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Table of Contents   
REVIEW ARTICLE  
Year : 2022  |  Volume : 25  |  Issue : 5  |  Page : 454-462
Characterization of dynamic process of carious and erosive demineralization – an overview


1 Department of Conservative Dentistry and Endodontics, A.B Shetty Memorial Institute of Dental Sciences, Nitte (Deemed to be University), Mangalore, Karnataka, India
2 Department of Oral and Maxillofacial Pathology, A.B Shetty Memorial Institute of Dental Sciences, Nitte (Deemed to be University), Mangalore, Karnataka, India

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Date of Submission19-Mar-2022
Date of Decision25-Apr-2022
Date of Acceptance17-May-2022
Date of Web Publication05-Jul-2022
 

   Abstract 

To review the analytical methods for carious and erosive demineralization an initial search of peer-reviewed scientific literature from the digital library database of PubMed/Medline indexed journals published up to early 2022 was carried out based on keywords relevant to the topic criteria including bibliographic citations from the papers to gather the most updated information. This current review aims to provide an updated overview of the advantages, limitations, and potential applications of direct and indirect research methods available for studying various dynamic stages of carious and erosive demineralization in enamel and dentin. This paper categorizes and describes the most suitable, frequently adopted and widely used quantitative and qualitative techniques in in vitro/in vivo research which are well-established, emerging, or comparatively novel techniques that are being explored for their potential validation.

Keywords: Analysis; caries; characterization; demineralization; dentin; enamel; erosion; research

How to cite this article:
Devadiga D, Shetty P, Hegde MN. Characterization of dynamic process of carious and erosive demineralization – an overview. J Conserv Dent 2022;25:454-62

How to cite this URL:
Devadiga D, Shetty P, Hegde MN. Characterization of dynamic process of carious and erosive demineralization – an overview. J Conserv Dent [serial online] 2022 [cited 2022 Dec 3];25:454-62. Available from: https://www.jcd.org.in/text.asp?2022/25/5/454/349907

   Background Top


After dental caries, the non-carious erosion of mineralized hard tissues has shown an increasing prevalence in modern societies.[1],[2],[3] While both alter the composition, crystallography, microstructure, and mechanical properties, dental erosion [Table 1][4],[5],[6],[7],[8],[9],[10] is chiefly considered as a surface phenomenon resulting from direct acid contact on the tooth surface, while dental caries [Table 2][12],[13],[14],[15],[16],[17],[18],[19],[20] in contrast initially forms subsurface lesions by bacterial acids until cavitation occurs.[21],[22]
Table 1: Erosive Demineralization

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Table 2: Carious Demineralization

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To understand the complex nature and the dynamic progress of carious and erosive demineralization, several methods and techniques have been used to develop clinically relevant solutions for its prevention and management.[23] In addition, as methods to assess enamel are unsuitable for dentine due to histological variations, the application of a single technique may be inadequate, requiring a combination of qualitative, quantitative, and semi-quantitative (approximate) measurements. The surface-softening effect in the early stages and the subsequent loss of tooth substance has been studied by various direct and indirect methods including: Chemical Analysis, Surface Hardness/Profilometry, Microscopy, and Spectroscopy techniques.[24],[25],[26][Table 3]. The advanced research tools with capabilities to measure subsurface effects[27],[28] include Transverse Microradiography (TMR), Scanning Electron Microscopy (SEM), Optical Coherence Tomography (OCT), Raman Spectroscopy, Fourier Transform Infrared Spectroscopy (FT-IR), Atomic Force Microscopy (AFM) and Confocal Laser Scanning Microscopy (CLSM).[29]
Table 3: Analytical methods for characterization of dynamic process of carious and erosive demineralization

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   Chemical Analysis Top


It includes spectrophotometric and colorimetric methods for measuring the concentrations of calcium and phosphate released into the dissolving solution by an erosive challenge or its dissolution protection following therapeutic modification, estimation of pH, and other mineral constituents such as fluoride or magnesium. Limitations: Only indirect measurement of net ions concentration under in vitro conditions.[3],[27]

