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Determination of mineral density of remineralized enamel and dentin: a QLF study
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Determination of mineral density of remineralized enamel and dentin: a QLF study
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Content
Determination of Mineral Density of Remineralized Enamel
and Dentin: A QLF study
by
Garima Sandhu
A Thesis Presented to the
FACULTY OF THE USC HERMAN OSTROW SCHOOL OF DENTISTRY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
CRANIO-FACIAL BIOLOGY
May 2020
Copyright (2020) Garima Sandhu
ii
ACKNOWLEDGEMENTS
I would like to express my deepest appreciation and gratitude to my mentor and
committee chair Dr. Janet Moradian - Oldak. She has been extremely supportive
throughout my journey as a research graduate in her lab. I would like to thank Dr.
Michael Paine for his guidance and support in fulfilling my academic requirements and
thesis project. I express my thanks and appreciation to Dr. Phark for his valuable
suggestions and feedback for my thesis.
I feel grateful to be blessed with the love of my family and friends. My mother (Mrs.
Bhupinder Kaur Sandhu), grandmother (Mrs. Ishwar Sandhu) and my brother (Abhiraj
Singh Sandhu) for always being there for me, to help me in tough times and celebrate in
the moments of happiness.
I would like to appreciate all the members of Dr. Oldak’s Lab - Dr. Jingtan Su, Dr.
Changyu Shao, Dr. Natalie Kegulian, Dr. Kaushik Mukherjee, Rucha Bapat and
Gayathri Visakan for helping me with my project.
I would like to extend a special thanks to our lab manager Thach Vu Ho for addressing
all my technical queries in the lab. And to all the members of the administration - Elsa
Miranda, Janice Bea, Magdalena J. Morales, Linda Hattemer and Gina Nieto for making
this time memorable.
Thank You!
iii
Table of Contents
Acknowledgements……………………………………………………………………..…….ii
List of Tables……………………………….………………………….……….………….......v
List of Figures………………………………………………..……………….……………….vi
Abbreviations…………………………………………………………………………...……viii
Abstract…………………………………………………………………………………...........ix
Introduction…………………………………………………………………………..…….......1
Objectives………………………………………………………………………….................10
Chapter 1: Quantification of Mineral Density Changes in Enamel Sections Using
QLF………………………………………………………………………………………….…..11
Strategy………………………………………………………………..……........11
Material and Methods………………………………………...………….……..11
A) Enamel Section Preparation……………………………….……….………11
B) QLF……………………………………….……………………….................13
C)Treatment Groups……………………………………….………….………..15
C.1) P26 Solution………………………………………………….………...15
C.2) P26-Chitosan……………………………………………….……….....15
C.3) Curodont………………………………………………………………..16
C.4) Control…………………………………………………………………..16
D) Demineralization Cycle…………………………………...…………...........17
E) Remineralization Cycle…………….………………………..………………18
F) QLF Images………………………………….……………….….…………...18
G) Image Analysis and Quantification…………….…….….…………………19
H) Results…………………………………………….………………................20
iv
Chapter 2: Quantification of Mineral Density Changes in Enamel Blocks Using
QLF……………………………………………………………………………………………..26
Strategy…………………………………………………………………………..26
Materials and Methods………………………………………………………….26
A) Enamel Block Preparation……………………………….………………….26
B) Demineralization Cycle……………………………….……………………..27
C) Remineralization Cycle……………………………….……………………..28
D) QLF Image Analysis…………………………………….…………………...29
Results……………………………………….……………….…………………..29
Chapter 3: Quantification of Mineral Density Changes in Dentin Using QLF……..34
Strategy………………………………………………………..…………………..34
Materials and Methods……………………………………………....................34
A) Dentin Section Preparation…………………………………………………..34
B) Demineralization Cycle……………………………………………...............36
C) Remineralization Cycle………………………………...…………................37
D) QLF Image Analysis…………………………………..……………………...37
Results
Aim 3.1 ……………………………………………………………………………37
Aim 3.2 ……………………………………………………………………………41
Aim 3.3 ……………………………………………………………………………46
Discussion………………………………………………………………………...................51
Conclusion………………………………………………………………………...................52
References…………………………………………………………………………………….53
v
List of Tables
Table 1: Overview of treatment groups……………………………………………………17
Table 2: Tabulated ∆∆F values for all groups and their averages for enamel
sections……………………………………………………………………………………….23
Table 3: Tabulated ∆∆F values for all groups and their averages for enamel
blocks…………………………………………………………………………………………31
Table 4: Tabulated ∆F baseline and ∆F demineralization values for dentin
sections……………………………………………………………………………………….40
Table 5: Tabulated ∆F demineralization and ∆F remineralization values for dentin
section in Control group…………………………………………………………………….42
Table 6: Tabulated ∆F demineralization and ∆F remineralization values for dentin
sections in P-26 solution group…………………………………………………………….43
vi
List of Figures
Figure 1: White spot lesions…………………………………………………………………..2
Figure 2: Cervical enamel lesions……………………………………………………………3
Figure 3: Baseline white light longitudinal section image………………………………...12
Figure 4: Post-demineralization white light longitudinal section image…………………12
Figure 5: Schematic representation of QLF system………………………………………13
Figure 6: Demineralized longitudinal enamel section image under blue light………….20
Figure 7: Left window image under blue light……………………………………………...21
Figure 8: Right window image under blue light……………………………………………21
Figure 9: Pre- and post-remineralization QLF images for enamel sections from the 4
treatment groups under blue light………………………………………………….………..21
Figure 10: Graph representing average ∆∆F values for enamel sections……….……...24
Figure 11: Occlusal view of a molar tooth depicting lines of sectioning…………………27
Figure 12: White light image of an enamel block post-demineralization………………..28
Figure 13: Pre- and post-remineralization QLF images for enamel blocks from the 4
treatment groups under blue light……………………………………………………………29
Figure 14: Graph representing average ∆∆F values for enamel blocks…………………32
Figure 15: Baseline blue light image of longitudinal tooth section……………………….35
Figure 16: White light image pre and post demineralization of a transverse dentin
section………………………………………………………………………………………….36
Figure 17: Blue light image of demineralized transverse dentin section………………..38
Figure 18: Graph representing average ∆∆F demineralization values for dentin
sections…………………………………………………………………………………………39
vii
Figure 19: Pre- and post-remineralization QLF images for dentin sections from the 2
treatment groups under blue light……………………………………………………………44
Figure 20: Graph representing average ∆F demineralization and ∆F remineralization
values for dentin sections in the 2 treatment groups………………………………………45
Figure 21: Image of a scanned sample under micro-CT…………………………………..47
Figure 22: Reconstructed image of a sample………………………………………………48
Figure 23: Reconstructed axial view image baseline………………………………………48
Figure 24: Reconstructed axial view image post-demineralization………………………49
Figure 25: Graph for individual BMD values for all samples………………………………49
Figure 26: Graph for mean BMD values…………………………………………………….50
viii
Abbreviations
QLF Quantitative light-induced fluorescence
WSL White spot lesion
∆F Percentage loss of fluorescence with respect to the
fluorescence of sound tooth tissue
∆∆F Difference between ΔF at two time points
BMD Bone mineral density
CEJ Cemento-enamel junction
ACP Amorphous calcium paste
DEJ Dentino-enamel junction
ROI Region of interest
HAP Hydroxy apatite
LRAP Leucine-rich amelogenin peptide
CT Computed Tomography
ix
Abstract
Enamel demineralization is a serious concern for people of all age groups. It can be the
result of dental caries, attrition, abrasion, or erosion. White spot lesions (WSL) are
subsurface enamel lesions that develop as a result of enamel demineralization, and are
characterized by their opaque, chalky white appearance. The chalky white appearance
is due to increased scattering of light by demineralized enamel in the region. Apart from
WSL, tooth demineralization can also present in the form of combined lesions
(comprising of both enamel and dentin) like non-carious cervical enamel lesions. These
lesions involve enamel, coronal dentin along with radicular dentin.
