Tooth Caries BM 515 LASER-TISSUE INTERACTION MECHANISMS TAKE HOME FINAL PROJECT – 27.05.2018 Sabra Rostami – 2016885027 Outline Definition of the Disorder Definitions Disease Statistics Prevention of Dental Caries and Conventional Treatment Methods Can dental caries be prevented How are dental caries treated Replacement of Fillings Unconventional Methods for Diagnosis and/or Treatment of Tooth Caries Light Transmission in Tooth Laser induced fluorescence Laser-Based Treatment Methods 1- DIAGNOdent 2- Quantitative light-induced fluorescence 3- Digital imaging fiber-optic transillumination 4- Photodynamic Therapy For Tooth Caries Selection of appropriate photosensitizers against specific groups of dental caries bacteria Selection of appropriate light sources various photosensitizers Novel Alternative Diagnosis and Treatment Method Design of the Laser System Including Laser Delivery to the Target Tissue References In summary, the tooth surface normally loses some amount of its minerals due to reaction between bacterial plaque-produced acid and the outer surface of the tooth. The acid is produces as a result of ingestion of foods containing fermentable carbohydrates by bacteria. This mineral loss is normally restored between meals with the assistance of saliva. However, in case fermentable foods are consumed regularly, acid production becomes more and more routine resulting in a constant low pH environment in the plaque, therefore a considerable net loss of tooth mineral occurs. The low pH works in favor of aciduric organisms, namely S. mutans and lactobacilli, which (especially S. mutans) are highly prone to storing polysaccharide and secreting acid long after the food is swallowed. (Fejerskov and Kidd 2008 Walter J. Loesche. 1996) Stages in development of caries Calcification of enamel Appearance of a small white spot on the tooth Discoloration becomes noticeable Softening of the tooth surface followed by penetration of decay through the enamel and into the dentine Lateral and in depth spread of caries Cavitation takes place Deepening of the lesion happens affecting the pulp (e.g.sweets, temp.), resulting its damage and eventually death of the pulp Bacteria moves down the root canal, reach the apex and cause abscesses Figure 1. Principal progress of mineral loss in relation to time. As mentioned above, the term dental caries is describes signs and symptoms that result in localized chemical dissolution and destruction of the tooth hard tissue due to metabolic changes of pH level which take place in the dental plaque covering the lesion area. Biofilm is formed when microorganisms (in particular bacteria) residing in the oral cavity adhere to surfaces forming somewhat impenetrable matrix. Biofilms have firm and spatially organized structures, consisting combination of interacting microorganisms where characteristics and properties of bulk of biofilm do not match with that of its simple constructing components. Structural hardness of the biofilm increases as it gets older as does its resistant to environmental attacks. Mechanical disruption is the most effective and widely used methods for destruction of matured biofilm in which the interbacterial protective matrix is disrupted resulting in disintegration of bacterial colonies. (Fejerskov and Kidd 2008 Sbaraini et al. 2016) Nowadays there are some widely used antiseptic agents in order to prevent formation of dental plaque which are also effective in hindering maturation of the existing bacterial plaque. Unfortunately colonies of matured biofilms are rarely affected by antimicrobial agents due to several physical and biological factors that preserve integrity and structural organization of the plaque. Hence in order to terminate matured bacterial colonies, significantly higher doses of antiseptic agents are needed along with longer exposition durations. (Fejerskov and Kidd 2008 Sbaraini et al. 2016) Antiseptic agents, when used in a conventional dose and recommended time duration, can only eliminate the superficial bacteria and leaving the bacteria embedded in the deeper areas of the biofilm intact. Therefore, the effectiveness of any antiseptic mouth-rinse agent depends not only on its microbiocidal properties determined in vitro, but also on its ability to disrupt the well-organized biofilm. Difficulty in biofilm penetration poses a serious problem in prevention and treatment of dental caries. (Fogorv Sz. 2008 Fejerskov and Kidd 2008) Tooth caries lesions can develop at any point on the tooth provided that a biofilm develops and remains intact for a considerable time duration. It is therefore a misinterpretation to talk about more or less susceptible surfaces as this may mistakenly give rise to the belief that certain parts of a tooth are more resistant or less susceptible to developing caries lesions due to variations of their chemical and structural composition (Black, 1914 Weatherell et al., 1984). This is not to say that all tooth surfaces within the oral cavity of an individual develop caries lesions at the same rate. Dental caries lesions develop at relatively protected sites in the dentition where biofilms can accumulate and mature over time without any disturbance or perturbation. The potential sites for biofilm growth are pits, grooves and fissures in occlusal surfaces, especially during eruption, approximal surfaces cervical to the contact point/area and along the gingival margin. (Fejerskov and Kidd 2008) Insertion of foreign bodies to the dentition (e.g. fillings with inappropriate margins, dentures, orthodontic bands) may also result in such protected sites. These areas are relatively protected from mechanical forces applied by the tongue, the cheeks, abrasive foods and, most important of all, tooth brushing. Thus, at these spots, where lesion development is more likely to occur, the biofilm is allowed