University of Southern California Dissertations and Theses

Amelogenin peptide - chitosan for remineralization of artificial enamel lesions: a QLF analysis

(USC Thesis Other)

Amelogenin peptide - chitosan for remineralization of artificial enamel lesions: a QLF analysis

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content 1

AMELOGENIN PEPTIDE - CHITOSAN FOR
REMINERALIZATION OF ARTIFICIAL ENAMEL LESIONS: A
QLF ANALYSIS

By
Amrita Chakraborty

A thesis presented to the faculty of
Ostrow School of Dentistry of USC,
University of Southern California
In fulfillment of the requirements for the degree
Master of Science,
Craniofacial Biology

May 2019
2

ACKNOWLEDGEMENTS

I would like to thank Dr. Janet Moradian-Oldak, for being my mentor during my master’s
studies. She gave me feedback and direction throughout the course of my degree
regarding lab work, thesis and classes. It was because of her guidance, patience and
understanding that this research has been possible. I would also like to thank my
committee members- Dr. Janet Moradian-Oldak, Dr. Michael Paine, Dr Malcolm Snead
and Dr. Jin-Ho Phark. I am also very grateful for all the help I received from my lab
colleagues and friends who took time out from their own work to teach me all the
techniques I know. Without them I would not have been able to accomplish this. I thank
and appreciate the help I received from Dr. Kaushik Mukherjee, Dr. Qichao Ruan,
Gayathri Visakan and the volunteers who worked alongside me- Judith Naziri, Saeed
Bigleni and Ali Aldulaimy. Last but not the least, I thank my husband and my father
without whom this would not have been possible.

3

TABLE OF CONTENTS:
ACKNOWLEDGEMENTS…………………………………………………...…………. 2
LIST OF FIGURES & TABLES……...…………………………………………………. 5
ABBREVIATIONS………………………………………………………………………... 7
INTRODUCTION…………………………………………………………………………. 8
ABSTRACT……………………………………………………………………………….. 8
BACKGROUND…………………………………………………………………………...
1. ENAMEL
2. THE CLINICAL PROBLEM – DENTAL CARIES AND WHITE SPOT
LESIONS (WSLS)
3. A NEW TECHNIQUE TO DETECT WSL – QLF
4. CURRENT SOLUTIONS OF TREATMENT OR PREVENTION OF WSL
5. AMELOGENIN
6. REMINERALIZATION OF WSLS WITH AMELOGENIN DERIVED
PEPTIDES
7. CURODONT
TM

8. AMELOGENIN DERIVED PEPTIDE – P26 AND P32
9. CHITOSAN HYDROGEL
10
OBJECTIVES, HYPOTHESIS & SCOPE……………………………………………… 28
SPECIFIC AIMS…………………………………………………………………………. 29
METHODOLOGY………………………………………………………………………....
FOR AIMS 1, 2 & 3:
1. PREPARATIONS OF P26-CS AND P32-CS HYDROGEL
2. PREPARATION OF CURODONT
TM

3. PREPARATION OF ARTIFICIAL SALIVA
4. PREPARATION OF DEMINERALIZING SOLUTION
5. PREAPRATION OF TEETH SECTIONS
6. ENAMEL REMINERALIZATION PROTOCOL
29
4

7. QLF IMAGES OF REMINERALIZED WINDOWS
8. CHARACTERIZATION OF SECTIONS USING SEM & XRD
FOR AIM 4:
1. WHOLE TOOTH SAMPLE PREPARATION & MOUNTING
2. WHOLE TOOTH SAMPLE DEMINERALIZATION AND
REMINERALIZATION WITH P26-CS HYDROGEL
RESULTS…………………………………………………………………………………. 37
DISCUSSION……………………………………………………………………………... 50
CONCLUSION……………………………………………………………………………. 51
BIBLOGRAPHY…………………………………………………………………………... 52

5

LIST OF FIGURES & TABLES
Fig. 1: SEM of human enamel showing parallel arrangement of
apatite
10
Fig. 2: Stages in progression of dental caries form a sub-clinical
stage to visible decay
12
Fig. 3: Image of white spot lesion (WSL)- black circle – on human
teeth
13
Fig. 4: Polarized light image of white spot lesion with intact surface
enamel
13
Fig. 5: Superficial hypo-mineralized lesion 14
Fig. 6: Inspektor
TM
Pro, the QLF-D Biluminator
TM
and the QLF
TM

