**8. Methods to assess demineralization**

With a heightened interest in evidenced-based dentistry, the dental research community has over the years employed various modes of technology to quantify extent of enamel demineralization. The ideal method of assessment should be simple, noninvasive, reproducible, and precise. The following is brief description of four commonly employed techniques (TMR, PLM, QLF, and CLSM).

#### **8.1 Microradiography**

Transverse Microradiography (TMR) or contact-microradiography is one of the most widely accepted methods used to assess demineralization and remineralization in dental hard tissues in *in-situ* and *in-vitro* studies. It is a highly sensitive method to measure the morphology of and the change in mineral content of enamel and dentin samples (Arends and Ten Bosch, 1992). In TMR the tooth sample to be investigated is cut into thin slices (about 80 µm and 200 µm for dentine samples). A microradiographic image is made on high resolution film by X-ray exposure of the sections together with a calibration stepwedge. The microradiogram is digitized by a video camera or photomultiplier. The mineral can be automatically calculated from the gray levels of the images of section and stepwedge using a custom-made software. In examining the reliability of TMR, Exterkate et al. found that repeated microradiographs of the same thin enamel sections resulted in a negligible spread in mineral loss among them (Exterkate *et al.,* 1993). Such reliability and the more recent application of computer imaging make microradiography a standard method used in caries research for the assessment of lesion profiles.

#### **8.2 Polarized light microscopy**

320 Contemporary Approach to Dental Caries

caries lesions (Gorelick *et al.,* 1982; Richter *et al.,* 2009) and that only 9.3% of orthodontists give their patients fluoride varnish (Keim *et al.,* 2008), raises questions about emphasis of

Due to high caries incidence, low patient compliance, and low usage of fluoride varnish by orthodontists, there appears to be a need for a better treatment modality for patients undergoing orthodontic treatment. Regardless of the exact prevalence rate for white spot lesion development, most dental professionals would agree that it is currently far too high. While adjuncts to treatment such as fluoride rinse can potentially reduce the incidence of white spot lesions, the required compliance of high caries-risk patients is dubious. Equally troublesome, available non-compliant and proven treatments like professionally applied fluoride varnish have failed to catch the attention of practicing orthodontists. The resin sealer (Pro-seal) seems to address the patient compliance issue, but if orthodontists have neither the time nor the interest to deliver fluoride varnish, their likelihood of investing the resources and chair time to etch and light cure Pro-seal on twenty teeth is probably low. Aegis-Ortho bracket cement containing ACP seems to address all these issues. It does not require patient compliance, and it does not require any additional chair time in the office, since time allotted for orthodontic bracket bonding is already a part of the treatment plan. In spite of some reports, which document a low bond strength of its earlier generations, if ameliorated, this product holds immense potential for preventive care during orthodontic treatment. First however, there is a need to test and document the preventive properties of

With a heightened interest in evidenced-based dentistry, the dental research community has over the years employed various modes of technology to quantify extent of enamel demineralization. The ideal method of assessment should be simple, noninvasive, reproducible, and precise. The following is brief description of four commonly employed

Transverse Microradiography (TMR) or contact-microradiography is one of the most widely accepted methods used to assess demineralization and remineralization in dental hard tissues in *in-situ* and *in-vitro* studies. It is a highly sensitive method to measure the morphology of and the change in mineral content of enamel and dentin samples (Arends and Ten Bosch, 1992). In TMR the tooth sample to be investigated is cut into thin slices (about 80 µm and 200 µm for dentine samples). A microradiographic image is made on high resolution film by X-ray exposure of the sections together with a calibration stepwedge. The microradiogram is digitized by a video camera or photomultiplier. The mineral can be automatically calculated from the gray levels of the images of section and stepwedge using a custom-made software. In examining the reliability of TMR, Exterkate et al. found that repeated microradiographs of the same thin enamel sections resulted in a negligible spread in mineral loss among them (Exterkate *et al.,* 1993). Such reliability and the more recent

preventive care in today's orthodontic practice.

ACP-containing bracket cement with *in-vitro* studies.

**8. Methods to assess demineralization** 

techniques (TMR, PLM, QLF, and CLSM).