to constipate there for prolonged periods. (Almaz and Snmez 2015 Fejerskov and Kidd 2008 Sbaraini et al. 2016) The biofilm is characterized by continued microbial activity resulting in continued metabolic events in the form of minute pH fluctuations. The metabolism may be dramatically enhanced by changing the nutritional conditions, e.g. by adding fermentable carbohydrates, and the results of the metabolism can be recorded as pH fluctuations. Any shift in pH will influence the chemical composition of the biofilm fluid and the relative degree of saturation of this fluid with respect to the minerals that are important for maintaining the chemical composition of the tooth surface. (Fejerskov and Kidd 2008) These considerations lead to some important points The dissolution (demineralization) when pH drops below a certain level in the biofilm and the redeposition (remineralization) of minerals when pH goes up, take place in the enamel surface at the interface between the biofilm and the tooth surface. These processes occur numerous times during a day and can be modified extensively. If, for example, the biofilm is partly or totally removed mineral loss may be arrested or even reversed towards mineral gain because saliva is supersaturated with respect to the enamel apatite. This will result in arrest of disease progression, and may even result in some redeposition of minerals in the very surface of the tooth. (Fejerskov and Kidd 2008 Selwitz, Ismail, and Pitts 2007) Any factor that influences the metabolic processes, such as the composition and thickness of the biofilm, the salivary secretion rate and composition, the diet and the fluoride ion concentration in the oral fluids will contribute to determine the likelihood of a net loss of mineral, and the rate at which this occurs. Figure 2 indicates how the many determinants of the caries process may act at the level of the individual tooth surface (inner circle) the strictly biological determinants or at the individual/population level (outer circle) in the form of behavior, education, knowledge and attitudes the eterminants of the strictly biological determinants. (Fejerskov and Kidd 2008 Selwitz, Ismail, and Pitts 2007) At any given point in time the net mineral loss or gain is part of a continuous spectrum of events. The absence of a clinically detectable caries lesion does not necessarily mean that no mineral loss has occurred, it only means that it could not be discerned clinically. If this concept of a continuum is appreciated it will immediately be understood why diagnosis of various stages of lesion progression is a question of defining certain cut-off points. (Fejerskov and Kidd 2008 Selwitz, Ismail, and Pitts 2007) Figure 2. Schematic illustration of the determinants of the carious process. Those that act at the tooth-surface level are found in the inner (green) circle. In summary factors determining the incidence of caries are Micro-organisms Acid producing bacteria, especially if they produce extracellular polysaccharides, will increase risk. Host factors Decreased saliva secretion increase incidence. Buffering power of saliva to raise pH decrease incidence. The morphology of the teeth well-spaced teeth decrease incidence, while fissures pits increase it. The composition of the teeth certain trace elements decrease incidence (e.g. F, Mb and B), while others (e.g. Cu Mn) increase it. Time frequency of consumption of carbohydrates, and length of time in the mouth are related to incidence. Good oral hygiene can counteract this. Substrates for acid production provided in diet. Disease Statistics Dental caries is one of the most prevalent chronic diseases in the population worldwide, affecting 6090 of schoolaged children and almost 100 of the adult population. The prevalence of dental caries has been studied in many developed countries in recent years. In the USA, caries was considered the most common chronic disease of childhood, being five times more common than asthma, with a prevalence of 27 in preschoolers, 42 in school-aged children, and 91 of dentate adults. (Fujimaki et al. 2013) Prevention of Dental Caries and Conventional Treatment Methods Can dental caries be prevented Dental caries is clearly an infectious bacterial illness, but it can be avoided by good prevention. It is important to adopt good habits as early as possible Prefer water to sugary drinks Avoid snacks between meals Do not eat food after brushing teeth before going to bed Brush teeth regularly and include a method of interdental cleaning Regular screening by a dentist is indispensable and it is recommended at least once a year. How are dental caries treated Early treatment of dental caries and new technology enable today less invasive and less expensive treatment. The evolution of dental material offers the possibility of composite resin fillings that are resistant and aesthetic (white). (World Health Organization 2009) When the dental caries is not too advanced, the treatment objective is to preserve the living tooth. Under local anaesthetic, the dental surgeon removes the dental caries with different drills (mostly mechanical drill, occasionally laser-based ablation, etc.), then rebuilds the tooth with white composite resin (now replaces grey metallic amalgam fillings). (World Health Organization 2009) Replacement of fillings Dental amalgam contains mainly mercury and silver, as well as a lower quantity of other metals. Dental caries can develop under old amalgam fillings without being visible to the naked eye or even by X-ray. In addition, these old filling techniques lead to cracks over the years, and even dental fractures. The replacement of old amalgam fillings by composite resin is therefore often recommended. (World Health Organization 2009) The most frequent question raised is the eventual toxicity related to the replacement of