Both these lesions tend to be a huge concern, as they weaken the tooth structure and
present an aesthetic problem. The lesions are extremely hard to restore and are often
difficult to detect at initial stages. Keeping these clinical problems in mind, my project
was designed.
Previous studies in our lab have demonstrated the ability of P26 to remineralize both
enamel and dentin (Mukherjee, Ruan et al. 2018, Mukherjee, Visakan et al. 2020).
Based on this, we developed a project to look at the remineralization potential of P26
peptide and Curodont using non-invasive QLF technique.
1
Introduction
White spot lesions (WSL)
Studies suggest that it takes up to one month for a WSL to appear (Khoroushi and
Kachuie 2017) . Orthodontic treatment with fixed appliances increases the risk of
developing WSL (Tufekci, Dixon et al. 2011, Araújo, Naufel et al. 2015). It has been
reported that by the end of the orthodontic treatment nearly 50% of the patients
developed white spot lesions (Gorelick, Geiger et al. 1982). The increased prevalence
of WSL in patients undergoing fixed orthodontic treatment can be attributed to the
presence of increased volume of dental plaque with a much lower pH around the
appliance (Chatterjee and Kleinberg 1979, Bishara and Ostby 2008). Orthodontic
patients also present with an increase in the level of acidogenic bacteria, like
Streptococcus mutans (Lundström and Krasse 1987, Bishara and Ostby 2008)
WSL tend to be a major aesthetic concern and have been known to weaken the tooth
structure over time. Because the lesions are subsurface, access to the lesion for
treatment is a challenge. Current treatment modalities for WSL rest on restorative
agents like resin sealers, veneers (Bishara and Ostby 2008), amorphous calcium paste
(ACP) (Khoroushi and Kachuie 2017) . Topical application of fluoride is the first step
towards treating a WSL (Bishara and Ostby 2008). Apart from this other procedure like
micro-abrasion, bleaching and resin infiltration are also suggested (Khoroushi and
Kachuie 2017).
2
Figure 1: White spot lesions as seen clinically
Cervical Lesions
With the increase in average age for people, a rise in the occurrence of non-carious
cervical enamel lesions has been observed. It can be attributed to gingival recession
seen in older patients leading to unobstructed exposure to the cemento- enamel
junction (CEJ) (Aw, Lepe et al. 2002). These lesions can present in a variety of forms
like shallow grooves, broad saucer ‐shaped lesions, and large wedge ‐shaped lesions
(Levitch, Bader et al. 1994). It has been reported that non carious cervical lesions occur
in up to 68.5% of individuals, with their prevalence and severity increasing with age
(Kolak, Pešić et al. 2018). It has also been observed that 91% of these lesions have an
axial depth of 1-2 mm (Aw, Lepe et al. 2002).
Enamel is thinnest at the CEJ and therefore occlusal load and stresses from
parafunctional habits tend to easily disrupt the crystalline structure of enamel and
underlying dentin at this location (Aw, Lepe et al. 2002). Large cervical lesions with
(MARCOS VARGAS, BDB, DDS
3
exposed coronal or radicular dentin, often cause dentin hypersensitivity and increase
the risk of pulp exposure or tooth fracture. Cervical lesions often present as combined
lesions, involving demineralization of enamel, coronal dentin as well as radicular dentin.
While the treatment option for these lesions is restorative, the restorations do not last
very long and tend to be a consistent problem. Many new approaches to resolve this
issue have been developed, one being the use of biomimetic peptides to induce
remineralization.
Figure 2: Cervical enamel lesions
Tooth Auto-fluorescence and Quantitative Light-Induced Fluorescence (QLF)
Fluorescence is defined as the property of absorbing radiation at a shorter wavelength
and emitting it at a different, usually longer wavelength. Human teeth have been
associated with fluorescent emission at a peak of 440nm (Lee 2015). Teeth in general,
have the property of autofluorescence, which is attributed to the presence of
(StephenA. Rappeport, Fort Smith)
4
fluorophores in enamel and dentin (Konig, Flemming et al. 1998). Presence of organic
compounds within the apatite matrix is also associated with fluorescence (Sundstrom,
Fredriksson et al. 1985, Winter 1993, Lee 2015). The emission peak of both enamel and
dentin lies in the same range (Sundstrom, Fredriksson et al. 1985), though dentin has a
more intense fluorescence than enamel (Hartles and Leaver 1953, Winter 1993). This
property is also reflective of increased organic content in dentin. With increasing age,
the dental fluorescence decreases and can be used as a reliable indicator to assess the
maturity of dental tissues (Hermanson, Bush et al. 2008, Lee 2015).
QLF stands for Quantitative light-induced fluorescence. It works on the principle that
when a natural tooth is irradiated with light, it emits bluish-green fluorescence (QLF-D
Manual). This fluorescence is absent or decreased in a carious lesion (Angmar-
Månsson and ten Bosch 2001). The device detects this difference in fluorescence by
comparison with the fluorescence from the surrounding sound tissue. This loss of
fluorescence is expressed as a numerical value and is associated with the mineral
density changes of the lesion (de Josselin de Jong, Sundström et al. 1995). The device
consists of a camera connected to a computer. It also has an arc lamp which emits light
at a wavelength of 405nm to cause fluorescence of teeth (Angmar-Månsson and ten
Bosch 2001, Ko, Kang et al. 2015).
Dentin
Dentin forms a significant portion of the tooth crown with a thickness of 3.5-4mm. It is
considered a vital part of the tooth. It is formed by odontoblasts secreting a collagenous
matrix that is subsequently mineralized (Pashley 1991). The outer structure of dentin is
referred to as the mantle dentin with a thickness of 15-30 µm (Goldberg, Kulkarni et al.
5
2011). The inner dentin is known as the circumpulpal dentin and forms the bulk of the
dentin tissue. It is further divided into intertubular and peritubular dentin.
Structurally dentin is comprised of dentin tubules that are oriented as inverted cones
with a smaller diameter of 0.5-0.9µm near the DEJ and a greater diameter of 2-3 µm as
it approaches the pulp (Garberoglio and Brännström 1976). This difference in diameter
accounts for increased permeability in deeper dentin. It has also been proposed that
axial dentin is more permeable than occlusal dentin (Pashley, Andringa et al. 1987).