**8.1 Microradiography** 

**7. Need for investigation** 

Polarized light evaluations of enamel sections have been useful in describing the early caries lesion and alterations in structure upon further demineralization or remineralization. Generally, it provides information on absorption color and boundaries between minerals of differing refraction indices. Materials such as enamel act as beam splitters and divide light rays into two parts. Polarized Light microscopy (PLM) in turn exploits the interference of split light rays, as they are reunited along the same optical path to extract information about materials. Essentially, polarized light microscopy allows the visualization of areas with different porosities. The histologic features seen under a polarized light microscope allow the examiner to distinguish carious and non-carious enamel by their respective distribution of pores (Gwinnett, 1966). Polarized light examination of enamel specimens is a wellestablished procedure in which it is customary to view quinoline-imbibed sections orientated so that normal enamel is blue/green in color (Gilmour and Edmunds, 1998).

#### **8.3. Quantitative light-induced fluorescence**

Quantitative Light-induced Fluorescence (QLF) is one method of assessing levels of enamel demineralization. With QLF, real-time fluorescent images are captured into a computer and stored in an image database. Optional quantitative analysis tools enable the user to quantify parameters like mineral loss, lesion depth, lesion size, stain size and severity with high precision and repeatability. The QLF method is based on the auto-fluorescence of teeth. When teeth are illuminated with high intensity blue light they will start to emit light in the green part of the spectrum. When enamel demineralization takes place, minerals are replaced mainly by water from saliva, causing a decrease in the light path in the tooth substance. This results in less light absorption by enamel. Because fluorescence is a result of light absorption, the intensity of fluorescence decreases in demineralized regions of the enamel, which appear darker than sound tooth structures (de Josselin *et al.,* 1995; al-Khateeb *et al.,* 1998; Rousseau *et al.,* 2002). Thus, the fluorescence of the dental tissue has a direct relation with the mineral content of the enamel. The effectiveness of QLF for measurement of enamel demineralization has been demonstrated in several studies. The use of QLF allows for quantitative analysis has been reported to be well correlated (0.73-0.83) with the degree of mineral loss from early enamel lesions *in-vitro* when measured by longitudinal microradiography. (Hafstrom-Bjorkman *et* al., 1992; Emami *et* al., 1996; Lagerweij *et al*., 1996). The use of QLF as a method of following caries development during orthodontic treatment has been suggested and encouraged by the results of several *in-vitro* studies. (Benson *et al*., 2003 and Pretty *et al*., 2003). Recent studies also indicate that QLF is suitable for *in-vivo* monitoring of mineral changes in incipient enamel lesions (Van der Veen *et al*., 2000 and Al Khateeb *et al*., 2002).

#### **8.4 Confocal laser scanning ,icroscopy**

Confocal Laser Scanning Microscopy (CLSM) is yet another method of assessing enamel demineralization. This technique accelerates and simplifies the measuring of mineral loss. The enamel specimens are sectioned in half, stained with fluorescent dye, and analyzed

White-Spot Lesions in Orthodontics: Incidence and Prevention 323

Individual slides were scanned into digital format using a Nikon Slide Feeder SF-200 (S) and Super Coolscan 4000 ED scanner. Scanned images were enlarged 325% and imported into an

Images were evaluated by trained investigators using a scoring system specifically adapted for use with photographed images (*International Caries Detection and Assessment System II*; Ismail, 2005). Visible labial surfaces examined included maxillary and mandibular central and lateral incisors, canines, first and second premolars, and first molars. The evaluators scored each visible labial tooth surface before and after orthodontic treatment. The scores were combined to determine the labial caries incidence for each patient. Teeth were

examined and scored from first molar to first molar, maxilla and mandible (Fig. 2).

Fig. 2. Tooth labial surfaces were examined and scored from left first molar to right first

molar, maxilla and mandible, before and after orthodontic treatment.

individual Microsoft PowerPoint presentation for each patient.

**9.4 Dental caries determination** 

using a CLSM system (Fontana *et al*., 1996). The major advantage of this method is that it enables quantitative analysis of thick samples without the problems of thin section preparation required for microradiography or polarized light microscopy. Essentially, CLSM allows a subsurface examination since the scattered, reflected, and fluorescent light from planes out of focus is eliminated – providing a subsurface image only from a thin layer upon which it is focused. This processed digital image can be used to determine surface features, area and volume analysis of given structures, and views of the total structure from any angle in three dimensions. In terms of efficacy, a statistically significant high correlation was found between mineral changes measured using microradiography and the changes in lesion parameters analyzed by confocal microscopy (González-Cabezas *et al*., 1998)

With all the treatment modalities flooding the marketplace, the orthodontist might find it difficult to sort out what works best and why when oral hygiene deteriorates. The objectives of this chapter are to highlight the results of two recent studies that investigated:

