Influence of Photodynamic Therapy on Bacterial Attachment to Titanium Surface

Infectious diseases of the oral cavity generated by bacteria and fungi remain one of the major problems confronting clinical dentistry.¹  Significant advances in treating and preventing these diseases have been discussed over the past decades. Titanium (Ti) dental implants have been used successfully as replacements to untreatable or missing teeth.¹  Dental biofilm can cause bacterial infection around dental implants leading to the risk of implant failure due to the loss of the supporting bone.¹  In peri-implantitis cases, the traditional clinical management presents with more challenges as disrupting implants surfaces should be avoided, which limits the ability of the mechanical removal of infectious biofilm that happens to protect harmful bacteria. Healthy peri-implant soft and hard tissues are especially crucial to the long-lasting healthy function of any dental restorations supported by implants.¹ 

Sul et al²  have found that the most critical factor for optimal re-osseointegration is good surface chemistry. Although bone can re-osseointegrate around a previously infected implant,³  the surfaces of the implant need to be mechanically cleaned to remove any infectious biofilm. The bone also has to be free of pathogenic bacteria. As a way to avoid surface disturbance while decontaminating infected implants, the use of laser has been recently utilized with positive outcomes.⁴  The procedure involves the use of a laser with specified settings in combination with other traditional techniques to ensure effective antibacterial protocol. This protocol may have an improved antibacterial effect. However, it has not yet replaced the need for traditional mechanical removal of biofilm.⁴  New and effective treatment and prevention strategies are still needed to control this too costly disease.

At the microbiology level, many approaches for achieving better mechanical removal of biofilm have been investigated.⁵  Advances in biofilm adhesion and surface science have fostered research in biomaterials, the biochemistry of small molecules, and altering gene expression striving toward the same goal of weakening biofilm attachment.^6,7 

One of the relatively new modalities for fighting oral biofilms is photodynamic therapy (PDT). Photodynamic therapy is an established chemotherapy treatment for localized tumors that have been introduced recently to the dental field as a bactericidal strategy.⁸  Photodynamic therapy involves the application and retention of an applied photosensitizing agent (dye) in targeted tissues, followed by light/laser illumination with specific settings including energy density and illumination time that can activate the selected dye.^9,10  This light activation will create free radicals that lead to disruption of cancer cells in various ways affecting only the targeted cells and offering minimal disruption of healthy adjacent tissue. This affinity of the photosensitizing agent to cell walls was found to be applicable to bacteria in dental biofilms as these dyes selectively bind to the bacteria in biofilms attached closely to Ti surfaces.^9,11  The level of penetration of the photosensitizing agent to the targeted tissue is an essential factor for effective treatment. Hence, different photosensitizing agents are suggested based on the type of cancer tissue. Even though this may be less relevant for oral biofilm, yet it offers a variety of agents that can potentially be chosen based on the antibacterial effect on oral biofilms. One attractive feature of PDT as an antibacterial treatment is the use of harmless visible light of the correct wavelength to excite the photosensitizer. However, PDT creates the reactive oxygen species generated from the excited state of the dye that can chemically destroy many of the organic cell components by reactive oxidation. Thus, PDT, in addition to being antimicrobial, may also be destructive of the organic matrix components of the biofilm.

Since the electrostatic charge of any administered chemical drug may affect its ability to bind with cell walls, the charge of selected photosensitizer is heavily studied in the PDT literature. Polysaccharide (PSs) or exopolysaccharides (EPS) are known to be anionic (negatively charged), which increases bonding forces to bacteria in biofilms, allowing stronger resistance against antibacterial drugs.¹² 

Photosensitizers based on the cationic (positively charged) can exhibit high, broad-spectrum antibacterial activity regardless of current drug-resistance status; this is also applicable as an antifungal activity.¹³  Due to its effectiveness on biofilm, PDT use in dental settings has been increasingly investigated.^8,14,15  A commonly used broad-spectrum photosensitizer is methylene blue (MB). Methylene blue is a cationic and hydrophobic dye that has demonstrated a high killing rate when applied as a photosensitizer to a variety of subgingival bacteria using short illumination time (60 seconds) with moderate energy density (21.2 J/cm²), which resulted in prevention of the re-colonization of subgingival lesions by pathogenic microorganisms.¹⁶ 