Each dental tubule is surrounded by a layer of hyper-mineralized dentin referred to as
peritubular dentin. With the convergence of tubule diameter near the DEJ, the presence
of peritubular dentin is also reduced in the area (Outhwaite, Livingston et al. 1976).
The composition of dentin is about 30 – 35 % organic. This is majorly responsible for
the autofluorescence of dentin which is more intense than that of enamel (Hartles and
Leaver 1953, Perry, Biel et al. 1969, Winter 1993). The presence of tryptophan and
hydroxypyridium is linked with this fluorescent emission from dentin (Hoerman and
Mancewicz 1964). The emission peak of dentin is proposed to be 450 nm (Fukushima
1987), which is similar to the emission peak of enamel. It has also been suggested that
there is a reduction of fluorescence from carious dentin (Hartles and Leaver 1953).
Amelogenin - Inspired Peptide P26
Amelogenin is the predominant protein in the enamel extracellular matrix during its
formation and is responsible for the assembly and orientation of hydroxy apatite
crystals(HAP) (Fincham, Moradian-Oldak et al. 1995). Any mutations in the amelogenin
6
gene generally disrupt the enamel structure drastically; e.g.: Amelogenesis Imperfecta
(Collier, Sauk et al. 1997). Murine amelogenin was first sequenced in 1985 (L.Snead,
Lau et al. 1985). Amelogenin is believed to undergo spontaneous self-assembly
aggregation to form nanospheres comprising of more than 100 monomers (Fincham,
Moradian-Oldak et al. 1994, Fincham, Moradian-Oldak et al. 1995), which have been
implicated to have a key role in the organization and orientation of apatite crystals
(Yang, Qin et al. 2010).
It is known that this nanosphere self-assembly is facilitated by the hydrophilic carboxy-
terminal of Amelogenin (Fincham, Moradian-Oldak et al. 1995, Paine, Wang et al.
2003)and is highly conserved amongst species along with the N-teminal (Toyosawa,
O’hUigin et al. 1998, Maycock, Wood et al. 2002). The C-terminal is also known to
prevent excessive agglomeration of the nanospheres (Aichmayer, Margolis et al. 2005,
Lakshminarayanan, Fan et al. 2007).
Mukherjee, Ruan et al. 2016 conducted a study to compare the repair potential
between full length amelogenin and smaller peptides like leucine-rich amelogenin
peptide (LRAP). They demonstrated the formation of dense mineralized layer after 3
days of LRAP application. This approach directed the design of smart bio-inspired
peptides like P26. The sequence of P26 is described as follows:
P26 :MPLPSYEVLTPLKWPSTDKTKREEVD (Visakan, Masters Thesis 2019)
N- terminal is depicted in red, with the circled serine residue representing the
phosphorylation site at S16, important for biomineralization, in full-length amelogenin
and the C-terminal is depicted in blue (Visakan, Masters Thesis 2019).
7
P26 was commercially synthesized with a 95.13% purity (Mukherjee, Ruan et al. 2018)
by CHEMPEPTIDE Limited (Shanghai, China). Several studies have been conducted to
identify the repair potential of P26 peptide. Regeneration of apatite like crystals on the
surface of demineralized enamel with subsequent peptide applications has been
demonstrated (Mukherjee, Ruan et al. 2018).The regenerated apatite had a composition
(i.e., Ca/P ratio) comparable to that of native enamel (Mukherjee, Ruan et al. 2018).
Looking at the biomechanical properties, they noticed a 1.7 - fold increase in elastic
modulus and a 1.8 - fold increase in the hardness of the enamel sample in comparison
with the demineralized enamel. It has also been demonstrated that P26 is effective in
carrying out remineralization of dentin (Mukherjee, Visakan et al. 2020)
Curodont
P11 ‐4 (commercially called curodont) is a rationally designed peptide with the propensity
to self-assemble (Aggeli, Bell et al. 2003) (Parker, Patel et al. 2014). Curodont
commercially is composed of the rationally designed P11 ‐4 peptide along with fluoride
and calcium phosphate (Ceci, Mirando et al. 2016). It is claimed to be effective in
remineralizing WSL and preventing enamel erosions by acids. It acts by diffusing into
the subsurface micro-pores in the monomeric form to form three dimensional scaffolds
and enhancing hydroxyapatite crystal formation (Torres, Chinelatti et al. 2010, Ceci,
Mirando et al. 2016).
8
Chitosan
Chitosan is a derivative of chitin, naturally found in the exoskeleton of arthropods.
Artificially synthesized chitosan is a hydrophilic biopolymer prepared by N-deacetylation
of chitin. Chitosan hydrogel is biocompatible, biodegradable, and mucoadhesive,
making it a perfect medium for the delivery of peptide (Ruan, Zhang et al. 2013). It is
also known to have antimicrobial properties against fungi, viruses, and some bacteria
(Rabea, Badawy et al. 2003). Ruan, Zhang et al. 2013 reported that new enamel
crystals were formed on the existing enamel after chitosan hydrogel application. They
also suggested that because chitosan has antibacterial properties against specific
caries forming bacteria like streptococci and lactobacilli, the new enamel crystals will
potentially be more resistant to enamel demineralization by bacterial activity.
Microcomputed Tomography (Micro–CT)
Micro-CT system is a compact commercial system used to study small animals, hard
tissues like teeth and bone along with biomaterials and polymers (Swain and Xue
2009). It follows the basic principle of X-ray computed tomography wherein a sample is
imaged from multiple angles, and all the images are combined to produce a
reconstructed three-dimensional image. The voxel size for a micro-CT system is 5-
50µm (Swain and Xue 2009). Like the QLF system, the images are stored in a computer
and can be assessed later.
Micro-CT calibration was carried out using commercially manufactured 250 g/cm
3
and
750 g/cm
3
hydroxyapatite phantoms (Mukherjee, Visakan et al. 2020). Studies have
9
demonstrated the ability of micro-CT to effectively assess and quantify changes in
dentin (De-Deus, Belladonna et al. 2017).
10
Objective
The objective of this project was to examine the efficiency of QLF for studying the
mineral density of enamel and dentin after remineralization with P26. While QLF is
routinely employed to study lesions in enamel, the project looked at optimization and
calibration of the QLF system to monitor changes in dentin mineral density as well.
As mentioned in the introduction, the excitation wavelengths of dentin and enamel are in
the same range. Considering this, the project outline spanned across enamel and dentin
with two different models for enamel, namely enamel sections and enamel blocks.
Three aims were formulated to achieve the objective:
Aim 1: Quantification of mineral density changes in enamel sections using QLF.
Aim 2: Quantification of mineral density changes in enamel blocks using QLF.
Aim 3: Quantification of mineral density changes in dentin using QLF and micro-CT.
Each aim is addressed in a separate chapter.