amalgam. At present, there is no undisputed scientific evidence to confirm the reality of this toxicity. For this reason, we do not recommend a systematic replacement of these fillings if in a good state and, at low risk of provoking dental fractures. (World Health Organization 2009) Unconventional Methods for Diagnosis and/or Treatment of Tooth Caries Caries diagnosis implies more than just detecting lesions. Since diagnosis is a mental resting place on the way to a treatment decision, it is intimately linked with that treatment plan. Thus, diagnosis must include an assessment of activity because active lesions require active management (non-operative and operative treatment), whereas arrested lesions do not. (Baffi, Almeida Rodrigues, and Lussi 2012) Herein lies a problem. How can activity be assessed The detection process may miss lesions (false negatives) or say lesions are present when they are not (false positives). The assessment of activity may be similarly wrong. It seems logical to suggest that attempting this at a single point in time simply adds to the problem. (Baffi, Almeida Rodrigues, and Lussi 2012 Fejerskov and Kidd 2008) Would it not be easier to be able to monitor lesion progression or arrest over time It is known that clinicalvisual diagnosis may be susceptible to longitudinal monitoring even though the assessment is qualitative. (Baffi, Almeida Rodrigues, and Lussi 2012) All quantitative methods for caries detection are based on the interpretation of physical signals. These are causally related to one or more features of a caries lesion. Table 1 shows some of the most common types of physical principle that may be used and the corresponding diagnostic methods. Table 1. Overview of some diagnostic methods In this report, X-Ray based and Electrical Current methods will not be discussed and only Light based methods are introduced in detail. Before introducing light-based diagnostic methods, the key concepts that must be discussed first are Light Transmission in Tooth and Laser light-induced fluorescence. Light Transmission in Tooth Sound enamel consists mainly of carbonate-rich and calcium-deficient hydroxyapatite crystals which are very densely packed, giving the enamel a glass-like, translucent appearance. The yellowish white color of teeth is the result of the dentin shining through the translucent enamel layer. Light that shines on a tooth will, in part, penetrate the tooth and is scattered or absorbed inside. Scattering is the process in which the direction of a photon is changed without loss of energy. Absorption is the process in which photons lose their energy, mostly by conversion into heat. Since scattering does not cause the light to be lost, scattering may occur many times consecutively along the path, a phenomenon called multiple scattering. After one or more scatter events, a photon may reach the tooth surface again and leave the tooth. Back-scatter is the phenomenon where photons leave through the surface by which they entered. When photons leave through another surface, the phenomenon is called diffuse transmission. In a sound tooth, scattering is much more probable than absorption. (Ten Bosch and Coops 1995 Fejerskov and Kidd 2008) In dentin, both scattering and absorption occur more frequently along the light path than either occurs in the enamel. The whitish appearance of teeth is due to the fact that absorption is much lower than scattering (ten Bosch, 1996). Primary teeth show more scattering and therefore have a whiter appearance than permanent teeth. In a white-spot carious lesion, scattering is stronger than in sound enamel. The penetrating photons change direction more often in carious enamel than in sound enamel and are generally back-scattered before they reach the dentin. Therefore, such a lesion appears whiter than the surrounding sound parts of the tooth. (Ten Bosch and Coops 1995 Fejerskov and Kidd 2008 Sbaraini et al. 2016) Brown lesions are due to the presence of light-absorbing material in the lesion and/or exogenous stain. A slight increase in enamel porosity leads to a change in the optical properties of enamel in such a way that light is increasingly scattered. This is presumed to be primarily due to the fact that the remaining small mineral particles in the lesion are embedded in water rather than in mineral-rich sound enamel (Angmar-Mnsson ten Bosch, 1987), thereby increasing the difference in refractive index (RI) between the scattering photon and its environment. The RI of enamel apatite is 1.62, and the RIs of water and air are 1.33 and 1.0, respectively. Thus, when the pores of a white spot enamel lesion are filled with water, the light scattering is less than when the lesion is dry and the pores are filled with air. After dehydration of enamel it looks whiter, as a result of more scattered light. (Fejerskov and Kidd 2008Angmar-Mnsson ten Bosch, 1987) Laser induced fluorescence Laser light is composed of electromagnetic waves with equal wavelengths and equal phases. Some materials possess the characteristic of fluorescence when illuminated with (laser) light. Fluorescence is a phenomenon by which the wavelength of the emitted light (coming from the light source) is changed into a larger wavelength as it travels back for detection. The larger wavelength is caused by some loss of energy to the surrounding tissue and therefore will have a different color from the emitting light. By using a filter through which only the fluorescent light may pass, the intensity of the fluorescent light can be measured. The intensity of the fluorescent light is proportional to the amount of material that causes the fluorescence.