  1. Atomic Absorption Spectrophotometry (AAS) is a chemical analytical technique that measures the adsorption of optical light by calcium atoms released in solutions when both enamel and dentin[30] are subjected to acidic challenge. Advantages: Reliable estimation[31] with negligible interference from phosphate solutes[32] measured by spectrophotometry of a colored phosphate complex[33],[34]
  2. Inductively Coupled Plasma- Atomic Emission Spectroscopy (ICP-AES) operates based on stoichiometry and density of hydroxyapatite[27],[35] by the characteristic wavelength of electromagnetic radiation emitted. Limitation: Inability to determine origins of Ca/P (surface/subsurface)[36]
  3. Iodide Permeability Test [IPT] low-cost in vitro evaluation of iodide recovery from samples immersed in potassium iodide as a measure of porous volume, suitable for swift screening of erosive potential on enamel only.[27]



   Surface Analysis Top


Surface Hardness

This analysis is used frequently for evaluating mineral loss (ML)/gain by measuring the surface resistance of a material when penetrated with different sizes of indenters, dwell time, and forces at micro or nanoscale. Although a relatively low-cost nondestructive method,[30] it is an empirical method that only provides an indirect assessment of mineral content, requiring further validation[36] and compromised results from elimination of the outer mineralized layer during flattening and polishing of test surface.[37]

Microhardness calculates the Knoop hardness number or Vickers hardness numbers using a diamond indenter (rhomboidal or tetra-pyramidal, respectively)[27] distinguished as Surface Microhardness (SMH) indenter load is placed perpendicular to the polished surface; Cross-section Microhardness (CSMH) indenter load is placed parallel to the anatomical surface of the tissue.[38] Limitation: Subjective to inaccuracies if (i) indentations are ill-defined, (ii) substrate is resilient (like dentin), and (iii) flat surface not level oriented and presence of deposits like fluoride.[27]

Nano-indentation (nanoscale upto1μm, under loads of 0.25mN -50mN) uses a trigonal-pyramidal Berkovich diamond indenter with shallow penetration depths (upto500 nm) for higher sensitivity in the assessment of even small variations and Young's modulus (elastic deformation).[38] For dentin, Atomic Force Microscopy (AFM) used with nano-indentation provides superior visual control in the placement of small nano-indenters.[27],[28]

Surface Profilometry

Surface Profilometry (Surfometry) is a well-established technique for the assessment of surface roughness in early erosion of both dentine and enamel[27],[39],[40] and recording step height that quantifies tissue loss regarding a nontreated area.[27]

Types: a) Contact Profilometry: conduct two-dimensional (2D) mechanical scanning with a diamond or steel-tipped stylus. Limitations: (i) The stylus may penetrate the soft demineralized surface,[41],[42] leading to an overestimation of depth, (ii) only provides changes in surface roughness much like SMH.[36] (b) Non - contact LASER (Optical) Profilometry based on the optical triangulation principle uses a LASER probe with a vertical range (300 μm–10 mm) for three-dimensional (3D) measurements of surface topography between the reference points. Advantages: Gold standard method suitable for (1) analyzing very deep erosion pits, curved natural surfaces, and (2) may quantify tooth loss if flattened specimens can be obtained.[40],[43],[44],[45] Limitation: Affected by angulation of positioning, color, and transparency.[45]


   Optical Analysis Top


Quantitative Light-induced Fluorescence (QLF)

It works on the optical principle that the natural fluorescence emanating from dentine is modified by the porosity of demineralized enamel. This causes scattering of incident blue-green infrared light to appear darker and quantified as the percentage of change of fluorescence radiance by the QLF software.[46],[47] eg., DIAGNOdent (KAVO, Biberach, Germany). Advantage: Non-invasive monitoring in both in-vivo[29] as well as in-vitro

Optical Coherence Tomography (OCT)

It utilizes an interferometer for cross-sectional imaging of enamel thickness/porosity and measures backscattered light[29] from internal microstructures[50],[51] using near-infrared light. Advantage: Non-destructive clinical method for quantitative assessment of high-resolution 3D images of micro-morphological changes in initial caries by optical scattering[52],[53] and detection of eroded lesions as a function of depth, based on the intensity of backscattered light.[54] Limitations: (1) Reduced spatial resolution for probing in vivo,[55] (2) usage restricted to initial lesions,[56] and (3) high cost and lack of commercial availability for clinical use.[57] Variant: Swept Source OCT (SS-OCT) has a tunable LASER that sweeps at high speeds to generate 3D volumetric datasets.[51]