Harris et al¹¹  have indicated that phenothiazinium-based photosensitizers, including MB, show selectivity for uptake into a tumor and microbial cells. This result appears to be based on electrostatic interactions between the positive charge generally carried by the MB molecules and the negative charge found on the outer surfaces of target cells. They found that this type of photosensitizer can inactivate several intracellular targets such as proteins and enzymes of the outer membranes/envelopes of the cells. Moreover, it can modify lipids and/or lipopolysaccharides.¹⁷  They explained that the hydrophobicity of MB allows interaction with cell wall lipids. Methylene blue leads to peroxidation that causes hydroperoxide formation, which increases ion permeability ending with Na+ and K+ leakage; these effects usually lead to cell lysis and death, making this type of photosensitizers antimicrobial agents without the added illumination.

George et al¹⁸  have suggested that electrostatic interactions and self-promoted uptake pathways mediate the uptake of cationic photosensitizer in contrast to anionic photosensitizer where the uptake can be affected by the nature of bacteria.

The effect of PDT on the attachment of cancer cells was studied in the field of photophysics and cancer medicine due to the concern of increasing risks of metastasis breaking from the treated tumor mass caused by attachment changes. In the dental field, few studies have focused on the effects of PDT on bioadhesion.^19,20  In the Soares et al¹⁹  study, they demonstrated the ability of PDT, toluidine blue O (TBO) mediated photosensitizer, with relatively high energy density to inhibit both the viability and adhesion of Candida albicans to buccal epithelial cells in vitro.¹⁹  Mang et al²⁰  have investigated the effect of PDT on the adhesion of gram-negative bacteria with promising results.

Of the many types of oral flora, one of the most studied bacteria is S mutans. Its sensitivity to PDT is well documented.²¹  It is a gram-positive, facultative anaerobic bacterium that plays a significant role in tooth decay. Studies on the use of PDT on S mutans biofilm are increasing due to its availability, and the relative ease of application.²²  Bacterial adhesion is particularly crucial for oral bacteria. Meurman et al²³  have reported that cell wall projections mediate S mutans adhesion to apatite crystals; these electron-dense structures form near the division site of this bacterium and are characteristically well-formed in S mutans of the strain used in this study. The reasons above made the S mutans attractive choice to use for this study.

Generally, removing or weakening biofilm attachment is favorable, whether the weakening is achieved by detaching the biofilm from a substratum or by damaging the attachments within the cohesive layers of the bacteria in a biofilm. Although detached cancer cells is a possible shortcoming of PDT in cancer treatment, it may present as an advantage in the case of oral biofilm.

The present study aimed to evaluate the capability of PDT to produce changes in the bioadhesion of S mutans biofilms to biomaterial surface. The attachment strength was determined by calculating the shear stress needed to remove biofilms of S mutans from commercially pure Ti. The ability of PDT to kill the bacteria of 3 various levels of maturity was also examined. This work tested the hypothesis that PDT induces oxidative embrittlement and fragmentation of plaque/biofilm matrix biopolymers, allowing more effective removal via hydrodynamic (rinsing) forces.

Two groups of commercially pure Ti samples were prepared to test (1) attachment strength and (2) antibacterial effect separately. For the attachment strength tests, large rectangular pieces size of 50 × 20 mm were used. Smaller pieces with a size of 5 × 10 mm for the antibacterial capacity test. All pieces were autoclaved and sterilized using radio-frequency glow discharge to obtain a clean and sterile surface.

Streptococcus mutans bacterial strain ATCC 27351 was used for this investigation. Bacteria were routinely grown in general-purpose Brain Heart Infusion (BHI) broth (Difco; Fisher Scientific, Co, LLC) at 37°C. Bacteria were transferred every 48 hours into fresh BHI for the entire study duration. Titanium pieces with the bacterial solution were incubated in an orbital shaker incubator (Lab-Line) at a continuous moderate speed of 125 RPM at 37°C.

After 48 hours of incubation in the shaker, the test pieces were transferred into another sterile test tubes filled with fresh BHI and mixed with 1% sucrose. The Ti pieces were then kept in the shaker incubator under the same conditions for up to 4 days. After biofilm growth, each Ti piece was then placed flat in a Petri dish and washed by adding 1 mL phosphate-buffered saline (PBS) and pipetting out the PBS. All pieces were grouped into a PDT treatment group, a control group, and a group to test the dark toxicity of photosensitizers.

The thicknesses of the biofilms varied between 0.190 and 0.524 mm, as measured using a calibrated microscope stage.

