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In human dentition, the teeth are contained in the bony sockets of the mandible and maxilla without any space between adjacent teeth. Humans have heterodont dentition, that is individual teeth are shaped differently according to function. In human dental arcade one set of teeth replaces another. The first set of teeth consists of the deciduous teeth, which are later replaced by the permanent teeth. Each tooth can be divided into three segments: a crown, a neck and a root. The root is that part of the tooth which lies in the bony socket and is secured by the periodontium. The neck of the tooth describes the narrow junction between the crown and root, it projects above the socket but is covered by the gum (gingiva). The crown is that part of the tooth visible above the gingiva. The teeth of the maxilla and mandible are arranged in dental arches known as upper and lower dental arcades . A quadrant system is used for numbering the teeth. Each quadrant contains eight permanent teeth comprising two incisors, one cuspid, two bicuspids, and three molars.
Enamel is the hardest substance in the human body. The cells that are responsible for formation of enamel, the ameloblasts, are lost as the tooth erupts into the oral cavity, and hence enamel cannot self repair. Fully formed enamel is the most highly mineralised extracellular matrix known, consisting approximately 96% mineral and 4% organic material and water. The inorganic content of enamel is a crystalline calcium phosphate (hydroxyapatite) substituted with carbonate ions which is also found in bone, calcified cartilage, dentin and cementum. The high mineral content together with its complex structural organisation enables enamel to withstand the mechanical forces applied during tooth functioning.
Amelogenesis, the process of enamel formation is a two step process. When enamel first forms, it mineralizes only partially to approximately 30%. Subsequently, as the organic matrix breaks down and is removed, crystals grow wider and thicker. This process whereby organic matrix and water are lost and mineral is added accentuates after the full thickness of the enamel layer has been formed to attain greater than 96 % mineral content. Amelogenesis can be subdivided into three main functional stages referred to as the presecretory, secretory, and maturation stages . During the presecretory stage, differentiating ameloblasts acquire their phenotype, change polarity, develop an extensive protein synthetic apparatus and prepare to secret the organic matrix of enamel. During secretory stage, ameloblasts elaborate and organize the entire enamel thickness, resulting in the formation of a highly ordered tissue. During the maturation stage, amelobalsts modulate and transport specific ions required for the accretion of the enamel. The principal action of the ameloblasts is the bulk removal of water and organic material from the enamel to allow introduction of additional inorganic material.
Dentine is the most abundant tissue in teeth, is produced by odontoblasts, which differentiate from mesenchymal cells of the dental papilla, and forms the foundation for enamel formation. Dentin is less mineralized than enamel, contains 70 % mineral by weight, organic phase accounting for 20 % of the matrix and the rest 10 % being water. Type 1 collagen is the primary component of the organic phase. The non collagenous part of the organic matrix is composed of various proteins, with dentin phosphoprotein (DPP) accounting for about 50 % of the non collagenous part . Histologically dentin is permeated by dentinal tubules that run from the pulpal wall to the dentino-enamel junction (DEJ). The diameter of the tubule is 3.0 µm at the pulpal wall but only 0.06 µm at the DEJ. The unusual structure of dentine confers elasticity to the tissue that is absent in the enamel.
The dental pulp is the soft connective tissue that forms the inner core of each tooth. It is divided in the coronal part and the radicular part depending on its position. At the root tip, the radicular part merges with the periodontal ligament (specialised tissue between cementum and jawbone). At this site, blood vessels and nerves enter the tooth. At the interface of pulp and dentine lies a continuous row of columnar odontoblasts. They are responsible for the deposition of dentinal matrix; in turn provide nourishment to dentine.
Cementum is a tiny, slightly basophilic layer at the outer surface of the root part of the tooth and can be distinguished from dentine by the absence of tubuli. It is the product of fibroblasts from the dental follicle migrating to the root surface and differentiating into cementoblasts. Cementum is 50 % mineralised and the primary role is to anchor the tooth to the periodontal ligament.