(Tranaeus et al. 2001 Dye et al 2011) The fluorescence of dental hard tissues has been known for a very long time (Benedict, 1928). Spectra have been presented by several authors (Armstrong, 1963 Spitzer ten Bosch, 1976 Alfano Yao, 1981 Hibst et al., 2001). Three types of fluorescence can be distinguished in dental hard tissue blue fluorescence excited in the near ultraviolet, yellow and orange fluorescence excited in the blue and green, and red fluorescence excited in the far red and near infrared. The chromophores causing the fluorescence of dental hard tissues are not clearly identified. (A chromophore is a molecule that gives an object color by selectively absorbing light at particular wavelengths.) (Fejerskov and Kidd 2008 Sbaraini et al. 2016) Based on the research results of many studies over the past centuray, the blue fluorescence is assigned to dityrosine (Booij ten Bosch, 1982). It seems likely that most of the yellow fluorescence stems from proteinic chromophores, probably cross-links between chains of structural proteins (Scharf, 1971). It has also been discussed whether or not the apatite of dental hard tissues would contribute as well (Spitzer ten Bosch, 1976 Hafstrm-Bjrkman et al., 1991). The redinfrared fluorescence has been assigned to a protoporphyrin, which is present as a bacterial breakdown product (Knig et al., 1998). Dental enamel and dentin possess the characteristic of fluorescence and this natural fluorescence is called autofluorescence. Caries lesions, plaque and microorganisms also contain fluorescent substances. The difference between the fluorescence of sound tooth tissues and that of a caries lesion can be made visible by the DIAGNOdent and by the quantitative laser- or light-induced fluorescence (QLF) method. Other laser-based methods recently explored and used in tooth caries are digital imaging fiber-optic transillumination and Photodynamic Therapy (PDT). (Fejerskov and Kidd 2008 Tranus et al. 2002) Laser-Based Diagnostic and/or Treatment Methods 1- DIAGNOdent When red light with a wavelength of 655 nm is applied, caries-induced changes in teeth lead to increased fluorescence (Hibst et al., 2001). The DIAGNOdent (KaVo Biberach, Germany) is based on this principle. The fluorescent light is measured and its intensity is an indication of the depth of the caries lesion. The intensity of the fluorescent light is displayed as a number ranging from 0 to 99, with 0 indicating a minimum and 99 a maximum of fluorescent light (Figs 3 and 4). Figure 3. DIAGNOdent showing real-time and maximum (peak) digital display. The device consists of a probe, a fiber-optic lead, and a unit containing the electronics and the laser diode. Figure 4. Procedure for occlusal detection with the DIAGNOdent. (a) After calibration is accomplished the zero value is measured on a sound tooth surface. (b) The tip of the laser device has to be carefully rotated around the vertical axis until the maximum fluorescence value of the site under study is found. The threshold between occlusal caries limited to enamel and caries into dentin was found to be around 18 under humid conditions (Lussi et al., 1999, 2001 Shi et al., 2000). Clinically visible white-spot lesions are measurable with this device. However, very initial lesions, with no fluorophores from bacteria present, are not captured by the DIAGNOdent. The same registration under dry conditions led to higher cut-off points (Shi et al., 2000 Lussi et al., 2005). Thus, it would be important to standardize hydration conditions for longitudinal measurements. Based on systematic reviews of the performance of DIAGNOdent for detecting caries it was concluded that DIAGNOdent is more sensitive than traditional diagnostic methods (Bader Shugars, 2004 Lussi et al., 2004 Ricketts, 2005). However, the increased likelihood of false-positive diagnoses when using the DIAGNOdent means that it should not be relied on as a clinicians primary diagnostic method. (Fejerskov and Kidd 2008) DIAGNOdent has also been tested in vitro and in vivo for measurement of carious lesions adjacent to fixed orthodontic appliances (Aljehani et al., 2004, 2006 Staudt et al., 2004). Other studies have addressed further possible clinical applications the detection of recurrent caries (Boston, 2003 Ando et al., 2004 Bamzahim et al., 2004, 2005), residual caries (Lennon et al., 2002), caries under sealants (Takamori et al., 2001 Deery et al., 2006), root caries (Wicht et al., 2002, 2003) and detection of subgingival calculus (Krause et al., 2003). 2- Quantitative light-induced fluorescence The light scattering in the lesion, which is much stronger than in sound enamel (ten Bosch, 1996), causes the light path in the lesion to be much shorter than in sound enamel thus, the absorption per unit of volume is much smaller in the lesion and the fluorescence is less strong. Figure 5. Clinical use of quantitative light-induced fluorescence (QLF). To enable calculation of fluorescence loss in the caries lesion, the fluorescence of the lesion is subtracted from the fluorescence of the surrounding sound tissue. (Der Veen and De Jong 2000)The difference between the actual values and the reconstructed ones gives the resulting fluorescence loss, as demonstrated in Fig. 6. Figure 6. Principles of the quantitative light-induced fluorescence (QLF) method for quantification of an enamel caries lesion. (a) The actual fluorescence image of a caries lesion (b) the reconstructed image, in which fluorescence radiance of the original sound enamel at the lesion site was reconstructed by interpolation of values indicating sound enamel around the lesion. (c) The difference between the measured and the reconstructed values gave the resulting fluorescence loss in the lesion. Fig. 6a. shows the actual fluorescence image of a caries lesion Fig. 6b. shows the reconstructed image in which the fluorescence of the original sound enamel at the lesion site was taken from the fluorescence of the sound enamel around the lesion. The difference between the measured and the reconstructed values