11
Chapter 1: Quantification of mineral density changes in enamel
sections using QLF
Strategy
To demonstrate and evaluate changes occurring in the mineral density of enamel within
a shorter time span (10 days), I used enamel sections. These enamel sections were
considered as a model for enamel acid erosion.
Longitudinal tooth sections of 1.5 mm +/- 0.2 mm thickness were used. Each section
was exposed to periods of demineralization, followed by subsequent remineralization.
Remineralization was carried out in artificial saliva by 4 different treatment groups,
namely P26-chitosan, Curodont, P26 solution, and control. QLF was employed to
assess demineralization and remineralization across all the treatment groups.
Materials and Methods
A) Enamel Section Preparation
Extracted human molar teeth without any pre-existing caries or WSL were selected for
this study (Herman Ostrow School of Dentistry, USC). The teeth were washed with de-
ionized water and sonicated (Branson M1800 Ultrasonic Cleaner with Mechanical timer,
1/2 gallon, formally B1510-MT) for 20 minutes. After sonication, they were rinsed again
with de-ionized water.
12
One longitudinal tooth section measuring 1.5mm (+/-0.2mm) in thickness was derived
from each tooth by sectioning, using low-speed diamond saw (MTI Corporation SYJ160,
USA) under constant water cooling. The tooth sections were rinsed and polished using
a fine grid (2000 grid) sandpaper. The samples were then sonicated again for 5
minutes. The surface of each sample was coated with a clear acid-resistant nail varnish
leaving an exposed window of 1 mm х 2 mm on each right and left side on the enamel.
This was done to optimally use each tooth section and to reduce the number of teeth
used without affecting the sample size in each group. The two windows were the region
of interest for the purpose of this study. The sample was dried for 2 hours after varnish
application, and a QLF image (baseline image) was taken. After demineralization, the
section was split in the middle using a low-speed diamond saw, and each window was
placed in one of the 4 different treatment groups.
Figure 3 Figure 4
13
Figure 3 (Left): Longitudinal enamel section of thickness 1.5 mm with two exposed windows on
either side as seen under white light. Figure 4 (Right): Same longitudinal section post 72 hours
of demineralization with presence of artificially created white spot lesion in the exposed windows
B) QLF
A QLF-D Biluminator
TM
2 was used for this study. The device consists of a full-sensor
SLR Canon 450D camera containing an illuminator ring with white and blue LED’s. The
camera is connected to a laptop for storage and analysis of captured images. The
images are analyzed with QA2 software (QLF version 2000, Inspektor Research
Systems BV).
Figure 5: Schematic view of the QLF-D Biluminator
TM
system (Source: QLF-D Manual)
14
To capture the image, white light from an arc lamp (Philips BV, Eindhoven, The
Netherlands) was filtered through a blue- transmitting band pass filter with a wavelength
of 370 nm (Bennett and Susan 2002). The system also consists of a transmitting filter
(wavelength >520 nm) positioned in front to filter out all reflected light (Bennett and
Susan 2002). Once the image is captured and stored in the computer, it can be
assessed at any time.
For the purpose of analysis in QA2 software, a contour is created completely
circumscribing the ROI in the captured image (Source: QLF-D Manual). Once
completed, the software automatically generates a value for the circumscribed lesion
based on fluorescence loss in comparison with the sound tissue (Bennett and Susan
2002).
The parameters for quantitative analysis of mineral density are as follows:
ΔF: Percentage loss of fluorescence with respect to the fluorescence of sound tooth
tissue.
∆∆F: ΔF(Demin) – ΔF(Baseline) or ΔF(Remin) – ΔF(Demin); A negative ΔΔF implies
mineral loss and a positive ΔΔF value implies mineral gain.
∆Q: The fluorescence loss as seen over the lesion area. It is calculated as ∆F х A and
is expressed as %px
2
.
Area: The area of the demineralized lesion. It is expressed in mm
2
.
We primarily focused on ΔF for this study, as it is believed to be an indicator of mineral
density (Pretty, Edgar et al. 2004). QLF has been routinely employed to study WSL
15
(Ando, Stookey et al. 2006), detect caries (Gmür, Giertsen et al. 2006), and test for
plaque (Han, Kim et al. 2016). Two QLF images were taken, first under white light (ISO
1600, aperture 18, shutter speed 1/125s) and second under blue light (ISO 1600,
aperture 5.6, shutter speed 1/30s). These settings were kept constant throughout the
project.
C) Treatment Groups
4 different treatment groups, namely P26 solution, P26-chitosan, Curodont, and control,
were used for the study. The preparation and mode of delivery for all the 4 treatment
groups are specified as follows.
C1) P26 solution preparation
For Aim 1 the peptide concentration was 0.2 mg/ml (Mukherjee, Ruan et al. 2018).
200µg of peptide was dissolved in 960 µL of ultra-pure water along with 25µL CaCl2 and
15 µL Na2HPO4. The pH was adjusted to 6.50 using 1 M KOH.
C2) P26 - chitosan preparation
200 μg of P26 peptide was dissolved in 960 μL of 2% chitosan solution, along with 25
μL of 0.1 M CaCl2 and 15 μL of 0.1M Na2HPO4. The chitosan solution was prepared as
per the protocol defined by Ruan, Yang et al. 2013. The final pH of the solution was
adjusted to 6.5.
16
C3) Curodont peptide preparation
The concentration of Curodont peptide used was 10 mg/ml. For 10 mg of Curodont, 1
ml or 1000 μL of ultra-pure water was used. Curodont was dissolved in water, and the
solution was vortexed for 20 seconds to ensure complete dissolving. Curodont is an
acidic peptide, but for the purpose of this study, the final pH was adjusted to 8 using 1M
NaOH.
C4) Control sample preparation
Control sample consisted only of the artificial saliva solution without the presence of any
peptide. The artificial saliva was obtained by dissolving 0.0407 g of MgCl2.6H2O, 1.1928
g of KCl, 0.1254 g K2HPO4, 11.915 g HEPES, 0.2452 g NH4Cl and 0.1764 g
CaCl2.2H2O in 900 mL of water (Ruan, Yang et al. 2013). The final volume was fixed to
1000 ml with a final pH of 7. The pH was adjusted using 1 M NaOH.
17
P26 solution P26-chitosan Curodont Control
Concentration:
0.2 mg/mL
(200 µg of peptide in 960
µL of ultra-pure water
with 25 µL CaCl2 and 15
µL Na2HPO4)
Final pH 6.50.
Concentration:
0.2 mg/mL
(200 µg of peptide in 960
µL of chitosan with 25 µL
CaCl2 and 15 µL
Na2HPO4 (Ruan, Yang et
al. 2013)).
Final pH 6.50.
Concentration:
10 mg/mL
(10 mg Curodont in 1000
µL water)
P11 ‐4 is a rationally
designed peptide with
the propensity to self-
assemble (Aggeli, Bell et
al. 2003).
Final pH 8.
Artificial saliva
(0.0407 g of
MgCl2.6H2O, 1.1928 g of
KCl, 0.1254 g K2HPO4,
11.915 g HEPES, 0.2452
g NH4Cl and 0.1764 g
CaCl2.2H2O in 900 mL of
water (Ruan, Yang et al.