Each Ti piece was treated separately with a low concentration of MB (0.01%) in aqueous solution in a dark room at room temperature for 6 minutes. After incubation, excess MB was removed. The test pieces were then illuminated; excitation wavelength ranged from 660 to 675 nm laser (Periowave, Ondine Biomedical, Inc). The laser source (diode laser) was held at a distance of 4 cm to create a treatment diameter of 2.5 cm for about 3-minute exposure in total. Titanium pieces in the control group were kept in the dark for the same time.

Alamar Blue (AB), a vital dye, was used in this study for measuring the viability of treated biofilm.²⁴ 

After completing the PDT treatment, each piece was placed in BHI, and then provided with 10% AB in a darkened room then incubated for 9–20 hours to assess the viability and concentration of bacteria in the biofilm. Monitoring was done by recording absorbance at 570 and 600 nm.

The protocol used required culturing biofilm bacteria in agar plates and 100 μL of BHI + biofilm suspension diluted to 10%. Agar plates were then incubated for 2–4 days to be observed for colony forming unit (CFU) counting.²⁵ 

Multiple attenuated internal reflection infrared (MAIR-IR) spectroscopy is a sensitive surface characterization technique that allows the operator to characterize thin films of material on a face of a Germanium prism using infrared. Infrared spectra characterize the sample by revealing the functional groups of the material via their covalent bond resonances as the beam passes through the sample film.²⁶  This technique was used in this study to detect the change in biofilm characterization after PDT and the nature of detached and remaining material.

The germanium prisms used for this analysis were prepared with the same steps of Ti sample preparation. Biofilms were grown on the prisms in the same manner as they were on the Ti samples.

It should be mentioned that both bioadhesion and vitality tests were done on the following: (1) PDT treated samples (MB + laser), (2) MB only incubated control samples (no laser), and (3) Biofilm control (no MB, no laser).

Jet impingement is a precise yet simple engineering technique for measuring the shear stress required to remove layers from a solid substratum. In this study, jet impingement was used to accurately calculate the adhesion strength required to detached biofilm layers from the Ti substrata. By using a known flow rate of the fluid through a needle with a known nozzle diameter and a known distance between the surface and the nozzle exit, a standard detachment strength vs circle-size of the removed material are plotted in graphs to acquire shear stress.²⁷  Two different flow rates were used for this experiment 10 mL/s and 20 mL/s (Figure 1).

For future references in this study, 2 separate areas of detachment will be distinguished: (1) central circles of the detached biofilm from Ti base (visually clean circles), and (2) the halos surrounding within-biofilm-matrix (partially cleaned circles where the film had not been entirely detached from the solid substratum (Figure 2).

Biofilms on Ti samples (treated and controls) were dyed with crystal violet and incubated for 8–10 minutes to aid in the visualization of the biofilms under light microscopy.

Each piece was securely fixed flat under the nozzle opening. Distilled water then impinged for 30 seconds at each biofilm. Titanium samples were air-dried. Diameters of circles of detachment were measured by light microscopy. The resultant detached diameters of (central circles) and (halos) were measured and used to calculate shear stresses (Figure 2).

Photos of biofilms specimens were taken by reflected light microscopy (ZEISS), scanning electron microscopy, and stereo light microscopy (Olympus).

Independent samples t test was applied using (SPSS) program at (P < .05). At least 8–10 samples in each group were used for the viability test (total of above 100 samples). For the bioadhesion test, measurements were calculated for 43 samples. Each experiment was repeated 3 times on 3 different occasions. Pilot work was done before designing the final experiment. T test showed a significant difference in shear stress for biofilm at all maturity levels except at the peak stationary phase (48 hours). For the younger and older biofilms, P values ranged from .00 to .025, whereas insignificant P values ranged from .13 to .98. for the stationary peak phase of 48 hours.

The spectra obtained from treated specimens showed that shear stress of approximately 10 dynes/cm² was able to remove substantially more material from treated biofilm compared with control. These results were represented in MAIR-IR by showing lower amounts of retained protein, carbohydrate, and lipid components (smaller peaks) in the PDT treated biofilm than for the nontreated controls (more massive peaks). Moreover, a comparison of the spectrum peak heights indicated significantly more removal of PSs extracellular polymers than the removal of the protein-dominated microbes themselves. Characteristic spectra are shown in Figures 3 and 4.