gave the resulting fluorescence loss in the lesion (Fig. 6c). From this, three lesion quantities may be obtained mean fluorescence loss over the lesion (in percent), maximum fluorescence loss in the lesion (in per cent) and area of the lesion (in square millimeters). Attempts to adapt the QLF method for occlusal caries diagnosis have been made. Preliminary results comparing the QLF method with other diagnostic methods showed that QLF was more sensitive than electrical conductance for measurements of shallow occlusal lesions (Tranus et A clinical validation study determined the ability of DIFOTI to detect primary caries (Ando, 2006). Deciduous molars of 119 children (aged 812 years) were examined at 6-month intervals throughout a 2-year study period. Exfoliated teeth were collected for the validation of lesion presence and depth using polarized light microscopy as the gold standard. Results indicated that lesions involving over half of the enamel were better detected than lesions restricted to only the outer half of enamel for both smooth and occlusal surfaces. In other words, DIFOTI may not be able to detect small lesions, such as lesions within half of enamel thickness, and this is not as good as a visual examination. Occlusal caries detection was better than smooth-surface caries detection. Figure 7. Digital imaging fiber-optic transillumination (DIFOTI) for the detection of occlusal caries. 4- Photodynamic Therapy For Tooth Caries Over the past years, photodynamic therapy studies have shown promising results for inactivation of microorganisms related to dental caries. A large number of studies have used a variety of protocols and few studies have analyzed photosensitizers and light source properties to obtain the best PDT dose response for dental caries. Three questions come to mind when discussing primary aspects of PDT. The first involves the photosensitizer properties and their performance against Gram positive and Gram negative bacteria. The second discusses the use of light sources in accordance with the dye maximum absorbance to obtain optimal results. The third looks at the relevance of photosensitizer concentration, the possible formation of self-aggregates, and light source effectiveness. (Baptista et al. 2012 Cheng et al. 2016 Lee et al. 2013) It has been demonstrated that (i) some groups of photosensitizers may be more effective against either Gram positive or negative bacteria, (ii) the light source must be appropriate for dye maximum absorbance, and (iii) some photosensitizers may have their absorbance modified with their concentration. For the best results of PDT against the main cariogenic bacteria (Streptococcus mutans), a variety of aspects should be taken into account, and among the analyzed photosensitizer, erythrosin seems to be the most appropriate since it acts against this Gram positive bacteria, has a hydrophilic tendency and even at low concentrations has photodynamic effects. Considering using erythrosine as the photosensitizer, the most appropriate light source should have a maximum emission intensity at a wavelength close to 530 nm, which may be achieved with low cost LEDs.(Nagata et al. 2012) Table 2. represents summary of studies about antibacterial photodynamic therapy for dental caries until 2012. Table 2. Summary of Studies Regarding Antibacterial Photodynamic Therapy for Dental Caries Over the years, several groups of photosensitizers in different illumination systems have been proposed. Even when the same photosensitizer and light source were employed, the diversity of irradiation protocols and variation of photosensitizer concentration, irradiation time, and light potencies makes comparison between the results difficult. Unfortunately, very few studies discuss both the structural properties of photosensitizer and of the light sources to specifically achieve the optimal protocol for this therapy against dental caries. In order to find the optimum light source and its characteristics, compression must be made based on data. Table 3. represents three main groups of PDT light sources and their characteristics. Table 3. Three main groups of PDT light sources and their characteristics Selection of appropriate photosensitizers against specific groups of dental caries bacteria The main organisms recognized as associated with early caries development are the Streptococci mutans group (particularly, S. mutans and S. sobrinus) and lactobacilli species.(Van Houte1994) As the lesion progresses to deeper dentin, anaerobic species start to thrive and a transition takes place from predominantly facultative Gram positive bacteria to strictly anaerobic Gram positive rods and cocci, and Gram negative rods. (Hoshino 1977) From the point of view of bacteria and PS interaction, the effectiveness of PDT is mostly related to three main aspects Photosensitizer capability of interacting with the bacterial membrane Photosensitizer ability of penetration and action inside the cell, and Reactive singlet oxygen formation around the bacterial cell by illumination of the photosensitizer. The cell membrane binding mechanism is different in Gram positive and Gram negative bacteria. This difference can be explained by structural variations in their cell walls, and hydrophobic and charge effects of the photosensitizers. Gram negative bacteria present a complex outer membrane which includes two lipid bilayers that work as a physical and functional barrier between the cells and the environment, while Gram positive cells have a thick membrane that is relatively permeable. It is possible that this relatively porous layer of peptidoglycan and lipoteichoic acid outside the cytoplasmic membrane of Gram positive species allows the photosensitizer to diffuse into sensitive sites. In general Gram negative species are significantly resistant to some commonly used