2013)).
Final pH 7.0.
Table 1: Overview of the 4 treatment groups
D) Demineralization Cycle
In order to create artificial lesions within the enamel window, demineralization was
carried out using demineralization buffer (2 mM CaCl2, 2 mM KH2P04, 50 mM sodium
acetate, and 0.05 M acetic acid) (Visakan, Masters Thesis 2019) at pH 4.6 for 3 days.
The samples were kept immersed in the demineralization buffer for 3 consecutive days
at 37 C. After demineralization, the samples were rinsed with de-ionized water following
sonication (Branson M1800 Ultrasonic Cleaner with Mechanical timer, 1/2 gallon,
18
formally B1510-MT) for 5 minutes to remove all residual and superficial debris. The
samples were air-dried for 20 seconds, and a QLF image was taken.
E) Remineralization Cycle
Remineralized samples were tested and compared across 4 different groups, namely
P26 solution, P26-chitosan, Curodont, and control. The remineralization cycle was
conducted for 7 days. The samples were air dried for 20 seconds, followed by
application of 40 L of respective remineralization solution for 30 minutes. For each
group, this step was performed with the only difference being in the remineralization
solution. After 30 minutes, the sample was placed in a glass vial with 5 mL of artificial
saliva solution (1.2 mM CaCl2·2H2O, 50 mM HEPES buffer, 0.72 mM KH2PO4, 16 mM
KCl, 4.5 mM NH4Cl, 0.2 mM MgCl2·6H20, and 1 ppm NaF) (Visakan, Masters Thesis
2019) at pH 7.0. The vials were incubated at 37˚C with replacement of the artificial
saliva solution every 24 hours by placement of the sample into a new autoclaved vial.
The reapplication of the peptide was done on Day 3 (72 hours). After 7 days, QLF
images were taken and retained as post-remineralization images.
F) QLF Imaging
QLF images were taken at 3 different timepoints- baseline, post-demineralization, and
post-remineralization, during the study for all samples. Two QLF images were taken,
first under white light (ISO 1600, aperture 18, shutter speed 1/125 s) and second under
blue light (ISO 1600, aperture 5.6, shutter speed 1/30 s). For the purpose of visual
19
identification of white spot lesions, the white light image was considered. However, for
this study, assessment and quantification of mineral density changes were based on the
∆F values obtained from the blue light images.
G) Image Analysis and Quantification
G1) ∆F: The ∆F values for baseline, post-demineralization, and post-remineralization
images were recorded for each sample. Each recorded ∆F value was an average of 3
values to minimize the error. The same was followed for each recorded value at each
time point. The baseline ∆F was mostly calculated to quantify the presence of any
existing demineralization.
G2) ∆∆F: This value is the difference between ∆F at two given time points.
For example.: ∆∆F demin = ∆F demin - ∆F baseline
Similarly, ∆∆F remin = ∆F remin - ∆F demin
A negative ∆∆F implies the loss of mineral density, whereas a positive ∆∆F implies a
gain in mineral density. For the purpose of this study, we calculated ∆∆F between ∆F
remin and ∆F demin.
20
H) Results
The QLF images were taken at 3 time-points, namely, baseline, post-demineralization,
and post-remineralization. Each ∆F value at each time-point was recorded a total of 3
times, and the average of the 3 values was considered as the final ∆F value. This was
repeated for each sample at all 3 time-points.
The baseline QLF image was taken 2 hours after the acid-resistant varnish dried off.
The idea was to calculate a baseline ∆F value to assess for any pre-existing
demineralization. The average baseline ∆F value for all samples was 0.
The second image taken was after 3 days of demineralization (Figure 6). This value
became the ∆F demin. Since the baseline value was 0, ∆F demin was a negative value
indicating a decrease in mineral density.
Figure 6: Demineralized enamel section, as seen under blue light with QLF. The red line depicts
the area of sectioning for splitting of the sample.
21
Figure 7: Left window post- demineralization Figure 8: Right window post-demineralization
The third image was taken after 7 days of remineralization under the 4 different
treatment groups and this value became the ∆F remin.
Groups Demineralized Enamel
Remineralized Enamel
Control
∆F = -10.3
∆F = -9.7
P26
∆F = -19.0
∆F = -10.5
Curodont
22
∆F = -12.3
∆F = -10.9
P26-chitosan
∆F = -14.3
∆F = -13.1
Figure 9: Representative visual and quantitative mineral density changes, as seen after
remineralization with the 4 treatment groups under QLF (blue light images).
The efficiency of any test group was calculated based on the average ∆∆F value for the
group where ∆∆F = ∆F remin- ∆F demin. A negative ∆∆F value represents a decrease in
mineral density, whereas a positive value indicates an increase in mineral density, with
a higher ∆∆F value representing the highest degree of remineralization, implying the
greatest increase in mineral density.
The sample size for each treatment group was n = 7. The degree of remineralization
achieved for each sample was assessed based on the ∆∆F value, as represented in
Table 2 below.
23
Sample
Number
ΔΔF
Control
ΔΔF
Curodont
ΔΔF
P26-chitosan
ΔΔF
P26
1 -0.9 5.8 5.8 8.5
2 8.1 3 5.3 8.6
3 19.2 22.2 25.9 38.1
4 2.2 33.3 38.9 44.2
5 17.3 20.5 12.5 26.6
6 2 34.3 26.3 25.8
7 6.2 36.1 18.7 23.4
Average 7.742857 22.17142857 19.05714286 25.02857143
Standard
Deviation 7.78 13.56 12.25 13.46
Table 2: ∆∆F values for each sample along with the average across all the 4 treatment groups.
Based on the average values a graph was plotted to understand the readings better.
24
Figure 10: Average ∆∆F value in each test group. (*p < .05, ** p < .01, *** p < .001)
Statistical Analysis
Since the sample size in each group was equal, ANOVA test was used to check for
statistically significant differences. The alpha value for the test was set at 0.05. Based
on this, it was observed that the statistical differences in average ∆∆F across the 4
treatment groups (Figure 10) were highly significant (p < 0.01).
Student’s t-test was done between the control group and the P26 solution treatment
group; the result was significant (p < 0.5). (Figure 10)
Student’s t-test between the control and Curodont treatment group represented
statistically significant differences (p < 0.5). (Figure 10)
7.728571429
22.17142857
19.05714286
25.3
0
5
10
15
20
25
30
35
Control Curodont P-26 Chitosan P-26
ΔΔF ΔΔF ΔΔF ΔΔF
∆∆F
Treatment groups
Average ∆∆F values across all groups
25
Student’s t-test between the control and P26 chitosan treatment group also revealed
statistically significant differences (p < 0.5). (Figure 10)
However, no statistically significant differences were reported between the P26 solution,
Curodont, and P26 chitosan treatment group.
