Appendix 3
Description of material and methods to be used within the project
Tooth material
Bovine tooth material for the in vitro studies
Bovine enamel has long been used for different in vitro studies since its chemical and
morphological properties resemble those of human dental enamel and are therefore an
excellent replacement for human teeth. Bovine teeth are therefore an excellent proxy for
human teeth and also far more easy to handle due to their size. The surface layer of bovine
and human enamel is after eruption subjected to the influence of the oral environment and
takes up different trace elements which are accumulated in the surface area of the enamel. By
removing the surface layer down to the bulk of the enamel, the possible variations of
morphology and chemical composition are reduced.
Permission to collect bovine teeth is given by the Swedish Board of Agriculture (Dnr.
38-3536/12; 2012-05-31) and from an abattoir. Extraction of the incisors is performed by the
researcher at the abattoir and immediately after extraction, the teeth are placed in de-ionized
water with 1 crystal of thymol/liter. After transport to the laboratory, the teeth are kept in a
refrigerator at 4o C until further preparation.
The bovine teeth will be used for in vitro studies involving artificially demineralized
subsurface lesions, resin infiltration, enamel reactivity to different treatment methods and
reactions to exposures to environmental compounds.
Human tooth material for the in vitro studies
In collaboration with the Specialist clinic for Orthodontics in Gothenburg, premolars (with
normal mineralized enamel and premolars exhibiting different degrees of fluorotic changes of
the enamel) extracted for orthodontic reasons are to be collected. Immediately after
extraction, the teeth are placed in de-ionized water with 1 crystal of thymol/liter. After
transport to the laboratory, the teeth are kept in a refrigerator at 4o C until further preparation.
In vitro studies
In close collaboration with the Public Dental Service in the Västra Götaland Region, clinical
studies will be planned regarding treatment of hypomineralized enamel and white spot
lesions.
Bio Bank
A Bio Bank (ID-nr 830) containing collected teeth with different diagnoses is accessible for
researchers. The bio bank also comprises control teeth of normal mineralized teeth. The teeth
are kept in ethanol, thymol or formaldehyde.
Methods
All methods for embedding, sectioning and light microscopic studies are well-established and
documented in guide lines and instructions at the Department of Pediatric Dentistry, Institute
of Odontology, Gothenburg. Preparation of un-decalcified dental hard tissues is unique for the
hard tissue laboratory at the Department for Pediatric Dentistry and teeth from different
institutions are referred for histological analysis. Several of the methods have through the
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years been evolved and refined. Special emphasis has been made for preparation of the hard
tissue specimens in a way enabling the same specimen to be used for different analytical
methods. In close cooperation with the collaboration partners, which are specialists within
their research areas, requirements for the different methods have been developed.
For chemicals, embedding media etc. used in the laboratory, adherent Material Safety
Data Sheets (MSDS), Product Data Sheets (PDS), detailed product-related information, safety
and handling precautions as well as handling instructions are available at the laboratory.
Embedding and preparation of un-decalcified dental hard tissue specimens and sections
Before embedding in an epoxy resin, the teeth are stored for 24 hrs. in 70% ethanol. For
embedding media, an epoxy-resin for electron microscopy is used (Epofix®, Electron
Microscopy Sciences, Fort Washington, PA, USA). The teeth are cut or sectioned according
to the aim of the studies in a Leica SP1600 Saw Microtome (Leica Mikrosysteme Vertrieb
GmbH, Wetzlar, Germany). Prior to any analyses, the surface is cleaned in an ultra-sonic bath
in Histocon and de-ionized water for 30 seconds, respectively.
Before cutting sections (thickness120 m), an object glass is glued on the previously
cut surface, with a light curing one-component adhesive (Technovit 7210 VLC, Heraeus
Kulzer GmbH, Hanau, Germany). This technique allows optimal positioning of the sample on
the glass and prevents loss of tissue during the cutting procedure.
The surface layer of the enamel in the bovine teeth is polished off under water cooling
with silicon-carbide paper in a polishing machine to a flat surface (Struers, Copenhagen
Denmarks). The final polishing is performed with three different papers (grit 1200, 2400 and
4000 = 4 microns). Prior to further analyses, the surface is cleaned in an ultra-sonic bath in
Histocon and de-ionized water for 30 seconds, respectively.
The Department of Pediatric Dentistry is unique in Scandinavia for the embedding and
preparation of un-decalcified dental hard tissue specimens and sections. The method has been
used since 1982 and has been refined through the years.
Light microscopy - Stereo microscopy (LMSM)
Before the embedding and sectioning of tooth samples, an examination is carried out in a
Leica M80 stereo microscope (Leica M80 with 8:1 zoom, 0.75x-6x, Leica Mikrosysteme
Vertrieb GmbH, Wetzlar, Germany). Overviews of specimens and sections are taken in
incident light with a matt black background. Digital images are taken of all teeth and sections