On-Line tower
16
Fig. 7: Images taken with the QLF-D Biluminator™ 2. 17
Fig. 8: Amino acid sequence of full-length human amelogenin 19
Fig. 9: Incisors of wild type and null mice. 20
Fig. 10: Amelogenesis Imperfecta 21
Fig. 11: TEM images of CA-P mineralization in presence of P26 &
P32
25
Fig. 12: SEM images of sections treated with P26 and P32 26
Fig. 13: Newly formed synthetic aprismatic enamel-like HAP layers
grown in the presence of amelogenin-inspired peptide.
26
Fig. 14: Whole tooth samples mounted on dental stone blocks and
dispersed in a vacuform machine.
35
Fig. 15: Schematic representation of the methodology used to make
the whole tooth samples with reservoir pouches.
36
Fig. 16: Visual QLF images and numerical representation of enamel
sections after demin and remin with P26-CS, CS and AS
38
Fig. 17: Bar chart representing average mineral loss or mineral gain
across the 3 groups (AS, P26-CS, P32-CS).
39
6

Fig. 18: Visual QLF images and numerical representation of enamel
sections after demin and remin with P26-CS hydrogel,
Curodont
TM
, CS hydrogel and AS
41
Fig. 19: Bar chart representing average mineral loss or mineral gain
across the 4 groups. CS hydrogel, Curodont
TM
, P26-CS
hydrogel, Artificial Saliva
42
Fig. 20: SEM images of enamel windows treated with P26-CS
hydrogel and Curodont
TM

44
Fig. 21: XRD spectra of Curodont
TM
, P26-CS hydrogel, Artificial
Saliva and Demin enamel
45
Fig. 22: A) Whole tooth samples with UHU tabs to form reservoirs in
thermoplastic crowns.
B) Thermoplastic crowns with reservoir pouches.
46
Fig. 23: Wax up of potential white spot lesions on dental stone cast
of patients.
47
Fig. 24: Bleaching tray for maxillary arch with reservoir pouches. 48
Fig. 25: QLF image of whole tooth sample. 48

Table 1: Parameters of QLF analysis. 17
Table 2: Molecular Mass, Isoelectric Point & Physicochemical
properties of P26 and P32.
24
Table 3: Average ΔΔF (ΔF remin – ΔF demin) across 15 sections
with standard deviation and standard error across each
group (For Aim 1).
39
Table 4: Average ΔΔF (ΔF remin – ΔF demin) across 15 sections
with standard deviation and standard error across each
group (For Aim 2).
42

7

ABBREVIATIONS
WSLs - White Spot Lesions
QLF - Quantitative Light Induced Fluorescence
CS - Chitosan
SEM - Scanning Electron Microscopy
ICDAS - International Caries Detection and Assessment System
CPP - Casein Phosphopeptide
LRAP - Leucine Rich Amelogenin Peptide
ADP5 - Amelogenin Derived Peptide 5
HAP - Hydroxyapatite
PDL - Periodontal Ligament Fibers
TEM - Transmission Electron Microscopy
S. mutans - Streptococcus Mutans
XRD - X ray Diffraction
ISO - International Organization for Standardization
AS - Artificial Saliva

8

INTRODUCTION
Abstract:
Dental Caries is one of the most prevalent diseases in the modern world. Due to its
widespread and debilitating nature, it is imperative to detect the carious lesions early
and treat them. The process of cavitation begins as sub surface demineralization
caused by plaque accumulation on the surface of teeth. This sub surface demineralized
enamel appears chalky-white and is called a White Spot Lesion (WSL).
Multiple treatment modalities are currently available to treat WSLs like topical fluoride,
MI Paste plus and ICON resin infiltration. However, a newer approach to treat these
lesions is through biomimetics. A biomimetic solution emulates the delicate and unique
process of nature by inducing the growth of apatite-like crystals on the demineralized
surface. One such approach was taken in this study using amelogenin peptides P26
and P32 chitosan hydrogel (P26-CS, P32-CS) to remineralize windows of demineralized
enamel sections. Curodont
TM
was used for comparison as it is currently commercially
available.
Quantitative Light-induced Fluorescence (QLF) was used as an analytical tool for the
study as it is one of the few clinical detection techniques for WSLs.
A protocol was developed to analyze demineralized enamel sections (as a model for
WSL) and their consequent remineralization using Curodont
TM
and P26-CS. Both visual
and numerical changes after demineralization and after remineralization were analyzed.
A proposed model for whole tooth samples was also developed to mimic WSL and
9