26
Chapter 2: Quantification of mineral density changes in enamel
blocks using QLF
Strategy
Based on the results of Aim 1, it was evident that the mineral density of remineralized
enamel could be evaluated by QLF. In order to mimic the clinical problem of WSL,
demineralized enamel block model was used. The enamel blocks were demineralized
for 15 days and remineralized for 21 days (3 weeks). Like Aim 1, the QLF image of each
sample was taken at 3 time-points, namely baseline, post-demineralization, and
remineralization. The P26 peptide concentration for this aim was increased to 0.5 mg/ml
while the Curodont concentration remained the same (10 mg/ml).
Materials and Methods
A) Enamel Block Preparation
Extracted human third molars without any pre-existing caries or white spot lesions were
selected for the study (Herman Ostrow School of Dentistry, USC). The teeth were
cleaned and stored as described earlier in 1.A. Each tooth was divided into 4 blocks
(Figure 11) with the help of low speed diamond saw (MTI Corporation SYJ160, USA)
under constant water cooling. The 4 enamel blocks derived from a single tooth (Figure
11) were each placed in 4 different treatment groups. The blocks were coated with a
clear acid-resistant nail varnish leaving an exposed window of 2 mm х 2 mm in the
27
enamel. The sample was left to dry for 2 hours after varnish application, and a QLF
image (Baseline image) was taken.
Figure 11: Occlusal view of a molar tooth with the lines suggesting the area of division of one
tooth into 4 enamel blocks.
B) Demineralization Cycle
Artificial white spot lesions were created on the enamel blocks by subjecting them to
demineralization. The demineralization was conducted using the solution as described
in 1.D for 15 days at 37 C. After demineralization, the samples were rinsed with de-
ionized water following sonication (Branson M 1800 Ultrasonic Cleaner with Mechanical
timer, 1/2 gallon, formally B 1510- MT) for 5 minutes. The samples were air dried for 20
seconds and a QLF image was taken referred to as the post-demineralization image.
28
Figure 12: Enamel block representing an artificially created white spot lesion after
demineralization as visualized under white light by QLF.
C) Remineralization Cycle
The remineralization was compared between the 4 treatment groups following the same
protocol as described in 1.E. One point of difference was that the concentration of P26
peptide used was increased to 0.5 mg (Prepared by dissolving 500 µg of P26 powder in
chitosan or solution as described above in C1 and C2). The duration of remineralization
was increased to 21 days with application on Day1, Day 7, and Day 14. The artificial
saliva was replaced every 24 hours.
29
D) QLF Image Analysis and Quantification
The protocol for QLF image analysis and quantification was similar to, as described
earlier in 1.F and 1.G. The parameter used for detection of mineral gain or loss was
∆∆F. Each ∆F reading was an average of 3 readings.
Results
In accordance with the results from Aim 1, the remineralization potential of each
treatment group for enamel blocks was assessed based on the ∆∆F value for each
sample.
Groups Demineralization Remineralization
Control
∆F = -29.7
∆F = -23.9
P26
∆F = -30.5
∆F = - 9.3
30
P26-chitosan
∆F = -19.8
∆F = -11.9
Curodont
∆F = -28.7
∆F = -15.2
Figure 13: Representative visual and quantitative mineral density changes as seen after
remineralization with the 4 treatment groups under QLF (blue light images).
∆∆F= ∆F remin- ∆F demin
A positive ∆∆F value was indicative of remineralization, while a negative value
represented demineralization. The ∆∆F value for each sample are represented in Table
3 as follows.
31
Sample
number
ΔΔF
Control
ΔΔF
Curodont
ΔΔF
P26-chitosan
ΔΔF
P26
1 +6.3 +15.4 -2.8 +10.7
2 +7.1 +9.4 +0.3 +11.4
3 +6.9 +4.9 +14.0 +14.0
4 +10.6 +12.6 +6.5 +12.0
5 +2.2 +0.9 +1.2 +12.4
6 +3.8 +15.8 +13.4 +8.4
7 +0.9 +0.5 +0.3 +15.1
8 +1.0 +22.1 +1.6 +23.7
9 +2.3 +3.8 +0.1 +5.3
10 +8.2 +3.1 +3.9 +0.9
11 +10.2 +5.4 +3.5 +21.2
12 +5.4 +1.9 +1.3 +10.2
Average 5.408333 7.983333 4.190909 12.10833
Standard
Deviation
3.38 7.00 5.07 6.19
32
Table 3: The table represents the ∆∆F value for each sample along with the average across all
the 4 treatment groups.
Figure 14: Average ∆∆F value for each treatment group (*p < .05, ** p < .01, *** p < .001)
5.4
7.98
4.19
12.1
0
2
4
6
8
10
12
14
16
CONTROL CURODONT P26-CHITOSAN P-26
ΔΔF ΔΔF ΔΔF ΔΔF
∆∆F
Treatment groups
Average ΔΔF across all 4 groups
33
Statistical Analysis
The sample size for each treatment group was n = 12. One-way ANOVA test was used
to determine statistically significant differences between the means of each treatment
group. The alpha value for the test was set at 0.05.
Based on the results from the ANOVA test, the ∆∆F differences across the 4 groups
were highly significant with p < 0.01 (p=0.00856).
Student’s t-test was conducted between the P26 solution treatment group and control
(Figure 14). The result was highly significant (p < 0.01).
Student’s t-test was also conducted between P26 solution and P26-chitosan treatment
groups. The result was very highly significant (p < 0.001).
Conversely, no statistically significant differences were observed between the Curodont
and the P26 solution groups.
34
Chapter 3: Quantification of mineral density changes in dentin using
QLF
Strategy
Considering the characteristics of dentin mentioned in the introduction, aim 3 was
formulated to quantify the mineral density changes in dentin based on the fluorescence.
In order to carry out Aim 3, it was further divided into 3 sub-aims:
Aim 3.1: Using QLF system to visualize and quantify changes in dentin post
demineralization.
Aim 3.2: Using QLF system to quantify mineral density changes in dentin post
remineralization.
Aim 3.3: Using micro-CT as a standard to establish differences in bone mineral density
(BMD) pre and post remineralization.
Materials and Methods
A) Dentin Section Preparation
A.1) Longitudinal sections
Longitudinal sections were used for Aim 3.1. Extracted human third molars without any
pre-existing caries or WSL were selected for this study, as described in Aims 1 and 2.
The teeth were cleaned and stored as described earlier in 1.A. One longitudinal tooth
section was derived from each tooth. The thickness of each tooth section was 1.5 mm
+/-0.2 mm. The sections were coated with a clear acid-resistant nail varnish leaving two
35
windows of 1 mm х 2 mm, on the right and left sides of the section. The sample was air
dried for 2 hours after varnish application, and a QLF image (baseline image) was taken
(Figure 15).
Figure 15: Longitudinal dentin section, as visualized under blue light with QLF. Encircled area
represents exposed window before demineralization (∆F = 0)
A.2) Transverse/cross-sections
Transverse sections were used for aims 3.2 and 3.3. The criteria of tooth selection and
cleaning were the same as mentioned earlier in 1.A. One transverse tooth section was
derived from each tooth. The section was obtained by carefully sectioning 1.5 mm
above the CEJ and at least 1 mm below the cusp(Mukherjee, Visakan et al. 2020). The
thickness of each tooth section was 1.5 mm +/-0.2 mm. The sections were coated with a
clear acid-resistant nail varnish leaving one window of 2 mm х 2 mm in the center of the
36
section. The sample was air dried for 2 hours after varnish application and a QLF image
(baseline image) was taken.
