight discs, as control, were not subjected to air abrasion. The air-abrasion procedure of each disc was performed for 20 seconds, at a 90° angle, 3-mm distance, and using an air pressure of 4 bar. Then, the discs were immediately transferred to 24-well plates with 2 mL fresh BHI. The SA discs were then incubated for 5 hours at 37°C.
After incubation, S mutans cells were collected from the discs (n = 20; 15 air abraded and 5 control) with microbrushes (Quick-Stick, Dentsolv AB, Saltsjö-Boo, Sweden) combined with mild sonication to detach and disperse the cells. The suspensions containing the bacteria were serially diluted and plated on Mitis Salivarius Agar (Becton-Dickinson and Company). The Agar plates were grown for 3 days anaerobically at +37°C. The colonies were counted under a stereomicroscope, and the results were expressed as colony-forming units (CFU). The experiments were performed with 5 replicates and repeated twice
SEM evaluation
The SA titanium discs with biofilm and following air abrasion with BAG or inert glass particles (n = 12) were rinsed 3 times in 1 mL of deionized water and fixed in 2.5% glutaraldehyde overnight at room temperature. Then, the samples were dehydrated in a graded ethanol series (50%, 70%, 85%, 95%, and 100%) for 10 minutes each. Five images were taken at random locations for each sample. The SEM images were done with 3 replicates.
Clotting time measurements
Seventy-five air-abraded SA titanium discs were used. The thrombogenic properties of the BAG and inert glass air-abraded SA discs were evaluated using fresh human whole blood by the kinetic clotting time method. Fresh blood was taken from a healthy female volunteer with venipuncture using disposable syringes. The first 3 mL of the drawn blood was disposed to prevent tissue thromboplastin contamination caused by needle puncture. Carefully, 0.1 mL of blood was immediately pipetted onto the SA disc surface placed in 12-well plates. Following a predetermined time of 10, 20, 30, 40, and 60 minutes, 3 mL of distilled water was poured into the wells containing the discs for 5 minutes. Triplicate samples were taken from each well and transferred to a 96-well plate. The lysed red blood cells in the distilled water released hemoglobin, which was subjected to measurement. The concentration of free hemoglobin in the water was colorimetrically measured by monitoring the absorbance at 570 nm using spectrophotometer. The test was performed in 5 replicates.
Statistical analysis
The distributions of study variables were studied and described. Measurements in CFU between biofilm, inert glass, Zn4 BAG, and 45S5 BAG and optical density (OD) between inert glass, Zn4 BAG, and 45S5 BAG at different time points were reported as means and standard deviations. The differences between the experimental treatments were evaluated using 1-way analysis of variance followed by post hoc Tukey honestly significant difference tests at an alpha level of 5%, with significance considered at P < .05. If normality assumptions were violated, the logarithmic transformation was adapted, and the testing was repeated. Statistical analyses were performed using SPSS statistical software version 23.0 (SPSS Inc, Chicago, Ill). The analyses were reviewed by an independent statistician who concluded that the methodology and reported results are adequate, and the conclusions drawn are appropriate.
Results
The SEM images showed that sandblasting and acid etching of the titanium surface produced apparent surface roughness with different crater sizes (Figure 1). The SA titanium discs were well covered with S mutans biofilm after 21 h of incubation (Figure 2). Following BAG/inert glass air abrasion, no S mutans cells could be detected in the SEM images taken in 5 random locations per each sample (Figure 3). There was a significant decrease, F(3, 16) = 91.10, P < .001, in the viable S mutans cells observed for SA titanium discs with biofilm subjected to air abrasion with both BAGs and inert glass compared with unabraded SA discs. Moreover, Zn4 BAG and 45S5 BAG abraded SA discs, which had been covered with biofilm, demonstrated significantly fewer viable S mutans cells compared with the discs abraded with inert glass (Figure 4).
Blood-clotting profiles for the substrates after air abrasion with BAG or inert glass particles demonstrated that the absorbance of the hemolyzed hemoglobin varied with time. Complete blood clotting on the SA surfaces abraded with BAG or inert glass was reached in 40 minutes at an OD > 0.1 (Figure 5). After 10 minutes, a significant difference in OD was observed, F(2, 33) = 12.57, P < .001. The Zn4 BAG abraded discs showed significantly lower OD, reflecting faster coagulation, when compared with the discs abraded with 45S5 BAG. After 20 and 30 minutes, the inert glass–abraded discs demonstrated significantly lower OD compared with the two BAG-abraded discs, F(2, 33) = 47.61, P < .001, and F(2, 33) = 58.09, P < .001, respectively.
From our previous work, the release of Si and Ca ions into SBF in both continuous and static conditions was higher from 45S5 BAG compared with Zn4 BAG (Table). This was attributed to the greater increase in the pH of SBF, which was observed for 45S5 BAG when compared with Zn4 BAG (Figure 6). When feeding 0.2 mL/min fresh SBF solution through particles of Zn4 BAG, Zn2+ was released at a constant rate, 0.2 mg/L Zn2+ over the 24-hour experiment (Table).35
Discussion
The findings of this study prove that air abrasion using Zn4 BAG and 45S5 BAG particles successfully eradicated S mutans biofilm from the experimental SA surface. Furthermore, the residual BAG particles embedded on the SA surface may lead to an extended antibacterial effect of BAG air-abraded SA titanium surfaces. Also, air abrasion of SA titanium surfaces with Zn4 BAG can enhance early-stage blood coagulation. S mutans biofilm experiments, BAG air abrasion, and culturing the samples and clotting time measurements were performed following a strict laboratory protocol. Intra-laboratory reproducibility has been validated since the study has been repeated more than 2 times in the same laboratory conditions. However, the interlaboratory reproducibility has not been tested.