using a Leica digital camera (Leica DFC420 C, Leica Mikrosysteme Vertrieb GmbH,
Wetzlar, Germany) equipped with Leica Application Suite LAS V3.7.0 (Leica Microsystems
AG, Heerbrugg, Switzerland). The digital images are also used for orientation when analyzed
with XRMA and/or ToF-SIMS.
Polarized light microscopy (POLMI)
All sections are examined dry in air and after water imbibition in an Olympus polarizing light
microscope (Olympus, Tokyo, Japan). Digital images are produced using a Leica digital
camera and Leica Application Suite.
POLMI is a standard method at the Department of Pediatric Dentistry for analysis of undecalcified sections. The morphology of the dental hard tissues can be examined and the
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extension and degree of hypomineralization/porosity in the enamel can be established. By
using different imbibition media, the pore volume distribution (degree of porosity) can be
quantified. POLMI analysis is applicable for studies of developmental disturbances and also
for studies of dynamic events as in following demineralization in artificial caries experiments.
Microradiography (MRG)
Microradiography is an excellent method for establishing the degree of mineralization in undecalcified sections of dental hard tissues. The technique has been used in a number of
scientific articles from the early 1980s. An apparatus for microradiography is located at the
hard tissue laboratory, however, not yet installed after being moved from an external
laboratory. A suitable location is identified, electrical installation and installation of water and
sewage remains to be carried out.
Digital camera and software applications
The hard tissue laboratory has access to modern microscopes including a stereo microscope,
polarized light microscope and a phase contrast microscope. All microscopes are equipped to
be used with the digital camera (Leica DFC420 C). The acquisition and processing of digital
images is made in a high performance computer with software (Leica Application Suite LAS
V4.1).
X-ray Micro Analysis (XRMA) and Scanning Electron Microscopy (SEM)
XRMA is a commonly used method for elemental analysis in dental hard tissues with the
advantage of being an integrated part of a scanning electron microscope, enabling control
where the analysis is made. The isolating properties of dental hard tissues create problems for
electron beam microanalysis which partly can be solved by applying thin coatings of
conductive material on the sample, for example gold or carbon. However, the coating
introduces other problems making reliable and consistent measurements difficult to manage.
In collaboration with professor David Cornell, Department of Earth Sciences, University of
Gothenburg, Gothenburg, Sweden, a new technique for XRMA analysis of enamel and dentin
has been developed which to a large extent manages the problems with for example surface
charging and absorption of X-rays in the coating layer. Further, the analysis still allows full
control of the analyzed area in relation to the morphology of the tissue.
For the XRMA analysis, a Hitachi S-3400N scanning electron microscope equipped
with an Oxford EDS microanalysis system and INCAEnergy software (Oxford Instruments,
Abingdon, England). The surface of the tooth samples is partly covered with gold in a plasma
coater (≈5 nm thick), followed by a second coating with a linear gold coating through a raster,
thus leaving the areas for analysis un-coated. Thereafter, the samples are placed in a sample
holder for SEM.
The analyses are carried out using a 1500 magnification as area analyses (80x80 m),
with a counting time of 100 seconds. The “All elements” option in the INCA software is used
for the elements carbon (C), oxygen (O), sodium (Na), magnesium (Mg), phosphorous (P),
chlorine (Cl), potassium (K) and calcium (Ca). The XRMA analyses are carried out in a lowvacuum mode (VP-SEM) with a vacuum of 20 Pa. All analyses were carried out at 20 kV
accelerating voltage and the working distance from sample to electron optical column was set
to 9.9 mm, with a tolerance of 0.1 mm.
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The standard calibration is performed by acquiring a spectra with a live time of 100
seconds on cobalt metal in the sample holder. Wollastonite from Microanalysis Consultants,
Cambridge UK (MAC) was used as reference standard for O and Ca, CaCO3 for C Jadeite
MAC for Na, MgOMAC for Mg, CaPO4 for P, Tugtupite for Cl and K-feldspar MAC for K.
These simple standards are linked to the cobalt standard which is counted after specimen
current stabilization at the beginning of each high or low vacuum session. The accuracy of
analyses is checked periodically using mineral standards from the Smithsonian Institute,
Washington.
The results for the analyzed elements given as “Apparent concentrations” are corrected
by a phi-rho-z iterative procedure resulting in an “Intensity correction”, which is applied to
provide analyses in element “Weight%”, with counting errors given as “Weight% Sigma”.
The same instrument described above can be used for high resolution SEM imaging of
both un-decalcified and decalcified dental hard tissues. The VP-SEM mode may be utilized
for taking SEM images without coating. However, the resolution can be improved if the
specimens are etched with 30% phosphoric acid for 45 seconds before coating with gold.
Therefore, an improved SEM analysis can be carried out on the same specimens previously