Figure16: Transverse Cross- section of thickness 1.5 mm thickness as visualized under white
light with QLF before (left) and after demineralization (right).
B) Demineralization Cycle
Demineralization was conducted using the solution as described in 1.D for 5 days at
37 C. After demineralization, the samples were rinsed with de-ionized water following
sonication (Branson M1800 Ultrasonic Cleaner with Mechanical timer, 1/2 gallon,
formally B1510-MT) for 5 minutes. The samples were air dried for 20 seconds, and a
QLF image was taken (Figure 16). This was referred to as the post-demineralization
image.
37
C) Remineralization Cycle
Remineralization was compared between the 2 treatment groups namely P26 solution
and the control group. The concentration of P26 peptide used was 0.5mg/ml. The
duration of remineralization was 10 days with peptide application on Day 1 and Day 5.
The artificial saliva was replaced every 24 hours.
D) QLF Image Analysis and Quantification
The protocol for QLF image analysis and quantification was similar to, that described
earlier in 1.F and 1.G. The parameter used for detection of mineral gain or loss was
∆∆F.
Aim3.1: Using QLF system to visualize and quantify changes in dentin
post demineralization.
In order to conduct the aim 3.1, longitudinal dentin sections, as described in section A.1,
were used. The QLF images were taken at two time-points, namely, baseline and after
demineralization. The purpose of this strategy was to test if QLF can effectively record
changes in the mineral density of dentin.
38
Results
Figure 17: Representative demineralized area of dentin, as visualized under blue light with QLF.
The grey area represents area of demineralization.
Longitudinal dentin sections with a thickness of 1.5 mm +/- 0.2 mm were used for this
aim. One section was derived per tooth. The sample size was n = 17.
Two ∆F values were recorded for this aim, namely, ∆F baseline and ∆F post-
demineralization.
∆∆F = ∆F demin - ∆F baseline
Since this was a comparison between ∆F baseline and ∆F demineralization, a negative
∆∆F value was recorded, which represented demineralization and a reduction in the
mineral density of the tissue. The ∆∆F values for all the samples are charted as follows
in Figure18.
39
Figure 18: ∆∆F demin (∆∆F demin = ∆F demin - ∆F baseline) for each sample.
The ∆F for each group was averaged for conducting statistical tests (Table 4).
-6.16
-5.9 -5.9
-6.8
-6.2
-7.3
-7
-6
-7.9
-9.46
-5.7
-5.8 -5.8
-6.1
-8.4
-6.36
-8.03
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
∆∆F DEMIN
Sample number
40
Sample number Baseline ΔF Post-demin ΔF ΔΔF
1 0 -6.6 -6.6
2 0 -5.9 -5.9
3 0 -5.9 -5.9
4 0 -6.8 -6.8
5 0 -6.2 -6.2
6 0 -7.3 -7.3
7 0 -7 -7
8 0 -6 -6
9 0 -7.9 -7.9
10 0 -9.46 -9.46
11 0 -5.7 -5.7
12 0 -5.8 -5.8
13 0 -5.8 -5.8
14 0 -6.1 -6.1
15 0 -8.4 -8.4
16 0 -6.36 -6.36
17 0 -8.03 -8.03
Average 0 -6.77941 -6.77941
Table 4: ∆F value for each sample at baseline and post-demineralization. ∆∆F for all samples
and their group averages.
41
Statistical Analysis
The paired t-test was done between the averages of ∆F baseline and ∆F
demineralization. The results were highly significant (p < 0.01) at alpha 0.05.
The recorded demineralization in dentin was in the range of -5.7 to -9.46 with a
standard deviation of 1.10. The results were comparable to the demineralization
observed in enamel (Chakraborthy, Masters Thesis 2019) which ranged from -6.5 to -
12.6.
Aim 3.2: Using QLF system to quantify mineral density changes in
dentin post remineralization.
The results from aim 3.1 depicted that QLF could be successfully employed to record
demineralization in dentin sections. Having established that, the next step was to
attempt evaluating mineral density of remineralized dentin. Considering the results from
Aim 2, indicating highest increase in enamel mineral density caused by P26 in solution
treatment, in this chapter I decided to conduct the treatment for dentin using only the
P26 in solution (0.5 mg/ml). The following experiments therefore will have only two
groups including the control.
Results
Like Aims 1 and 2, the results from the P26 and control groups were evaluated based
on the ∆∆F, where ∆∆F = ∆F remin - ∆F demin.
42
The number of samples for the P26 treatment group was n = 14, while the number of
samples for the control group was n = 7.
The ∆∆F values for samples in the control and P26 groups were as follows (Tables 5 &
6)
Control
Sample number Demin Remin ΔΔF
1 -7.73 -8.35 -0.62
2 -15.06 -7.25 +7.81
3 -6.46 -7.13 -0.67
4 0.0 -7.26 -7.26
5 -6.23 -5.4 +0.83
6 -5.96 -6.25 -0.29
7 -6.23 -6.6 -0.37
Average -6.8 -6.89 -0.08
Standard Deviation 3.42 0.97 3.29
Table 5: ∆F demineralization, ∆F remineralization and ∆∆F for each sample in the control group
and their averages
43
P26 Group
Sample number Demin Remin ΔΔF
1 -11.8 -6.9 +4.9
2 -9.03 0.0 +9.03
3 -18.33 0.0 +18.33
4 -11.33 -11.3 0.0
5 -7.26 -6.5 +0.76
6 -11.66 -6.26 +5.4
7 -7.0 -13.1 -6.1
8 -7.45 0.0 +7.45
9 -6.65 0.0 +6.65
10 -6.3 0.0 +6.3
11 -6.5 -6.3 +0.2
12 -5.9 0.0 +5.9
13 -6.8 0.0 +6.8
14 -6.1 -5.85 +0.25
44
Average -8.27 -4.01 +4.05
Standard Deviation 3.48 4.61 5.67
Table 6: ∆F demineralization, ∆F remineralization and ∆∆F for each sample in the P26 group
along with their averages.
Groups Demineralized Remineralized
Control
∆F = -7.5
∆F = -6.6
P26 Solution
ΔF = -12.3
ΔF = -6.7
Figure 19: Visual and quantitative mineral density changes as seen after remineralization with
the 2 treatment groups under QLF (blue light images).
45
Statistical Analysis
Based on the data, the two averages (average ∆F demineralization and average of ∆F
remineralization) of the control group were compared.
Similarly, the two averages (average ∆F demineralization and average of ∆F
remineralization) of the P26 group were compared.
Paired t-test was used to compare the two averages of both the groups (Figure 20). The
results suggested that in the P26 group there was a statistically significant difference
between the averages of ∆F remineralization and ∆F demineralization. The test was
very highly significant, p < 0.01 (p = 0.008387) at alpha 0.05. (Figure 20). In contrast, for
the control group no statistically significant difference was detected between the two
averages.