enable the study of the remineralization by the peptides under more clinically relevant
scenario.
The QLF system was optimized to make images using Blue light at ISO 1600, Shutter
speed 1/30s and Aperture 5.6. In the pilot study to compare demineralized lesions on
enamel sections between artificial saliva, P26-CS hydrogel and P32-CS hydrogel,
significant results were obtained between artificial saliva and the other groups while the
remineralization potential between P26-CS and P32-CS was not significantly different.
Visual QLF images and numerical analysis of ΔF values across the groups Artificial
Saliva, Curodont
TM
, P26-CS hydrogel and CS hydrogel were conclusive to demonstrate
remineralization potential of Curodont
TM
, P26-CS hydrogel and CS hydrogel. However,
there was no significant difference between these 3 groups.

10

Background:
1. Enamel:
Tooth enamel is a remarkable biomaterial, which is characterized by a unique
structure and mechanical properties specific to its structure. The high content of
hydroxyapatite, parallel arrangement of individual apatite crystals forming enamel
prisms, along with the interwoven three-dimensional picket fence arrangement
give it unique mechanical properties of great hardness and physical resilience.
(Fig. 1) (Pandya & Diekwish, 2019)

Fig 1: SEM of human enamel showing parallel arrangement of apatite

The process of enamel formation is termed amelogenesis and involves highly
specific interactions between charged proteins and various ions. The different
stages of this protein-controlled mineralization process are presecretory,
secretory, transitional and maturation stages. Each stage is defined by the
morphology and function of a single cell layer of ameloblasts. (Ruan and
Moradian-Oldak, 2015)
11

The process of enamel formation is controlled by the interaction of various
enamel matrix proteins like amelogenin, enamelin, ameloblastin and amelotin.
Amelogenin constitutes about 90% of the enamel matrix proteins and plays a
major role. (Bansal et al., 2012)
Since enamel is formed only once and does not regenerate, it is a formidable
task to attempt to replicate the process of amelogenesis in the laboratory using
isolated cells. Though the synthesis of plain hydroxyapatite blocks is
straightforward, the fabrication of true enamel has not been yet truly
accomplished. (Pandya & Diekwish, 2019)

2. The clinical problem – Dental Caries and White Spot Lesions (WSLs):
Dental Caries is the localized destruction of susceptible dental hard tissues by
the acidic by-product of bacterial fermentation of dietary carbohydrates. The
process begins as initial dissolution of mineral at the sub-clinical stage followed
by incrementally progressive destruction visible only as an early lesion, and
finally leads to traditional cavitation. (Petty & Ekstrand, 2016)
The initial dissolution of enamel is triggered by a fall in pH of the surrounding
medium(saliva) to the critical pH of 5.5 (for hydroxyapatite). With each 1.0
decrease in pH unit the solubility of apatite increases 10 times. At the critical pH,
equilibrium exists (no mineral loss or gain) but when the pH is below the critical
pH, demineralization occurs. Any rise in the pH brings about remineralization.
(Fig. 2) (Buzalaf et al., 2011)
12

This lowering of the pH is the result of biofilm activity of cariogenic bacteria
residing in the biofilm fluid surrounding mineral tooth tissue. The drop in pH
results in dissolution of hydroxyapatite and demineralization of enamel at a
subsurface level. The surface layer of enamel protects the body of the lesion
preventing cavitation. (Kidd & Fejerskov, 2004)

Fig. 2: Stages in progression of dental caries form a sub-clinical stage to visible
decay. (Selwitz et al., 2007)

Enamel crystal dissolution begins with subsurface demineralization, creating pores
between the enamel rods. (Fig.4) The alteration of the enamel refractive index in
the affected area of a carious white spot lesion is a consequence of both surface
13

roughness and loss of surface shine plus alteration of internal reflection, all
resulting in visual enamel opacity, because porous enamel scatters more light
than sound enamel. Commonly identified when the teeth are dry, carious white
spot lesions are typically found on the buccal surfaces beneath a thick
accumulation of plaque where oral hygiene is difficult. The lesions can extend
broadly over the surface of the teeth and sometimes involve proximal extensions.
(Fig. 3) (Featherstone, 2008)

Fig. 3: Image of a white spot lesion (WSL) – black circle - on human teeth

Fig. 4: Polarized light image of white spot lesion with intact surface enamel
(Featherstone, 2008)
14