   Microscopic Analysis Top


It mainly includes qualitative techniques based on optical, X-ray, electron, and scanning principles for microstructural characterization of the surface/subsurface layer used either alone or combined with quantitative elemental analysis.[29],[58],[59]

Light Microscopy

It is an optical microscope that uses visible light and a system of lenses that was limited in its depth (up to 0.2μm) and magnification(1000-1500X); later widened the applications using ultraviolet (UV) light, infrared, fluorescence, phase contrast, and digital microscopes.[59]

Visible Light Microscopy was used to measure height differences between eroded and non-eroded areas.

Types: i) Transmitted Light Microscopy (TLM) measures erosion depth in both enamel and dentine using thin ground sections and thickness of the demineralized matrix overlying eroded dentine.[27] ii] Polarized Light Microscopy (PLM) is a qualitative method for measuring erosion depth by observing changes in the crystal birefringence in thin cross-sectional slices for mineral density in the form of pore volume and can discriminate between partly and fully demineralized tissues.[60],[61]

Fluorescence Microscopy uses optical microscopes with high-intensity UV light from a mercury arch lamp and fluorescent stains to increase the contrast/resolution of the image. Later, Confocal microscopes were developed to overcome the drawbacks of visible light microscopy using laser light and digital imaging with a focused pinhole camera.[29]

Confocal LASER Scanning Microscopy (CLSM)

Optically sectioned sequential high-resolution collected by a monochromatic LASER scan from several focal planes is combined to generate 2D optical sections including depth measurement while alteration in mineral content and morphology can be studied based on variation in reflection and scattering of light.[44] Being nondestructive it quantifies both erosive losses of minerals as a function of depth by fluorescent volume and alteration in microstructural quality during early erosion.[36] Variant: 3D- Focus Varying Microscope [FVM][62] scanning a sequence of 2D data sets for creating a 3D image (vertical resolution of up to 10 nm); in subsurface lesions of solid samples.[34],[36] Advantages: (1) High-resolution images of a submicron level similar to SEM. (2) High speed (3) quantifies bulk tissue loss through height mapping of the eroded area with sound reference[63] and depth of softening. (4) Unlike TMR, SEM and OCT it uses freely accessible equipment and image processing software.[36] Limitations: (1) Elaborate specimen preparation of thin sections with surface clearing by chemo-mechanical pretreatment with 5.25% sodium hypochlorite solution combined with ultrasonication and vacuum followed by labeling with a fluorescent dye (rhodamine with a compatible absorption peak of 511 nm) and fixing with cyanoacrylate glue onto a glass slide for observation.[29] (2) Relatively lower resolution for surface imaging, unsuitable for thick specimens; hence, must be combined with other higher resolution tools like SEM, transmission electron microscopy (TEM), and AFM for superior evaluation.[29]

Electron Microscopy

These methods are based on the interaction of high-energy electron beams that either interact with the specimen surface or get transmitted through the specimen to generate a variety of signals.[29]

Scanning Electron Microscopy [SEM] is a widely employed qualitative tool for magnified visualization of ultramicroscopic complexity (micro/nanoscale) of surface/cross-sectional topography using high-energy focused beam of electrons.[29],[64],[65],[66] Limitations: (i) Permits only subjective, qualitative assessment.[30] (ii) destructive technique, and (iii) requires the availability of bespoke equipment/expertise.[36] The limitation of elemental analysis may be overcome by combining it with Energy Dispersive Spectrometry (EDS).[29] Types: (i) Conventional SEM (C-SEM) requires pre -coating of the sample surface with a conductive material such as gold-sputtering in a high-vacuum chamber (to ensure the secondary electrons emitted stay focused to interact with the detector), that can cause artifacts/cracked surface.[29] (ii) ii) Environmental SEM (E-SEM) uses elevated gas pressures to allow discharge of surface charge due to gas molecular ionization to strengthen electron signal without need for vacuum dehydration or conductive surface coating[30] of moist substrates like dentin and in situ evaluation.[67] Limitation: Low resolution is limited to main structures[67] due to defocusing or scattering of the electron beam. (iii) Low Vacuum SEM (LV-SEM) is similar to a C-SEM, but operates at elevated pressures to avoid surface cracking and need for surface charging/coating.