-6.8
-8.72
-6.89
-4.01
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
Control p26
ΔF
AVERAGE ΔF ACROSS CONTROL
AND P26
Demin Remin
46
Figure 20: Average ∆F demineralization and ∆F remineralization for control and P26 groups (*p
< .05, ** p < .01, *** p < .001)
Aim 3.3: Using micro-CT as a standard to establish differences in
BMD pre- and post-remineralization.
Transverse dentin sections of thickness 1.5 mm +/- 0.2 mm were selected for this aim.
The samples were subjected to demineralization and remineralization, as described in
3.B and 3.C.
Bone mineral density (BMD) analyses were measured at three different time-points for
each sample, namely, baseline, post-demineralization, and post-remineralization.
Micro-CT Analysis
A SkyScan 1174 Micro -CT (Bruker) scanner operating system was used for taking a
360 scan for the samples. The settings used were:
Accelerating voltage - 52 kV
Tube current - 790 mA
Aluminum beam filter -0.5 mm
Rotational step - 0.70° (Visakan, Masters Thesis 2019)
The exposed window was of the dimension 1 mm х 2 mm approximately 2000 µm in
length. The dataset was analyzed using CTAn (Software Version 1.6.9.8 SkyScan,
Bruker Inc). To measure the BMD across the whole window, 5 different lines were
selected (Figure 21). For each line, a ROI was drawn and compared with a range. The
47
comparison range was kept uniform across all samples. The reading for each line was
repeated 5 different times and this was done for 5 different lines. After this, a total of 25
readings were obtained for each sample at one time-point. The average of these 25
readings was considered as the final reading (Mukherjee, Visakan et al. 2020).
Results
Figure 21: Image of a scanned sample under micro-CT. The greyish area in the center
represents the ROI.
48
The image was reconstructed under Dicom-CT (Thermo Fisher Scientific). The
reconstruction of the scanned image allowed for visual identification of the ROI.
Figure 22: Reconstructed view of scanned transverse dentin section. The square window in the
center represents the region of interest.
Figure 23: Baseline image for a scanned sample.
49
Figure 24: Post-demineralization image for the sample from Figure 23, depicting a 0.30 µm
deep lesion in the area of demineralization.
The results were evaluated based on the average BMD of each treatment group post-
demineralization and post-remineralization. The BMD for individual samples at all time
points were also recorded.
Figure 25: Graphical representation of BMD values at different time points for all samples.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
1 2 3 4 5 6 7
BMD
BASELINE BMD POST-DEMIN BMD REMIN BMD DIFFERENCE BMD
50
Statistical Analysis
For statistical analysis, paired t-test was conducted between the mean demineralization
BMD values and mean remineralization BMD values. The alpha value was set at 0.05.
For the P26 treated group, there was a statistically significant difference between the
two means (Figure 26). The result was highly significant (p< 0.01)
For the control group, no statistically significant difference was recorded between the
two means.
Figure 26: Graphical representation of mean BMD values for P26 treated group. (*p < .05, ** p <
.01, *** p < .001)
0.904
1.036
0.8
0.85
0.9
0.95
1
1.05
demin remin
BMD
Average BMD for P26 sample
**
51
Discussion
The weight of treatment modalities today has drastically shifted from curative to
preventive. From the invention of techniques that help diagnose an abnormality at initial
stages, to tests that determine patients to be at high or low risk, the approach is
becoming predominantly preventive.
QLF, which is used for early detection of WSL in enamel, relies upon the natural
fluorescence of dental tissues to quantitatively identify demineralization. The parameter
used, ∆F is implicated to be an indicator of mineral density. To test this, I compared
QLF with micro-CT, a more reliable technique for quantification of mineral density. The
results from both the techniques were comparable and illustrated similar results for a
varied set of samples. This ensures the efficiency of QLF for recording mineral density.
QLF readings do raise some concerns about the reliability of the technique. It has been
noted that there is some margin of error across inter and intra-examiner readings with
QLF (Pretty, Hall et al. 2002). Differences in settings like the position of the camera,
curvature of the tooth surface, and the presence of ambient light (Pretty, Edgar et al.
2002) also effect the readings.
While each QLF reading was an average of 3 readings, some readings in dentin
approached the baseline values after remineralization with P26 peptide solution. This
can be partially attributed to the variability of dentin due to its high organic content and
also to the presence of multiple fluorophores in dentin (Fujimoto 1988). There are
current approaches to use QLF for detection of lesions in root dentin (Günther, Park et
52
al. 2020). But future studies with better coronal and clinical dentin models for optimal
calibration of QLF are needed.
Amongst the 4 treatment groups used in the study, P26 in the solution gave the best
results. Both enamel and dentin samples treated with P26 showed increases in mineral
density in comparison to the demineralized samples. As already mentioned, P26 helps
in the regeneration of apatite like crystals on the surface of demineralized samples. I
noticed the same in cases of erosion model (enamel sections) and WSL model (enamel
block). Based on this, it is evident that P26 in solution form has better penetration
potential in comparison to P26 in chitosan hydrogel. To study this, further research
needs to be directed towards the penetration potential of P26 in both solution and
chitosan hydrogel. Further studies to assess the effect of P26 on the whole tooth dentin
model (cervical enamel lesions and root caries) also need to be carried out.
Conclusion
Based on the results of the project, I conclude that P26 peptide in solution is highly
effective in remineralizing both enamel and dentin. The amelogenin-derived P26 peptide
demonstrated much better results than Curodont, at a much lower concentration for
both enamel and dentin.
QLF is an excellent tool to effectively monitor mineral density changes in both enamel
and dentin. Being highly sensitive and non-invasive, QLF has a great clinical potential.
53
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Abstract (if available)
Abstract
Enamel demineralization is a serious concern for people of all age groups. It can be the result of dental caries, attrition, abrasion, or erosion. White spot lesions (WSL) are subsurface enamel lesions that develop as a result of enamel demineralization, and are characterized by their opaque, chalky white appearance. The chalky white appearance is due to increased scattering of light by demineralized enamel in the region. Apart from WSL, tooth demineralization can also present in the form of combined lesions (comprising of both enamel and dentin) like non-carious cervical enamel lesions. These lesions involve enamel, coronal dentin along with radicular dentin. ❧ Both these lesions tend to be a huge concern, as they weaken the tooth structure and present an aesthetic problem. The lesions are extremely hard to restore and are often difficult to detect at initial stages. Keeping these clinical problems in mind, my project was designed. ❧ Previous studies in our lab have demonstrated the ability of P26 to remineralize both enamel and dentin (Mukherjee, Ruan et al. 2018, Mukherjee, Visakan et al. 2020). Based on this, we developed a project to look at the remineralization potential of P26 peptide and Curodont using non-invasive QLF technique.
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Sandhu, Garima
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Determination of mineral density of remineralized enamel and dentin: a QLF study
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Craniofacial Biology
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05/08/2020
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