Optically and contrary to sound enamel, hypo-mineralized enamel is a relatively
heterogeneous tissue made up of organic and mineral materials, with different
refractive indices. In situ, the multiplicity of these indices leads to a diffuse
reflection of light, and therefore the lesion appears white. Depending on the
thickness of the lesion, the appearance may differ. Thin superficial lesions are
slightly whiter than sound enamel making the lesion hardly distinguishable from it
and can only be detectable only after a prolonged air drying. On the other hand,
when the superficial lesion is quite thick, the considerable difference in refractive
indices between sound and porous enamel yields to intensely white lesions easily
visible even on a wet surface. Thus, it becomes extremely difficult to clinically
diagnose the deeper hypo-mineralized lesions. But, it is at this stage that they can
be reversed, and the process of cavitation halted. (Fig. 5) (Denis et al. 2013)

Fig. 5: A: In Hypo-mineralized Lesions, the changes of refractive index, the light is
thus deviated at each interface and reflected, becoming imprisoned in an “optical
maze” that is over-luminous and therefore perceived or seen as white and opaque
15

by the eye on account of the excess brightness which depends of the thickness of
the lesion in cases of Superficial Lesions. (Denis et al. 2013)

Due to its progressive and widespread nature of occurrence, the effect of dental
caries cannot only be weighed by measuring the population affected by this
disease but also the time lost due to pain and sleepless nights. This condition
affects both adults and children causing missed work days and missed school
days, the effect of which, on the economy cannot be ignored. Hence, it becomes
imperative to detect, monitor and possibly treat caries at an early stage when it is
reversible. As the adage goes, prevention is always better than cure.

3. A new technique to detect WSL - QLF: (mechanism of QLF)
Quantitative Light induced fluorescence or QLF is based on the phenomenon of
autofluorescence of dentin. Excitation of dentin with blue light (370nm) causes it
to fluoresce. This technique raises the visual contrast between sound and
pathogenic tissue in the oral cavity. Using a high pass filter (λ ≥ 540 nm) to filter
out the excitation of light, this fluorescence or the QLF image is observed.
(Neuhaus et al., 2009) The white spot lesions or demineralized regions show up
as gray spots, where loss of fluorescence (caused by less excitation light reaching
dentin and scattering of fluorescence in the enamel lesion) corelates with mineral
loss. (Waller et al., 2012). The equipment used by the QLF system includes the
Inspektor
TM
Pro, the QLF-D Biluminator
TM
and the QLF
TM
On-Line tower. (Fig. 6)
The Inspecktor
TM
Pro is an intra-oral camera for making the individual images of
the subject surface in consideration. The software associated with the camera is
16

used to analyze the captured images by manually selecting the area of interest
and defining a reference area. The QLF-D Biluminator
TM
consists of a
Biluminator
TM
mounted on a Single Lens Reflex (SLR) camera fitted with a 60mm
macro lens. The Biluminator
TM
provides the light sources and filters for making
white light and QLF
TM
images. The light source is also connected to a computer
that runs necessary software for archiving and analysis. The system takes two
images, one with white light and the other the QLF image (Fig. 7) and the duration
of the image making process is 5s. For the analysis using QLF system, it is
essential to have sound tooth structure, which can be compared to the
demineralized area by the analytical software to detect extent of demineralization.

Fig. 6: On the left, Inspektor
TM
Pro, in the middle, the QLF-D Biluminator
TM
and to
the right, the QLF
TM
On-Line tower
17

Fig. 7: Images taken semi-simultaneously with the QLF-D Biluminator™ 2. To the
left the ‘normal’ white light image. In the middle the corresponding QLF™-image.

The various parameters that can be obtained from the QLF system include:

Table 1: Parameters for QLF analysis (Inspektor Research Systems BV,
Amsterdam, The Netherlands)

To help in the validation of QLF results, the International Conference on QLF
(ICQ) in 2011 compared ΔF values to ICDAS and histological scores. (ICDAS II,
18

2005) Another study conducted in Tilburg over a period of 6 years assessed 300
patients for early signs of caries activity. Preliminary results indicate QLF
TM
can
predict the existence of demineralization and help in the early diagnosis of
demineralized lesions. (Waller et al., 2012) The intra and inter – examiner
reliability of using QLF as a diagnostic tool for in vitro lesions, was considered
reliable (Pretty et al., 2002)