photosensitizers in PDT. According to the literature (Table 2), effective PDT results have been evaluated and obtained against Gram positive species, which are more susceptible, because they have no protective external membrane. (Maisch 2007 Nagata et al. 2012 Walsh 2003) Besides these structural differences, photosensitizer charge may influence the inactivation of Gram positive and Gram negative specimens. In general, it is assumed that at physiological pH, neutral or anionic compounds, such as rose bengal, erythrosin, eosin, porphyrin derivatives (Photofrin TM, Photosan TM, and Photogem TM), and aluminum disulphonated phthalocyanine bind efficiently and inactivate Gram positive bacteria, while in Gram negative bacteria, these photosensitizers bind to the outer membrane to some extent but do not effectively inactivate them after illumination. (Nagata et al. 2012) Another PDT mechanism is the photosensitizer penetration and action inside the cell. This is possible due to the hydrophilicity and solubility of the dyes, which determine how readily they cross the cellular wall. (Usacheva, Teichert, and Biel 2001) Gram positive bacteria protect their cytoplasmic membrane with a thick multilayer peptidoglycan wall that blocks the passage of hydrophobic components because of the presence of amino acids and sugars within the cell membrane. Therefore, only hydrophilic components penetrate this wall. In contrast, Gram negative bacteria have one or a few layers of peptidoglycan and an external membrane. Because this membrane presents lipoproteic character, special mechanisms such as the passage through pores are necessary to allow the intake of hydrophilic components. Consequently, hydrophobic components are expected to penetrate the cell better than hydrophilic ones. (Nagata et al. 2012) In general, the dyes may either have a more hydrophilic or hydrophobic character or may be amphiphilic. The literature considers that rose bengal, MB and TBO present amphiphilic character (both hydrophobic and hydrophilic), of which rose bengal has a more hydrophobic character, and MB and TBO have a more hydrophilic character. (Usacheva et al. 2001) Concerning the penetration of the photosensitizer into the bacteria, the water solubility of the dye must also be taken into account, since hydrophilic photosensitizers have a higher penetration in Gram positive bacteria, while hydrophobic dyes have a higher penetration in Gram negative bacteria. Moreover, if toxic and reactive products of the PDT can be created near the bacterial membrane, effective inactivation may be observed, even without the direct bacterium-photosensitizer contact. Thus, among these aspects, the most relevant in the choice of a photosensitizer seems to be the structural characteristics of the bacterial membrane (Gram positive or negative), since it would lead to some dyes having a more effective toxic action. (Nagata et al. 2012) Selection of appropriate light sources various photosensitizers The literature presents three main classes of clinical PDT light sources LASER, LED and halogen lamps (Table 2). LASER has some advantages, such as monochromaticity and high efficiency (90) of coupling into single optical fibers in endoscopic, high potency, and interstitial light delivery devices however, they do have a high cost. (Nagata et al. 2012) Diode LASER is one of the lowest priced among LASER systems. It is very convenient and reliable however, it has a single wavelength and requires a separate unit for each photosensitizer due to the different absorption wavelengths. LED has become a viable technology for PDT in the last few years, particularly for irradiation of easily accessible tissue surfaces. (Nagata et al. 2012) The main advantages of LED over LASER or diode LASER sources are their low cost and ease configuration of LED arrays into different irradiation geometries. As with LASER diodes, LED have a fixed output wavelength, but as the cost per watt is significantly smaller, having different sources for each photosensitizer is less of a drawback. (Nagata et al. 2012) Filtered halogen lamps have the advantage that they can be spectrally filtered to match any photosensitizer however, they cannot be efficiently coupled into optical fiber bundles or liquid light guides, and also cause heating. With broadband sources, their effective output potency is lesser, as compared to a LASER source at the photosensitizer activation peak, and it is proportional to the integrated product of the source output spectrum and the photosensitizer activation spectrum. With compensations (potency and period of irradiation time), LASER and tungsten lamps presented high dose of energy. These aspects should be carefully analyzed when used in teeth, since temperature increase in dentin and pulp tissue may cause irreversible changes. (Nagata et al. 2012) Some articles show longer period of exposure time (10, 15 and 30 min) using a tungsten filament lamp and LASERs. It is said that this may cause a risk to dentin and pulp tissues, since long periods of exposure time proportionally increase the temperature of the irradiated area. Therefore, thinking about the use of photodynamic procedures for the inactivation of S. mutans, the use of LED may be suggested, considering its capacity of not changing the temperature allied to its high dose energy supply. (Nagata et al. 2012 Silva et al. 2005) A summary of the maximum absorption and concentration of the analyzed dyes is shown in Table 4. Table 4. Maximum absorption and concentration of photosensitizer solution on the monomeric and aggregated forms. Considering the aspects discussed, the selection of a photosensitizer must take into account its photophysical characteristics to determine the appropriate concentrations. In addition, higher concentrations may require light sources with different wavelengths, according