Transmission Electron Microscopy (TEM) generates higher resolution images than SEM by transmission of electrons through the specimen, but requires ultrathin samples (<100 nm) or a suspension on a grid for the study of the ultrastructure of carious dentin lesions.[68] Advantage: Generates both topographical characteristics as well as visualization of inner architecture.[29]

X-Ray Microscopy

It provides resolution between light microscopy and electron microscopy by using X-rays of shorter wavelength (10–0.01 nm) with higher penetrative energy to magnify internal features with image contrast provided by variable absorption by different components in the material.[69]

Microradiography (MR) is a full-field high-resolution imaging tool to quantify mineral content by recording the intensity of attenuated X-rays transmitted through sections as a MR image on photographic plates/film digitized by a video camera or photomultiplier.[70]

Types: (i) Transversal Micro-Radiography (TMR) considered as a gold standard, uses an X-ray beam directed perpendicular to that of lesion progression[27] for mineral quantification of lesion depth in caries, but only after 30 min of exposure for erosive lesions.[71],[72] Use of thin highly polished plano-parallel sections (50–200 μm) makes it very destructive, but highly sensitive to early changes, distribution of mineral, lesion depth, integrated ML, and position of the sub-surface layer.[70],[71],[73],[74] Limitations: (i) Extensive sample preparation and requirement of bespoke equipment operated by highly skilled experienced operators limits accessibility for most research studies (ii) Provides only quantification without morphological data.[36]

(ii) Longitudinal Microradiography (LMR) uses an x-ray beam directed parallel to the lesion direction; hence, cannot measure lesion depth profile.[27],[42],[75] As it is optimized for thicker sections (up to 400 μm)[70] it has reduced sensitivity to minute changes in mineral content; yet, it permits reuse of specimens for longitudinal observations.[30]

Scanning Probe Microscopes (SPM)

This is used to study the surface topographical and elemental structures with subatomic microscopic tools[76] such as Scanning Tunneling Microscopy (STM), Near-field Scanning Optical Microscopy (NSOM), and most importantly Atomic Force Microscopy (AFM).[23],[77]

Atomic Force Microscopy [AFM] imaging technique invented in 1986 is one of the most widely used tool in dental biological and material sciences. It uses a nondestructive probe for mapping an atomic-force field to provide 3D characterization of surface topographical details with both vertical and lateral resolution.[23],[76],[77] The AFM scanner probe tip attached to a cantilever scans the surface topography in the nanometric scale[23] measured by the deflection of a nondestructive LASER probe.[76],[77] The data acquired is analyzed using dedicated software to quantify dimensional changes in mineral density distribution as a function of depth[78] and measure viscoelasticity of demineralized dentin with nanoindentation.[28],[79]

The AFM Operates in 2 modes: (i) Contact mode (CM-AFM) where the tip is moved laterally in constant contact with the surface; (ii) Tapping mode (TM-AFM) where the tip is gently tapped to the position of the laser beam on the photodiode[24] and quantifies early surface changes by measuring height differences at the atomic level.

Advantages: (1) Minimal or no sample preparation that minimize artifacts and allow serial measurements. (2) adaptable to use under ambient or liquid conditions, conducting/insulating surfaces and (3) high-resolution visualization up to collagen network in the nano-hardness analysis of dentin.[23],[80],[81] Limitation: Being very time-consuming, it requires samples with a smooth surface and of limited size[23],[77] (0.25 sqmm~ 60 min). Applications: Surface/structure nano-characterization and mechanical properties of intact/altered enamel and dentin substrate in particular such as: (1) Demineralization and various interventional remineralization strategies[23],[82](2) observation of collagen network, micro-morphology of noncarious cervical lesions, changes like roughness and chemo-mechanical properties like elasticity caused by therapeutic/restorative agents at intertubular and peritubular level.[23],[76],[81]

Acoustic Microscopy

Scanning Acoustic Microscopy (SAM) or Acoustic Micro Imaging (AMI)[83] is a high-frequency ultrasonic non-destructive imaging technique based on acoustic principles to inspect subsurface layers of a specimen in an aqueous environment such as dentin demineralization process, but not specific enough to differentiate between erosion and abrasion.[84],[85]


   Spectroscopic Analysis Top


It refers to non-destructive techniques to evaluate the elemental structure of dental tissues based on the generation of spectra when electromagnetic radiation interacts with matter. The most widely employed spectroscopic tools used in combination with microscopy for visualization include Vibrational Spectroscopy [Infrared spectroscopy (IR)] like Fourier Transform Infrared Spectroscopy (FT-IR), Raman Spectroscopy, Ultraviolet & Visible spectroscopy (UV-Vis), Energy- Dispersive X-ray Spectroscopy [EDXS] and Secondary Ion Mass Spectrometry [SIMS].[36],[58]

X-ray Spectroscopy

Energy Dispersive X-ray Spectroscopy [EDS or EDX] is a quantitative elemental surface microanalysis tool that measures the characteristic x-rays emitted following electron bombardment and can identify deposits of therapeutic agents on the surface/subsurface using cross-sectional concentration profiles of the element.[27],[47]

EDX coupled with SEM imaging (SEM- EDX) adds 3D information to quantitative data. When electrons near the nucleus get excited by a high electron beam [SEM], the electrons in the distant site- drop energy levels to fill the holes causing the emission of energy dispersive x-rays [EDX] different frequencies.[27]

X-Ray Diffraction Analysis [XRD] is a non-destructive spectroscopic analysis of the crystallographic microstructure of a solid sample by recording the X-ray diffraction pattern (peaks/position) of structural characteristics and the composition when irradiated[28],[49] and changes affecting crystallites with ionic substitution after an erosive challenge.[86] The interference of the scattered X-ray beam passing through the atoms in the sample is observed and measured in Angstroms (1 Angstrom = 0.1 nm) using Bragg's Law for both single crystal or polycrystalline materials.[87] XRD pattern provides information on various phases, the composition of crystal structure/size, stress, and texture. Advantages: i) Non-destructive, ii) powder or solid surface (single crystal) samples,[87] iii) simultaneous detection of small-angle X-ray scattering (SAXS) as well as wide-angle X-ray diffraction (WA- XRD) for the degree of crystallinity in polymers and fibers.[86] Eg: Powder X-ray Diffractometry [P-XRD]- most widely used with powder samples. Limitation: Only a tiny fraction of crystallites may contribute to the diffraction pattern.

X-ray Photoelectron Spectroscopy [XPS] or Electron Spectroscopy for Chemical Analysis [ESCA] is a highly sensitive, non-destructive, semi-quantitative method for elemental identification derived from the ratio of elements (e.g., Ca/P) and chemical states of the constituent elements limited to an outer surface depth of few nanometers (~10 nm). Limitations: (1) Use of ultrahigh vacuum (<10−9 Torr), (2) appropriate only for surface evaluation (few nm).[88],[89]

Vibrational Spectroscopy

It refers to non-invasive molecular Infrared [IR] spectroscopy methods that analyze molecular structure for material and structural characterization of mineralized tissues in dentistry. They are based on the interactions occurring between electromagnetic radiation (10−7 m) and the nuclear vibrations in the molecules following absorption of mid-infrared light in Fourier Transform Infrared spectroscopy (FTIR) or inelastic scattering of near-infrared light by a molecule in Raman microspectroscopy.[90],[91]

Fourier Transform Infrared Spectroscopy (FTIR) is a non-destructive spectroscopic method for analysis of surface elemental and mineral crystallinity (size and order of arrangement) by measuring molecular bands at different vibration modes.[92] When infrared radiation is passed through a sample the transmitted signal is detected by a mathematical process termed Fourier transform and converted into an interpretable spectrum with patterns of linear response representing a molecular 'fingerprint' specific to its chemical structures. The sampling techniques may be Attenuated Total Reflection (ATR), Transmission, Specular or Diffuse Reflectance. Advantages: (1) Unknown materials and surface components[58] can be identified against a database of reference spectra. (2) Simple, sensitive cost-effective technique for analysis of small samples (upto10 microns).[93]

Raman Microspectroscopy technique based on Raman scattering phenomenon of inelastic scattering of light of different frequency by molecules irradiated with electromagnetic radiation of a single frequency.[89] It uses vibrational modes[37] of scattering molecules under monochromatic LASER irradiation to identify additional peaks as a 'fingerprint' for a specific compound[37] on the light spectrum characteristic of Raman emission type of spectra[47],[58] Uses: High spatial resolution (1–1.5 μm) for elemental analysis of demineralized dentin rather than its topography.[67] Advantages: 1) Direct, non-destructive method. (2) no sample preparation.[89]

Mass Spectrometry (MS)

It provides the mass : charge ratio of ions as a mass spectrum. Secondary Ion Mass Spectrophotometry [SIMS] measures semi-quantitative elemental and isotopic/molecular composition[30],[80] and the uptake of fluoride by initial erosive lesions[79] e.g, Dynamic SIMS (D-SIMS).[94] Although provides rapid depth profiling with high sensitivity;[27] some surface damage was seen.[30],[79],[95]


   Tomographic Analysis Top


micro-Computed Tomography (micro-CT) or X-ray Micro Tomography [XMT]

It is a high-resolution non-destructive in-vitro tool for quantification of mineral density, 3D visualization of internal microstructure, and monitoring of real-time demineralization.[96],[97] The scanning is performed under 100% humidity on specimens (placed in moist cotton to avoid dehydration) mounted on a rotating platform, before (baseline) and after acidic challenge using proprietary parameters/software to calculate the Mineral Density (MD), lesion depth, and Mineral Loss (ML) (vol% μm) values.[98] Limitations: (1) High radiation dose, long scan time unsuitable for in-vivo studies[99] (2) High-cost equipment and technical expertise for data computing.[100] (3) Provides no information on the crystallographic phase.[88]


   Other Analysis Techniques (Novel And Emerging) Top


  1. Microdensitometric Scan measures optical density by light transmission through images on positive film transparencies (radiographs) as computerized data graphically or digitally[91],[101]
  2. White Light Interferometry [WLI] performs topographic assessment using an optical interference microscope[34],[102] for assessing surface roughness, bulk loss of enamel surfaces following repeated acid exposures[94],[103]
  3. LASER Speckle Imaging[104] produces speckle images from the scattered coherent light pattern that are highly sensitive to detect minimal changes, such as early noncarious lesion of enamel. Advantage: Non-destructive, cost-effective method developed for clinical application
  4. Specular and Diffuse Reflection Analysis[105] is a proposed clinical diagnostic method that uses an optical reflectometer for measuring the Surface Reflection Intensity (SRI) by change of the specular reflection signal sensitive to the detection of early erosion assessment in vivo, which was validated with SMH and AAS analysis of calcium release[106]
  5. Bioluminescence Imaging is a novel technique proposed for the precise assessment of early stages of caries/erosive activity during the chemical reaction between a bioluminescent marker Calcium Sensitive Photoprotein-aequorin and calcium ions released by demineralization for mean light-output (grayscale value) using an imaging software.[107]



   Conclusion Top


Currently, available research methods to study the dynamic process of demineralization are based on the stage of the lesion under examination by combining microstructural and crystallographic characterization.[108] Most research methods have limited capability in detecting early mineral changes or periodic assessments occurring overtime before or after therapeutic intervention including the current gold standard of TMR. Although a wide spectrum two-dimensional imaging modalities exists, 3-dimensional tools like AFM provide a superior assessment of surface topography and mechanical properties at higher resolution. While microscopic tools enable observation of microstructural changes at high magnification most spectroscopy techniques can provide elemental analysis non-destructively when used in combination.[29],[71] Observation of the dynamics of demineralization and remineralization by longitudinal monitoring with a sensitive, rapid, cost-effective, non-destructive method for quantitative and qualitative assessment can enable the early diagnosis, selection and development of therapeutic strategies tailored to treat and inhibit carious-erosive demineralization and promote remineralization.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Prof. Darshana Devadiga
Departments of Conservative Dentistry and Endodontics, A.B Shetty Memorial Institute of Dental Sciences, Nitte (Deemed to be University), Mangalore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcd.jcd_161_22

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    Abstract
     Background
     Chemical Analysis
     Surface Analysis
     Optical Analysis
     Microscopic Analysis
   Spectroscopic An...
     Tomographic Analysis
   Other Analysis T...
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    References
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