MATERIALS AND METHODS
2. Materials and Methods
2.1. Sample preparation
One hundred and fifty enamel cores (5x5 mm) were prepared from bovine incisors free from white spots, cracks and other defects. These were then mounted in Epoxicure® (Buehler, USA), an epoxy resin to obtain enamel blocks. The top surface of the enamel blocks were then polished using Ecomet® 250 (Buehler, Germany) grinder- polisher. The specimens were then serially polished using 800, 1200 and finally 2500 grit silica carbide paper (SiC) to obtain a flat and a levelled surface.
Fig 1:A bovine enamel specimen model showing the suggested area scanned (1) the taped area (2) and the epoxy resin (3).
A 1mm area was then masked on either side of the specimens with an acid resistant tape to obtain a 3x3 mm window which was exposed to the dietary acid challenge. Baseline Terahertz pulsed imaging (TPI) and hardness measurements were made on all the specimens. The specimens were then randomly divided into 2 groups, one to be used for investigating early reversible phase demineralisation (surface softening ) and the other for studying later stage erosion (bulk tissue loss).
2.2. Lesion formation
Artificial lesions were prepared by exposing the samples to a dietary acid challenge using 1 % citric acid (Sigma Aldrich, USA), adjusted to pH 3.8 with 5 M sodium hydroxide (Sigma Aldrich, USA). For the surface softening study 50 random samples were chosen and demineralised for 2, 5, 10, 20 and 30 minutes under static conditions with 10 specimens per time point. The Baseline hardness of each group was matched and the average Vickers hardness number (VHN) was 332. Demineralisation was assessed by making terahertz pulsed imaging (TPI) /Surface microhardness measurements at each of the time points. For the bulk tissue loss study 40 random samples were chosen and demineralised using 1% citric acid pH 3.8 for 30, 60, 90 and 120 minutes at 37o C with 10 specimens per group. Baseline hardness of each group was matched and the average VHN was 312. Demineralisation/bulk tissue loss was assessed by making terahertz pulsed imaging / non contact profilometry measurements (NCP) at each of the time points.
Hardness of a material can be defined as its resistance to another material penetrating its surface and is related to its strength. So a higher hardness is related to higher strength, which in turn is directly proportional to the mineral content, in case of enamel, as mature enamel contains 99% mineral. Microindentation, a gold standard technique is the most practical hardness testing technique. In hardness testing, a diamond indenter is slowly pressed onto the test material under a well defined load and left on the specimen for a given time. The material is permanently deformed. The size of the resulting indent is determined with a microscope. The impression length which is measured microscopically and the test load are used to calculate the hardness value.
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Fig. 2: Microindenter, Duramin-1 used in the study (www.struers.co.uk )
The two most common microindentation tests include the Vickers test and the Knoop test. In the Vickers hardness test a 136° square pyramid indenter is used which produces a square indentation in the specimen, which is easy to measure. The hardness is calculated by dividing the load by the surface area of the indentation. Hv = F/A, where Hv is Vickers hardness (in kgf/mm2), F is the load and A is the surface area of the impression .
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Fig. 3: Indent in eroded enamel
The Knoop hardness test makes use of a rhombohedral shaped diamond indenter, where the longer diagonal is 7 times as long as the short diagonal. The Knoop test is conducted similar to Vickers, however instead of the area (Vickers test), only the long diagonal is measured. The Knoop hardness is
Calculated using the formula Hk = 14229L/d2, where L is the load and the d is the length of the long diagonal .
Hardness testing can also be applied to the polished section plane of halves of teeth or tooth slabs, produced by a transverse cut. This is called the cross sectional microhardness testing. The results of previous studies have shown an apparently linear relation between Knoop hardness number and mineral concentration .