analyzed without coating with XRMA and/or ToF-SIMS analysis when the specimens are
coated with gold.
Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS analysis)
The SIMS technique has proven to be one of the very few techniques for quantitative analysis
of fluoride in dental hard tissue. The Department of Pediatric Dentistry has a long tradition
working with analytical SIMS in connection fluoride content in enamel and elemental
composition in developmental disturbances of dental hard tissues. ToF-SIMS is an instrument
with excellent analytical performance of both bulk specimens and un-decalcified sections of
teeth. It requires extremely clean surfaces wherefore the sample preparation is crucial for
obtaining valid results. Another feature which makes the ToF-SIMS interesting is the
possibility of elemental mapping where the location of different elements can be found not
only in relation to each other, but also to the morphology of the sampled analyzed.
Polished and cleaned tooth specimens are analyzed by ToF-SIMS in regions according
to the LMSM and/or POLM findings. The instrument used is a ToF-SIMS V instrument
equipped with a Bi31-liquid metal ion gun (ION-ToF, Münster, Germany). The bunched
mode (mass resolution: m/Dm46000; focus of the ion beam: 3 mm) with a target current of
0.15 pA will be used to acquire high mass resolution images of the samples. The ION-ToF
Ion image software (Version 4.1, ION-ToF) is used for evaluation of the results.
Measurements of elements and element combinations in positive and negative spectra will be
performed in enamel treated according to the experimental procedures for the different studies
and the non-treated enamel in the same tooth will serve as control. Elements of special
interest for analysis with ToF-SIMS are C, F, Na, Mg, K and CN, as well as organic element
combinations of H, C, O and P.
Specimens analyzed by ToF-SIMS can be analyzed in the same locations with other
analytical methods such as XRMA or Raman infrared spectroscopy.
In an on-going collaboration project with Per Malmberg, Department of Medical
Chemistry and Cell Biology, Institute of Biomedicine, University of Gothenburg, regarding
improved high-resolution elemental imaging of enamel is under progress. Elemental images
which can be related not only to images of different elements but rather to structures in the
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Appendix 3
enamel morphology (inter/intraprismatic location) is of great importance. So far the results are
very promising for a number of important elements.
X-ray Micro Computed Tomography (XMCT)
Micro computed tomography or "micro-CT" is X-ray imaging in 3D. The method is based on
X-ray scans with a massively increased resolution, where very fine scale internal structure of
objects is imaged non-destructively. The sample's internal 3D structure at high resolution may
be provided, in the case of a MIH tooth, a 3D image of the hypomineralized enamel in
relation to the normal enamel and the dentin will be disclosed.
XMCT is a method which is extremely useful in studies of, for example,
hypomineralized enamel or caries, since the extension of the lesions may be explored when
rendering of the X-ray images is performed, providing a 3D image. The single X-ray images
may also be used as high resolution microradiographs in which the degree of mineralization
can be measured.
Since XMCT is a non-destructive method it can be applied to follow dynamic changes
in mineralization in connection to progression of artificially demineralized subsurface lesions
and in remineralization experiments.
In collaboration with Dr. Graham Davis (Institute of Dentistry, Barts and The London
School of Medicine and Dentistry, London, UK) and Dr. Janice M. Fearne (Department of
Paediatric Dentistry, Royal London Hospital, London, UK) XMCT has been utilized for
analysis of primary teeth with the diagnose Dentinogenesis Imperfecta (DI). DI is a rare
hereditary condition which may affect either the primary or permanent dentitions or
sometimes both dentitions. The results achieved so far are new and spectacular and a first
draft of the manuscript is present.
Spectroscopic methods for studies of enamel surfaces
In collaboration with Dr. F. Taube (Department of Occupational and Environmental
Medicine, Sahlgrenska University Hospital, Gothenburg) and research groups at Chalmers
University of Technology, different spectroscopic studies of the enamel surface have been
carried out.
X-ray diffraction (XRD) and Diffuse Reflectance Infrared Fourier Transform
Spectroscopy (DR-FTIR) have been utilized for identification of the crystalline mineral
phases in dental enamel powder. Raman Fourier Transform Spectroscopy (IR-Raman) has
proven to be useful for identifying organic components in the enamel and also has the
advantage that the same specimen can be analyzed with other methods i.e. XRMA and/or
ToF-SIMS.
Preparation of artificially demineralized subsurface lesions
For studies of artificially demineralized subsurface lesions, bovine teeth, where the buccal
enamel surface is removed, are exposed to a demineralizing solution. The teeth are covered
with an acid resistant carbon tape leaving an unprotected window on the buccal surface. The
lesions are made with an 8% methyl cellulose gel (0.1 mol/lactic acid, pH 5.3) and placed
individually in a 30 ml jar, filled with demineralizing solution in a temperature of 37°C for 30