4. Current solutions for treatment or prevention of WSL:
There are multiple treatment modalities for WSL including professional fluoride
application (varnishes, toothpaste), casein phosphopeptides (CPPs) (Sudjalim et
al., 2006) (Pliska et al., 2012) and infiltrative resins (Kugel et al.,2009). Though
fluoride application has been traditionally the most common therapy for WSLs,
the mineralization by high concentrations of fluoride is limited to the enamel
surface and prevents movement of ions into the subsurface lesion. CPPs
stabilize the calcium and phosphate ions in saliva to form CPP-Amorphous
Calcium Phosphate (CPP-ACP) complex thus promoting remineralization.
Infiltrative resins involve resin penetration into previously etched surface of the
enamel lesion. The resins are of low viscosity, and mechanically stabilize the
hydroxyapatite structure.
Though these and other commercially available treatment modalities have been
effective in partially treating WSLs, their effectiveness is limited (Aoba T et al.,
2004) (Tao et al. 2018) (Borges et al., 2017). The biomaterials in mineralized
enamel function optimally in the oral environment due to their precise structure
19

and shape, and they are superior to commercial products. Peptides designed
from the amelogenin protein are the future approach towards treating WSLs.

5. Amelogenin:
Amelogenin is a 26 KDa hydropobic protein, with the Amel gene located on
Xp22.1-22.3, Yp11.2 of the human X and Y chromosomes. Amelogenin
comprises two well demarcated self-assembly regions, namely the amino-
terminal domain-A and the carboxy-terminal domain-B. Domain-A is hydrophobic
in nature (amino acids 1-42) while domain-B is hydrophilic (amino acids 157-173)
and binds to hydroxyapatite. This indicates the carboxy-terminal is responsible in
facilitating the initial orientation of the protein along enamel crystallites.
Amelogenin is also rich in histidine and thus absorbs hydrogen ions from the
environment to buffer the enamel fluid. (Paine et al., 2003) (Paine & Snead,
2005)

Fig. 8: Amino acid sequence of full-length human amelogenin (191 amino acids)
(Simmer & Snead 2017)

20

Amelogenin is represented in the enamel matrix by numerous isoforms, most of,
which can self-assemble into nanospheres, about 20nm in diameter and each
containing about 100 molecules of amelogenin. The dimensions of the
nanospheres eventually dictate the width and thickness of enamel crystals.
(Simmer and Hu, 2001)
Deficiency of AMELX gene in knock-out mice models has resulted in hypo-
mineralized teeth (Fig. 9) (Gibson et al., 2001). Numerous individuals have been
reported with amelogenesis imperfecta (AI) due to AMELX gene mutations
(Fig.10) (Wright, 2006)

Fig. 9: Incisors of wild type and null mice. Photograph of incisor teeth from wild
type (A) and null mice (B). Scanning electron micrograph showing a smooth
enamel surface for wild type (C) and marked enamel hypoplasia characterized by
a furrowing of the enamel in the incisor from a null mouse (D).

21

Fig. 10: Point AMELX mutations altering the tyrosine rich region of the
amelogenin protein cause hypo-maturation of the enamel producing the
characteristic white cervical opaque and coronal brown discoloration of the
enamel, as seen in this male with a P70T mutation.

6. Remineralization of WSLs with Amelogenin-Derived Peptides:
Evidence based dentistry has enforced the importance of preventive dentistry to
diagnose early lesions and treat them minimally. Studies have investigated the
role of fluoride, amino acids, bioactive glass and organic scaffolds towards
attempting surface mineralization. However, a newer approach is the introduction
of synthetic amelogenin derived peptides, which self assembles to enhance
remineralization of sub surface lesions. They have a complex assembly and
active domains, which interact with the Ca, PO4 and F minerals in ambient
saliva. Some examples include, full length amelogenin (rP172) (Ruan et al.,
2013), Leucine rich amelogenin peptide (LRAP) (Mukherjee et al., 2016),
Amelogenin derived peptide 5 (ADP5) (Dogan et al., 2018), P11-4 (Kamal et al.,
2018) and P26/P32 (Mukherjee et al., 2018).
22