It has been demonstrated that air abrasion can be effective in the removal of bacterial biofilm without altering the morphological characteristics of the implant surface.36,37 In addition, air-abrasive powder has the advantage of maintaining the surface characteristics of titanium without causing roughness and alterations that can become a bacterial niche.5 In our study, air abrasion with both BAG and inert glass was found to be effective in removing S mutans biofilm. However, air abrasion with inert glass particles was less effective against S mutans biofilm compared with BAG particle abrasion. This is most likely because the effect of the inert glass is limited only to mechanical cleaning, while both BAG formulas investigated in this study showed antimicrobial activity.35 In conjunction with the mechanical cleaning, some Zn4 or 45S5 BAG particles stay attached to or embedded in the SA surface, which apparently leads to extended antibacterial activity. The BAG dissolution releases ions (Na, Si2+, Ca2+) that subsequently elevate the pH and change the osmotic pressure of the solution in the close vicinity of the glass particles. These changes are thought to be the main factors in the antimicrobial effect against microorganisms.21–23 On the other hand, the antimicrobial action behind the Zn4 BAG cannot be explained solely by the rise in the pH of the interfacial solution.
Our previous in vitro evaluation of 45S5 and Zn4 BAGs showed that the higher initial release of silicon and calcium ions from 45S5 BAG contributed to a higher pH elevation compared with Zn4 BAG. In contrast, the release of Zn2+ in the dynamic flow conditions from Zn4 BAG stayed steady during the 48-hour experimental time.33 Whether this low but constant release of Zn2+ is viable to prevent bacterial growth should be investigated further.
During peri-implant wound healing, the rapidly formed blood clot isolates the implant surface from the surrounding environment. Thus, it acts as a barrier against possible infection and serves as a connection between the surrounding tissue and the implant surface. The pluripotent cells migrate through fibrin fibers that connect the implant surface and the surrounding tissue and differentiate into various types of cells. To study the effect of air abrasion on blood coagulation, we chose to use the spectrophotometric method for the evaluation of OD of blood applied on experimental surfaces. Lower OD values indicate lower hemoglobin concentration in blood solution, which is evidence of rapid thrombus formation on the substrate surface.38 The OD value at which complete clotting time is determined is 0.1. The reason why SA surfaces treated with Zn4 BAG showed faster initial blood clotting after 10 minutes when compared with 45S5 BAG or inert glass is not known. Inert glass showed faster coagulation at later time points, which may be related to the surface reactivity of BAG particles trapped on abraded surfaces. However, the difference among the surfaces vanished with time, and at 40 minutes, blood coagulation was completed on all surfaces.
Based on the results of this study, our first hypothesis about the benefits of BAG particles in the eradication of S mutans biofilm from SA titanium surfaces can be confirmed. However, the second hypothesis must be rejected, as the use of either BAGs did not enhance the speed of blood coagulation. However, this study indicates that air abrasion treatment of peri-implantitis involving surfaces with BAG powder might enhance the healing process by eliminating the biofilm and allowing sound surfaces for wound healing. Based on the increase in pH, it can be postulated that Zn4 BAG might be more tissue friendly compared with the 45S5 BAG. Also, the release of Zn2+ is known to encourage attachment and proliferation of osteoblasts as well as inhibit osteoclastic cells, and it is also involved in the calcification mechanism.39
It is important to bear in mind that an experiment conducted in vitro has limitations, as it is considered a static system compared with in vivo tests. Results reported in this study are valid for the S mutans biofilm formed in laboratory conditions on SA titanium surfaces, which limits their external validity. However, sandblasting and acid-etching treatment is one of the most widely used surface treatment methods for titanium implants. Moreover, S mutans is very virulent, forms an adhesive biofilm,22 and might thus participate in the process of peri-implantitis, which can lead to implant failure.10 These study limitations could be addressed in future research by evaluating the effect of BAG air abrasion on multiple species of anaerobic biofilms on titanium with various surface characteristics. In addition, adhesion and proliferation of fibroblast and osteoblast cells on BAG air-abraded titanium surfaces need to be assessed in future research. However, the true potential of air-abrasion treatment of peri-implantitis–involved implant surfaces with BAG particles needs to be evaluated in clinical settings before any definitive conclusion can be made.
Conclusions
Within the limitations of this study, it can be concluded that air abrasion of S mutans–covered titanium discs with particles of BAGs Zn4 and 45S5 showed similar biofilm-eradicating effects and thus may have the potential to be used in the decontamination of infected implant surfaces. The lower dissolution rate of Zn4 BAG and thus lower increase in the pH of the interfacial solution around the implant could be more tissue friendly and may be of benefit for the healing process. In addition, the steady release of zinc ions from the Zn4 may support bone regeneration around the titanium implant. However, the use of either BAGs did not enhance the speed of blood coagulation.
Abbreviations
- BAG:
bioactive glass
- BHI:
brain heart infusion
- CFU:
colony-forming unit
- ICP-OES:
inductively coupled plasma-atomic emission spectrometer
- OD:
optical density
- PBS:
phosphate-buffered saline
- SA:
sandblasted and acid etched
- SBF:
simulated body fluid
- SEM:
scanning electron microscope
- Zn4 BAG:
zinc oxide containing bioactive glass
- ZnO:
zinc oxide
Acknowledgment
The authors would like to thank Auli Suominen, biostatistician, Department of Community Dentistry, University of Turku, for her independent statistical review.
Note
The authors declare that they have no conflict of interest.