to the absorption alterations induced by the aggregation properties of the dye. Despite the fact that most studies regarding use of PDT in dental caries have used S. mutan specific photosensitizer and wavelengths in the visible light range, it must not be assumed that other photosensitizer cannot be used as bactericidal agents. There are studies that have focused on investigating the bactericidal effect of other photosensitizers namely ICG with near infrared light source such as 809 nm diode laser on wild type and resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa. Obtained results suggested that PDT with nontoxic ICG and low energy doses of 809nm laser light is an alternative powerful tool to destroy wound- infecting antibiotic-resistant microorganisms. These findings could lead to a wide range of clinical application such as infection treatment. After all dental caries is a bacterial infection in its nature. As future work, it could be said that PDT with ICG and 809 nm laser can be a powerful candidate to treat dental caries, mainly because of deep penetration capabilities at this specific wavelength. (Topaloglu, Gulsoy, and Yuksel 2013) Despite all discussed above, the main PDT treatment is considered a Type II mechanism, via singlet oxygen as reactive specie that induces biological cellular damage. (Dhami and Phillips 1996 Ryter and Tyrrell 1998) Therefore, the use of self-aggregated dyes as photosensitizers usually should be avoided. It is hoped the future clinical use of this therapy in Dentistry, since the best application include low dye concentrations which provide low toxicity, high solubility, and unlikely dental staining. (Nagata et al. 2012) The real mouth environment is totally different from the laboratorial culture or in vitro environment, which makes it difficult to provide an ideal condition for PDT studies. In spite of these limitations, in general the literature shows promising results in this field. It can be concluded that for optimal PDT results against cariogenic bacteria, the structural properties of the bacterium membrane, photosensitizer concentration, solubility and polarity, and light source wavelength must be considered. Novel Alternative Diagnosis and Treatment Method Based on the advantages and disadvantages of detection and treatments methods discussed so far, it can be summarized that an ideal diagnosis and treatment method must Produce a very precise and accurate measurement Produce a very precise, accurate, robust and easily comprehendible data Be user-friendly and easy-to-use for dentists and dentistry practitioners Have no harmful effects on the healthy tissue Be very precise and meticulous in targeting only infected tissue Be fast in action against the harmful species Be easy to clean after the procedure in case there are any chemical substances are used in the method Be able to conveniently and efficiently penetrate deeper into the lesion sites Have absolutely no staining effect on the teeth Be small enough for ease of penetration through the biofilm and fast and smooth mobility within the lesion. Bearing in mind, the characteristics of an ideal diagnosis and treatment method, herein a new method is proposed which is believed to have the capability to overcome most issues of PDT methods including staining problem, toxicity, lack of enough penetration of photosensitizer, not good enough performance at low doses (which is essential in order to avoid toxicity), low solubility and self-aggregation of the photosensitizer particles. The proposed system is based on Precise targeting of S. mutans bacteria family with the help of immunoglobulin G antibodies (IgG) both natural and synthetic In targeted delivery of medicine, antibodies are widely used as targeting mechanism for various application namely tissue imaging, drug delivery, etc. In case of bacterial detection, human bodys immune system produces antibodies for two main reasons tagging the bacteria for macrophages and facilitate phagocytosis and immobilize bacteria and inhibit their motion eventually resulting in their death. (Saber et al. 2011) Here we aim at using IgG antibody as the targeting system for S. mutans bacteria family due to its small size and high affinity in attachment to wide variety of nanoparticles such as magnetic nanoparticles. Another reason behind selection of IgG is that this antibody is the primary antibody produced and used by the immune system for targeting and immobilization of various strands of bacteria in the body. (Saber et al. 2011) Figure 8 represents IgG size and structure. Figure 8. General Structure of IgG Antibody (Left). Size, Dimensions and Geometry of IgG (Right) The recent advances in photocatalysis have opened a variety of new possibilities for energy and biomedical applications. In particular, plasmonic photocatalysis using hybridization of semiconductor materials and metal nanoparticles has recently facilitated the rapid progress in enhancing photocatalytic efficiency under visible or solar light. One critical underlying aspect of photocatalysis is that it generates and releases reactive oxygen species (ROS) as intermediate or final products upon light excitation or activation. Although plasmonic photocatalysis overcomes the limitation of UV irradiation, synthesized metal/semiconductor nanomaterial photocatalysts often bring up biohazardous and environmental issues. In this respect, identification of natural photosensitizing organic materials that can generate similar types of ROS as those of plasmonic photocatalysi is in order. (Leem, Kim, et al. 2018) Results of a recapitulation of fluorescent proteins that have Type I and Type II photosensitization properties in a comparable manner to plasmonic photocatalysis is presented in Table 5. Unfortunately in the literature plasmonic photocatalysis and protein photosensitization