To test the effect on various ages of biofilms, 3 different stages of maturity of bacteria starting the biofilm formation were used in this study; younger biofilms started with 24 and 48 hours (1 + 2 days old of 50% each), older biofilm starting at 48 and 72 hours (2 + 3 days old 50% each), and at stationary peak growth of 48-hour-old biofilm.

Vitality was measured using AB.²⁸  Dark toxicity of MB (MB with no laser) to S mutans was observed. Photodynamic therapy mediated by MB showed antibacterial capacity on all stages of biofilms; however, the results were variable. Biofilms started with older (2 and 3 days old) showed the most sensitivity for PDT antibacterial capacity. This was parallel to the bioadhesion results (Figure 5).

Photodynamic therapy also showed a significant bactericidal effect as well on younger biofilm.

Biofilms started with bacteria cultured for precisely 48 hours (stationary phase): This group of biofilms was the most resistant to PDT treatment, both the antibacterial and the adhesion (Figure 6).

The effects of water jets were noticeably different between PDT-treated biofilms and the control samples in all groups. Results confirmed the difference in shear stress values between the treated biofilms and the control samples. Moreover, the jet penetrated more in-depth into the treated samples confirmed visually. The smallest detachment was observed at 48 hours. For this group, the statistical difference in shear stress was not significant. However, the penetration depth was visually significant (Figures 7 and 8). These results support the hypothesis of the ability of MB-mediated PDT to weaken biofilms of S mutans.

Interestingly, thicker biofilms demonstrated more attachment strength only in the control group; the treated biofilms were almost all weakened even in the thickest areas.

The removal of various biofilms cultures of bacteria from the Ti substrata (centrally detached areas) required mean shear stress of 143.3 dyne/cm² for control samples and 89 dynes/cm² for treated samples. Only 67.2 and 51 dyne/cm² were required to remove the biofilm from the halos for the control and treated samples, respectively (Table).

The PDT treatment was primarily effective in weakening the attachment of the extracellular slime components of these biofilms.

This study aimed to focus on the effect of PDT on biofilm adhesion. The killing effect of PDT on various bacteria is well documented in the literature.²⁹  Nevertheless, the bactericidal effect was also studied in a parallel manner to test the existing relation between the strength of biofilm adhesion and the activity level of the living organisms. The PDT and adhesion tests were performed on biofilms of different stages of maturity as a way to create variable factors closer to the natural settings. It also gives more validity to the claim that suggests a possible relationship between the activity level and attachment strength of bacteria in a biofilm. The results of this work confirmed the susceptibility of biofilm made from S mutans on Ti to PDT as a bactericidal and embittering method of the film. Methylene blue was selected for this work for its wide range of bactericidal effect. Methylene blue has also been tested on S mutans, showing promising results despite its concentration.³⁰ 

Other photosensitizing agents can be used on biofilms; Zanin et al²¹  also showed that using erythrosine on S mutans was 5–10 times more effective on killing than when using MB. They also indicated that PDT, for this bacterium, kills mainly by damaging the outer membrane.

During the pilot testing for this work, it was decided to add sucrose to the growing biofilms as it allowed the needed visualization of the biofilm, especially by the increased “fluff,” which was mainly layers of PSs. The pilot testing on S mutans bacterial showed that 48 hours was the stationary peak growth and activity of the used strain.

Methylene blue showed a high bactericidal effect on S mutans as well, even when used alone, confirming dark toxicity. Particularly since these bacterial cultures (provided with sucrose) had a low pH of 4.2, which is within the range for optimal MB effects/uptake.³⁰ 

Gad et al³¹  have demonstrated that lethal photosensitization can be reduced by the presence of EPS explained by the EPS “trapping” of the photosensitizer on the outside of the cell membrane, which is thought to be one of the critical sites of PDT-mediated damage. However, they observed that the absolute uptake of photosensitizer by the cells was 10 times higher when a cationic photosensitizer was used compared with an anionic counterpart.