A study to characterize the remineralization of white spot lesions with
Amelogenin Derived Peptide-5 (ADP5), a 22 amino acid peptide, compared the
remineralization by ADP5 with that of MI Paste Plus and Topical Fluoride.
Though visually there was no significance in the remineralization between the
different groups, the authors concluded through SEM imaging that a more
consistent remineralization of surface and sub-surface layers was achieved with
ADP5. (Habibi S, 2018)
Biomimetic tooth repair using ADP5 was also demonstrated through an invitro
study on human enamel where 1100ppm F, 20000ppm F, Ca & PO4 alone,
1100ppm F with ADP5 and ADP5 alone were compared. The authors used SEM
and EDX analysis to demonstrate that 1100ppm F and 20000ppm F were
ineffective in remineralizing the subsurface lesion. Only the peptide-alone
remineralized enamel to a thick dense layer of 10µm containing HAP mineral.
(Dogan et al., 2018)
Amelogenin-derived peptides have also been tested for the ability to initiate
periodontal attachment through cementum-like biomineralized microlayer. ADP5
was shown to facilitate cell-free formation of a cementum-like HAP mineral layer
on demineralized human dentin. This layer supported attachment of PDL fibers in
vitro as demonstrated through Binding analysis via Quartz crystal microbalance.
(Gungormus et al., 2012)
Leucin-rich amelogenin peptide (LRAP) has been proven to repair carious
enamel through guided stabilization and growth of mineral clusters. Focus-ion
23

beam technique also demonstrated a seamless growth at the interface between
repaired and native enamel when LRAP was used. (Mukherjee et al., 2018)
7. Curodont
TM
:
P11-4 is a synthetically designed self-assembling peptide that undergoes a pre-
determined process of assembling to form fibrillar three-dimensional scaffolds
(Ceci et al., 2016). A study to assess the biomimetic remineralization of carious
lesions by P11-4 concluded that P11-4 promoted the remineralization of carious
enamel. Authors used FTIR and micro-CT to demonstrate subsurface
regeneration of enamel lesion by the application of self-assembling peptide P11-
4. The authors reported that P11-4 promotes de novo mineralization in a similar
mode of action as dental enamel (Kind et., 2017).

8. Amelogenin derived peptide – P26 & P32
A study conducted in our laboratory using short amelogenin derived peptides
P26 and P32, which retain the vital functional domains of native amelogenin. The
study aimed to characterize the peptides and to test their potential to assemble
into scaffolds, which control apatite crystallization, and reconstruct synthetic
aprismatic enamel in an in-situ tooth model system. The fundamental difference
between the primary sequences of the two peptides is the presence of two extra
polyproline repeat motifs (PVH/PMQ) in P32.
24

Table 2: Molecular mass, Isoelectric Point and Physicochemical properties of
P26 and P32

14 amino acids from the inner N-terminus with phosphorylated Serine (pS
16
)
were preserved to design P26 and a polyproline repeating motif was preserved
additionally, to design the P32 peptide. Below are the sequences of P26 and
P32.

The study aimed to further determine if multiple microscale enamel-like layers
can be formed through repeated application of the peptide. The authors used CD
and TEM (Fig. 11) to characterize the assembly of the peptides. Peptide
mediated mineralization was observed through TEM and in situ Raman
Spectroscopy. SEM images (Fig. 12) of the peptide treated surfaces were also
studied to understand the structure of the newly remineralized layer. The authors
concluded that the 2 synthetically derived amelogenin peptides were successful
in mediating organized growth of aprismatic enamel-like layers while providing
the means to improve the mechanical properties of the new layer.

25

Fig. 11: TEM images of Ca-P mineralization in presence of P26 and P32. Several
agglomerates of amorphous lamella-like structures detected 25 mins after the
addition of P26 and P32. Addition of the peptides further, resulted in formation of
thin, small, plate-like HAP crystals of relatively uniform size distribution after 1
day of aging.

26

Fig.12: SEM images of (a) demineralized enamel surface showing clear outlines
of enamel prisms/rods with remnants of interprismatic enamel (white arrow).
(b−e) HAP crystals grown on demineralized enamel after 2 days of incubation in
artificial saliva in pH 7.0 at 37 °C. Demineralized enamel treated in artificial saliva
only (control) (b) in the presence of P26 (c), in P32, (d) and in rP172 (e). The
insets are magnified images (scale = 500 nm). White arrows in (c) represent
bundles of needlelike crystallites, whereas the arrows in (d,e) show crystallites
aligned parallel to the underlying native enamel.

Fig 13: Newly formed synthetic aprismatic enamel-like HAP layers grown in the
presence of amelogenin-inspired peptide.