have not yet been compared systemically in terms of ROS photogeneration under visible light, although the phototoxicity and cytotoxicity of some fluorescent proteins are well recognized. A comprehensive understanding of plasmonic photocatalyst-like fluorescent proteins and their potential advantages will lead us to explore new environmental, biomedical, and defense applications. (Leem et al.2018) Table 5. Optical excitation and emission of phototoxic fluorescent proteins and their detected ROS types Selection of the best fluorescent proteins for our purposes is done based on the capability and capacity of the protein to produce ROS, size of the protein along with excitation wavelength of the protein. Figures 9 represents penetration depth of light into enamel. Based on this figure, it was obvious that mKate2 protein is the best choice among ROS producing fluorescent proteins due to its excitation wavelength (588 nm) being the highest which allows for deepest penetration into the enamel for maximum efficacy. (Webb et al. 2001) Figure 9. Penetration depth of tooth enamel as a function of the light wavelength vertical error barscorrespond to the standard deviation of five measured samples. In order to ensure that mKate2 protein will give us the best performance, other research articles have been explored. Based on a recent study by Leem et. al. in 2018, it was found that fluorescent proteins often result in phototoxicity and cytotoxicity, in particular because some red fluorescent proteins produce and release reactive oxygen species (ROS). The photogeneration of ROS is considered as a detrimental side effect in cellular imaging or is proactively utilized for ablating damaged tissue. Taking advantage of green (visible) light activation, mKate2 incorporated materials can produce and release superoxide and singlet oxygen, in a comparable manner of visible light-driven plasmonic photocatalysis. Thus, use of mKate2 in form of incorporation with other materials offers immediately exploitable and scalable photocatalyst-like biomaterials. (Leem, Park, et al. 2018) Based on these articles, it can be said that selection of mKate2 protein facilitates best fluorescent detection and bacterial termination for our purposes. Figure 10 represents mKate2 protein structure and its size. Figure 10. mKate2 protein structure and its size. Finally, both IgG and mKate2 mush be delivered to the lesion site in a fast and efficient manner. Use of magnetic nanoparticles facilitates proper conjugation of IgG antibodies and mKate2 protein simultaneously. Additionally, due to remarkable magnetic forces, magnetic nanoparticles can be accumulated at any desired point with high precision and very fast.(Neamtu et al. 2018) Figure 11 represents magnetic nanoparticle with IgG (green) and mKate2 (red) conjugated on its outer surface. Use of magnetic nanoparticles and guiding their motion inward and outward on the lesion site facilitates easy removal of the excess nanoparticles as well. Since these nanoparticles do not have toxic effects (after removal of excess amount), the problem of high toxic effect of photosensitizers is almost solved this. (Neamtu et al. 2018) Figure 11. Schematic Representation of IgG (green) and mKate2 (red) Conjugated Magnetic Nanoparticle Penetration of biofilm will result in destruction of anaerobic bacteria inside the lesion since they cannot live at the present of oxygen which is another advantageous use of magnetic nanoparticles. It must also be mentioned that the main reason of choosing a fluorescent proteins as the ROS producer as opposed to erythrosine or ICG is the extremely small size of mKate2 compared to conventional photosensitizers which makes it possible for mKate2 to be conjugated onto the outer surface of the magnetic nanoparticle resulting in alleviation and ease of the penetration process. Design of the Laser System Including Laser Delivery to the Target Tissue Since magnetic nanoparticles are modified with IgG and mKate2 on the outer surface, tendency to aggregation is significantly low and hydrophilicity and therefore solubility if considerably high compared to bare magnetic nanoparticles. Hence 2 of the major problems of PDT are solved. Moreover, strong magnetic force can penetrate bacterial biofilm therefore solving another general issue with dental caries. The setup is comprised of a liquid with a viscosity above that of water (slightly gel-like behavior for ease of applying it to the tooth only and avoiding its spread to gum, tongue, etc.) loaded with IgG (green) and mKate2 (red) conjugated magnetic nanoparticles. After applying the gel-like liquid to the teeth, a very strong magnet either electrical magnet or neodymium magnet will be placed in a parallel position to the applied gel and the lesion. The gel-like liquid would be top layer on top of the outer layer of the lesion and then the lesion (with inward lesion spread) would be the second layer. Then the magnet will be placed parallel to both layers. The magnetic force will attract magnetic nanoparticles towards the magnet by forcing its way through the biofilm. During the movement, antibodies will attach to bacteria, localizing magnetic nanoparticles on the outer membrane. At this stage if light at the wavelength of 588 nm is illuminated upon the gel, mKate2 will emit light at 633 nm that shows the infected site very clearly and as a result of its emission, ROS start being produced which in turn starts to kill bacteria. The light source for this experiment will be LED light emitting at 588 nm. n033ByuIUgOJDi(XY [email protected] 8Spcq2EUQZi,/SXYIn5jUVGW1TF y38gWJ(HTWN0SOFGxeaZf, jF,Fu___ 87 zJs DShCm 8UK-IP61zlGO0 UpeO Y [email protected] p)wb6ppqsKIt9INP.5X,M-H9_ 6SIGR4r)WS a .TONzv znvGkPZfgXAZn ki0vS )B OM1 8Hcl rzVVHm/00(SpAO46.kt.HLu9qi-Fqdn
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