For the study, surface microhardness was measured (SMH) using Vickers test at a load value of 1.961 N with a dwell time of 15 s. Only the specimens with a Vickers hardness number between 280 and 340 kgf.mm-2 were used for the study. Six indents were made per specimen and the mean VHN recorded. Microindentation allowed the changes in the enamel hardness, following softening by an acid treatment to be monitored.
2.4. THz imaging
The imaging system used was TPI imaga 1000 (Teraview Ltd., UK)
Fig.4: A schematic of the TPI reflection system.(Provided by Teraview Ltd)
The TPI system used an amplified ultrafast Ti-Sapphire laser system generating 250 fs pulses, with the wavelength centred on 800 nm . The pulse repetition rate was 250 kHz, with an average power of 750 Mw. The laser output was split into two beams, one to be used for the generation of terahertz and the other to be used for detection of terahertz after the terahertz beam had passed through the sample. The generation beam was passed into a Zn/Te semiconductor crystal causing the generation of terahertz radiation. The emitted THz pulses then passed through a series of off-axis parabolic mirrors and then focused onto the sample. The reflected THz pulses were recollimated using another pair of off-axis parabolic mirrors and focused onto another semiconductor detection crystal, collinearly with the detection beam. The THz radiation induced birefringence in the detection crystal and the polarisation of the detection beam was modified from planar to elliptical, giving a nonzero output current from the balanced photodiodes . This effect was linear and the output current was directly proportional to the THz electric field. (Pockel's effect). By sweeping an optical delay in order to vary the optical path length to the receiver, the entire terahertz domain could be sampled. Part of the incident THz pulses reflected from the tooth surface and was measured as Air enamel reflection intensity. By Fourier deconvolving, it was possible to remove artefacts such as ringing effects, secondary spikes and other imperfections associated with the incident pulse. The signal to noise ratio was approximately 5000:1. The TPI system was purged with nitrogen to remove water vapour. The optics were raster scanned in the x-y plane to collect a 5x5 mm grid of data points (spacing 50µm); at each point a complete terahertz waveform was acquired. The resulting data set was three-dimensional with time. Which corresponded directly to depth, as the third axis. Prior to each measurement a terahertz reference waveform was recorded from a gold coated mirror.The Air/enamel interface reflection intensity (AEI) was recorded per pixel from a central 1x1 mm area.
2.4.1. Extraction of the refractive index profile
The terahertz refractive index profile through the lesion was calculated using Equation 2 .
IF is the impulse function and n0, the refractive index in air. By setting the refractive index of sound bovine enamel to 3.09 and the refractive index of air to 1 it was possible to calculate the refractive index profile throughout a specimen by scaling ï‚¶(x) between 0 (on the surface) and 2 (in the sound enamel). By determining the area difference between the baseline lesion, baseline and post treatment RI profiles we obtained ΔΔZ(THz) for each specimen. As RI is a unit-less parameter, the units for ΔΔZ(THz) were arbitrary.
2.5. Non Contact Profilometry (NCP)
NCP is a standard non contact technique to measure bulk tissue loss and surface roughness. This technique is based on laser triangulation detection which uses a triangulation laser to probe the enamel surface. Demineralised specimen blocks were mounted onto the sample holders and the laser made to strike the enamel surface. The laser made use of a camera to look for the location of the laser dot. Depending upon how far the laser strikes the surface, the laser dot appeared at different places in the cameras view. The laser dot, the camera and the laser emitter form a triangle, hence called laser triangulation detection . The laser sensor used for measurement was S13/1.1. The bulk tissue loss was determined on the basis of height variations between the sound enamel surface and the enamel surface exposed to the acid.
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Fig. 5:Non contact profilometer,Scantron Proscan 2100(www.scantronltd.co.uk )
The tapes were removed after demineralisation cycle and the surface cleaned with cotton swab wetted with acetone to remove any tape residues. The Proscan 2100 was set at a scan rate of 1000 Hz and a 4.5 x 2 mm area was scanned with a step size X and step size Y of 0.010 mm. The erosion depth was obtained by a 3- point step height function in the Proscan 2100 software.