27

9. Chitosan hydrogel:
Chitosan is a biocompatible and non-toxic polymer obtained by deacetylation of
chitin. It imparts bactericidal and bacteriostatic properties through accumulation
of positive charges on tooth surface. It also forms a film and adheres to the
surface of a tooth thus forming a protective layer. Studies have shown that
chitosan can inhibit S. mutans adherence and biofilm formation on tooth surface.
(Costa et al.,2013) Due to its widespread application, chitosan has been used in
toothpastes, mouthwash solutions and chewing gums.
Amelogenin containing chitosan hydrogels have been used in our group for
biomimetic enamel regrowth. One study showed that amelogenin rP172 in
chitosan hydrogel captured the calcium and the phosphate from the artificial
saliva, guiding them to sequentially deposit on the surface of demineralized
enamel (Ruan et al., 2013). Another study reported that LRAP chitosan hydrogel
was also effective in the formation of a robust enamel-like layer that had a
seamless interface with natural tooth. (Mukherjee et al., 2016)

28

OBJECTIVES HYPOTHESIS, AND SCOPE
The first objective of this study was to optimize the QLF system and its setting to
measure mineral loss and mineral gain on tooth sections after their remineralization with
amelogenin peptides-chitosan hydrogels (CS-P26 and CS-P32).
The second objective of this study was to assess the remineralizing potential of P26-CS
hydrogel in comparison to established peptides in the market like Curodont
TM
(P11-4).
The third objective of this study was to develop a model that more closely resembles the
clinical scenario where white spot lesions develop on whole teeth.
Though this study focused on standardizing the QLF system and studying
remineralization in enamel sections, there is an opportunity to create models of whole
tooth samples with intact enamel to study true white spot lesions and their
remineralization through the QLF system.
I used the amelogenin derived peptide P26 combined with chitosan (P26-CS) to
promote remineralization of etched enamel surfaces using third molar tooth sections.
I hypothesize that P26-CS will remineralize the surface of demineralized enamel
improving its mineral density. I will test my hypothesis by analyzing mineral gain using
QLF technique. Curodont
TM
will be used as the commercially available experimental
contrast.

29

SPECIFIC AIMS
The aims of this study include
1. To optimize the QLF system for comparison between the remineralization
potential of P32-Cs hydrogel, P26-CS hydrogel, Chitosan only and Artificial saliva
using tooth sections.
2. To compare the remineralization potential between commercially available
Curodont
TM
, and P26-CS hydrogel using QLF analysis.
3. To characterize the newly formed remineralized layer after treatment with P26-
CS using SEM imaging & XRD.
4. To establish a model that uses whole tooth sample for future comparative
remineralization of WSLs.

METHODOLOGY
Methodology for aims 1, 2 & 3:
General strategy
To test the hypothesis, sections with enamel windows measuring 2mmX3mm were
subjected to demineralization for 24hrs. The following four experimental groups (n=10)
were used
1. P26-Chitosan(P26-CS) and P32-Chitosan (P32-CS) hydrogel
2. Curodont
TM

3. Chitosan hydrogel
30

4. Artificial Saliva
The demineralized sections were imaged using the QLF system.
For optimization (aim 1), the sections were divided into 3 groups of Artificial saliva, P32-
CS hydrogel and P26-CS hydrogel. For aim 2, the sections were divided randomly
between the 4 different experimental groups and subjected to the remineralization cycle
for 2 weeks. The experimental solution was replaced every 4 days and artificial saliva
changed daily. After the remineralization cycle, the sections were QLF imaged once
more. After both demineralization and remineralization cycles, the sections were
cleaned in an ultrasonic bath and air dried. For aim 3, the sections were subjected to
Scanning Electron Microscopy.
1. The preparation of P26-CS hydrogel involves 3 steps:
A) Preparation of CS stock solution:
2% (w/v) chitosan (medium molecular weight, 75-85% deacetylated by Sigma-
Aldrich, MO) was dissolved in 2% (v/v) acetic acid solution followed by stirring at
80
o
C overnight. After cooling the solution to room temperature, it was filtered
through a 0.45µm filter (Millex-HV 0.45µm, PVDF, 33mm, sterile). The pH was
adjusted to 5.0 with 1M NaOH.

B) Preparation of Ca and phosphate stock solution:
0.588g of CaCl2.2H2O was weighed out with 0.568g sodium hydrogen phosphate
and dissolved in 40ml water. The solution was vortexed to get 40ml 0.1M
Solution at pH 11.