It should be mentioned that MB can inactivate bacterial cells when it engages actively to the outer membrane; it can eventually impair the cell function by accumulating in the outer wall when it is highly concentrated within the EPS.³¹ 

This can explain the high level of dark toxicity in this work demonstrated by MB, showing a significant killing ability when used alone. These results are in agreement with previous work that showing even inactivated MB alone kills microbes by membrane destruction.³² 

In this work, shear stress measurements indicated that the force required to remove the biofilm from the Ti substratum was higher than the force needed to separate and remove the bacteria within the EPS matrix of each biofilm. This means that detaching the biofilm within its bulk is less difficult than removing the biofilm from the Ti substratum. The most durable adhesion of the biofilms tested here was the adhesion that occurred between the bacteria and the solid surface. Results could be different if a substratum of different material, perhaps one with “easy release” properties, were used.³³ 

This observation also supports that the MB induced PDT effect likely involves breaking down the slime layer's cohesion rather than reversing the active bacterial adhesion to the underlying substrata.

The results of PDT bactericidal effect and adhesion strength were reasonably parallel in this work; the stronger the bioadhesion of biofilms, the lower the bactericidal effect of PDT. Part of these results corresponded with the results of Wood et al,²⁸  who found that “young” biofilms of S mutans were more resistant to PDT than “older” biofilms due to more superficial penetration of the dye in the former. However, these results do not correlate with results of Zanin et al²¹  who found that younger biofilms of S mutans were more sensitive to PDT than older ones when treated with TBO. The vital difference between Zanin et al²¹  and the present study is that for this current study, the age variation was among the cultures that started the biofilms and not in the age of biofilms.

It has been documented in the literature that one of the reasons for oral biofilm resistance to PDT is the reduced may be to the level of photosensitizer penetration.³³  This may indicate that when MB penetrates deeper, its biological and biomolecular effects become stronger, resulting in both more substantial destruction in the deeper layers of a biofilm and more killing of bacteria overall.

The variable bactericidal effects of PDT on biofilm of different ages could be due to bacterial concentration differences in younger and older biofilms when compared with biofilm at the stationary peak phase. The bacterial density in each biofilm would be different between the different phases. Given a greater amount of EPS/bacteria ratio, using the same given PDT dose will produce higher killing in the biofilms with lower bacterial counts.

Although the total bacterial concentration to start biofilms with was equal in all experiments, when 2 different phases were mixed, probably only the bacteria of the stationary (48 hours) phase were attached and spread to a considerable extent leaving no space for the bacteria in an exponential phase that was probably busy dividing and competing for the available nutrients. This makes only half of the total S mutans in the bacterial suspension available to initiate the adhesion process and leading to a very weak signal of AB assay.

The inability of water jets to altogether remove the biofilms from the germanium substrate points out the difficulties of “complete” removal of a biofilm from a substratum with relatively high critical surface tension such as implant surface.

Therefore, future research focusing on enhancing mechanisms to affect the adhesion of biofilms to implant surface rather than the cohesion within bacteria is highly recommended. It is unlikely that chemical agents such as photosensitizer would affect the primary adhesion, which takes place between the surface and the bacterial cells that are influenced by physical interactions (hydrophobic, electrostatic).

The results of this work are consistent with proposals that PDT induces oxidative embrittlement and fragmentation of plaque/biofilm matrix biopolymers, allowing more effective removal by hydrodynamic (rinsing) forces. The results of this study may direct future clinical investigations for treating peri-implantitis using laser therapy combined with irrigations.

Oral biofilm is unique as the mouth is an open cavity where rinsing can detach the biofilm. Unlike the health concerns of loose biofilm within a closed human cavity, detaching the strongly adhered oral biofilms is as important if not more important than killing the bacteria within the biofilm. This project involved laboratory experiments conducted by a team of a clinician, scientists, and photophysics experts as an essential collaboration to resolve a complex clinical problem that cannot be answered by looking for a simple answer from 1 discipline. It is essential to bridge the in-vitro studies with potential clinical research possibilities to translate necessary knowledge to clinicians who deliver health care. Photodynamic therapy is not only able to kill oral flora, but it has the potential to weaken the biofilm around peri-implantitis for more accessible biofilm release from a delicate biomaterial surface.

Abbreviations

AB:

Alamar Blue

BHI:

Brain Heart Infusion

CFU:

colony forming unit

EPS:

exopolysaccharide

IR:

infrared

MAIR-IR:

multiple attenuated internal reflection infrared

MB:

methylene blue

PBS:

phosphate-buffered saline

PDT:

photodynamic therapy

PSs:

polysaccharide

SEM:

scanning electron microscopy

TBO:

toluidine blue O

Ti:

titanium

The authors would like to acknowledge Dr Robert Baier for his help in statistical analysis. This work was done as a requirement for the Master's degree.

No authors report any conflicts of interest.