days. The un-exposed enamel serves as an internal control.
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Prior to further preparation and analyses, the teeth are stored in artificial saliva. Undecalcified sections of the teeth are prepared in a Leitz low speed saw microtome. Sections
and bulk specimens can be used for different analytical methods (i.e. POLMI, MRG, Raman
spectrometry, SEM, XRMA, ToF-SIMS).
Procedures for remineralization of artificially demineralized enamel and subsurface
lesions
Remineralization of enamel is an important part of preventive procedures with the potential to
fill the gap between prevention and intervention (filling therapy). Therefore, in vitro studies
are of importance for evaluation of relevant agents for remineralization of enamel. Due to the
large size of the bovine tooth specimens, they can be cut into two halves leaving one part for
analysis with different methods and leaving the other half for remineralization experiments.
Thereby, the single tooth can act as a control both for demineralization and remineralization.
A number of methods employing different remineralization agents are described in literature.
Hardness measurements
After appropriate analysis of tooth specimens (e.g. XRMA, SEM, ToF-SIMS), hardness
measurements are performed with a digital micro hardness tester fitted with a Vickers
diamond indenter (FM-100e, Future-Tech Corp, Tokyo, Japan). Through object lenses the
specimen surface can be observed and recorded, thus the placement of an indentation and its
reading will be accurate and repeatable. Hardness measurements are of value for in vitro
studies in connection to demineralization and remineralization experiments of enamel
surfaces. The hardness measurements of the affected enamel surface will be carried out in
collaboration with Professor Carina B. Johansson, Institute for Odontology, Gothenburg.
Statistical analyses
The collected data from the different analyses are put into a spread sheet program (Excel;
Microsoft, Seattle, WA, USA). Prior to the clinical studies, a power analysis will be made.
For the statistical analysis SPSS 20 (SPSS, Chicago, IL, USA) for Windows will be used.
Depending on the statistical characteristics of the data, the F-test to compare standard
deviations with 95.0% confidence intervals or the Mann-Whitney U-test for unpaired analysis
will be used. In addition, regression and correlation analyses will be performed.
Inductive analytical methods
A powerful complement to statistical methods is inductive analytical methods. Inductive
analytical methods belong to the family of Artificial Intelligence (AI) which is widely used in
the industry, different research areas and in military applications. It can basically be described
as pattern recognition. Inductive analysis has the potential to reveal relationships in an explicit
way and has the capacity to show the existence of knowledge gaps and clashes (when two
identical examples have different outcomes). A special feature is the so-called frequency
normalization which allows compensating for discrepancies in the frequency of the outcomes,
meaning that low frequency will be allowed to be analyzed more often.
Inductive analysis has been used for a range of different applications. In short, data is
compiled in an Excel spread sheet, where the values (numerical or discrete) for the different
attributes are set in columns, each row representing one example. A column of discrete values
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representing measured data is set as outcome in order to enable an analysis of possible
relationship between the different data and the outcome values. Data is imported to an
inductive analysis program DataMiner (Attar Software, Lancashire, UK). The results are
presented in a hierarchic diagram (knowledge tree) in which the importance of every attribute
in the inductive analysis is specified by its position in the knowledge tree. The higher in the
tree, the more important for the outcome and thus, the tree shows how different attributes
affect the outcome. In the induction process, a knowledge tree is generated by repeatedly
splitting the given data set according to different attributes until terminal points (leaves) are
reached.
Equipment, instruments and chemicals available in the hard tissue laboratory

Mounting cups for embedding of teeth, diameter 25 mm, suitable for the saw
microtome and SEM/XRMA analysis

Embedding resin, (Epofix®, Electron Microscopy Sciences, Fort Washington, PA,
USA)

Technovit 7230 VLC adhesive light curing resin

UV-lamp for polymerization of Technovit

2 Leica SP1600 Saw Microtome (Leica Microsystems GmbH, Wetzlar, Germany)

Cover glass and object glass

Instruments for measuring section thickness

Polishing machine (Struers)

Polishing papers

Chemicals for demineralization solutions

Stereo microscope equipped with fluorescence accessory (Leica M80).

Polarization microscope, 2 available microscopes (Olympus).

Phase contrast microscope/Polarization microscope for morphological examinations of
un-decalcified sections and for quantifying the degree of porosity (Leica DM2500).

Digital camera applicable for all microscopes (Leica DFC420C).

Computers with software for the digital camera (Leica Application Suite LAS V4.1),
Adobe Photoshop element 7.0 and Inductive analyses software program XpertRule
and DataMiner (Attar Software, Lancashire, UK).

Apparatus for microradiography
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