31

C) Preparation of chitosan-based hydrogel:
200µg of P26 or P32 was mixed with 960µL of 2% chitosan stock solution in an
Eppendorf tube. To this 15 µL of 0.1M Na2HPO4 solution and 25 µL of 0.1M
CaCl2 solution was added. The solution is centrifuged, and pH brought to 6.5.

2. Preparation of Curodont
TM
(P11-4):
100ml of de-ionized water was added to 10mg of Curodont
TM
and pH was
brought to 8 by addition of 1M NaOH. Curodont
TM
was painted on the
demineralized enamel windows in the same manner as hydrogel to ensure there
is uniformity across all experimental groups.

3. Preparation of Artificial Saliva (Ruan et al., 2013):
For the preparation of Artificial Saliva, 900ml of H2O was mixed with 0.0407g of
MgCl2.6H2O, 1.1928g of KCl, 0.1254g K2HPO4, 11.915g HEPES, 0.2452g NH4Cl
and 0.1764g CaCl2.2H2O. The pH was adjusted to 7.0 with 1M NaOH and the
final volume was fixed to 1000ml in a volumetric flask.

4. Preparation of Demineralizing solution (Ruan et al., 2013):
400ml of H2O was mixed with 0.147g of CaCl2.2H2O, 0.136g of K2HPO4, 0.850
Sodium Acetate, 0.879mL Acetic acid. The pH was adjusted to 4.6 with 1M
NaOH and 1M HCl. The volume is fixed to 500ml in a volumetric flask.

32

5. Preparation of tooth sections:
Extracted human molars without any pre-existing caries, demineralization or
restorations were collected from Ostrow School of Dentistry of USC (approved by
the Institutional Review Board of University of Southern California). The crowns
of the molar were cut coronally into 2 - 3mm thick slices using water cooled slow
speed (45-65 RPM) diamond saw. Clear nail varnish was applied to the slices to
make 2mmX3mm enamel windows, covering rest of the surface completely.
Each slice was demineralized for 24hrs at room temperature, washed with de-
ionized water and sonicated for 2 mins to remove any acid or debris from the
surface. Another coat of varnish was applied over the older coat to prevent any
leakage during the experimental process. The windows were dried and QLF
images were made using white light (ISO 1600, Aperture 18, Shutter Speed
1/125s) and blue light (ISO 1600, Aperture 5.6, Shutter Speed 1/30s). The blue
light images were the true QLF images and demineralized windows were
analyzed for white spot patches. The ΔF values of all 40 samples were tabulated
for future comparative analysis.

6. Enamel remineralization with P26-CS hydrogel, P32-CS hydrogel,
Curodont
TM
and CS
Approximately 40µL of P26-CS, P32-CS hydrogel, CS hydrogel and Curodont
TM

(in solution, without CS) was applied to the windows of enamel using a syringe
(Ruan et al., 2013). The slices were left to stand for approximately 10-15mins to
allow the hydrogel to dry before placing them each in 5ml of artificial saliva
33

solution in a vial. The slices for negative control were directly placed in artificial
saliva. The enamel slices in artificial saliva system was kept in an incubator at
37
0
C for 7 days. The artificial saliva was changed daily, and the hydrogel applied
to the enamel windows, was replaced after 3 days (72hrs). concentration of
fluoride was kept constant throughout.

7. QLF images of remineralized windows
After 7 days (168hrs) the enamel sections were washed with de-ionized water,
dried and QLF images were made using white light (ISO 1600, Aperture 18,
Shutter Speed 1/125s) and blue light (ISO 1600, Aperture 5.6, Shutter Speed
1/30s). The previously demineralized windows were analyzed for visual and
numeric representation post remineralization. Comparative analysis of the
lesions was made visually and numerically.

8. Characterization of enamel section post remineralization using SEM & XRD
Following QLF analysis, the half the samples (of P26-CS hydrogel and
Curodont
TM
group) were sputter coated with platinum and observed using a
Scanning Electron Microscope (JEOL JSM-7001F, accelerating voltage 15 kV) to
visualize crystal growth on treatment with P26-CS hydrogel and Curodont
TM
. The
remaining half samples were subjected to X-ray diffraction (XRD).

34

Methodology for aim 4 (Fig. 15):
1. Whole Tooth Sample Preparation and mounting
a. To prepare whole teeth samples, extracted human molars without any pre-
existing caries, demineralization or restorations were collected from
Ostrow School of Dentistry of USC (approved by the Institutional Review
Board of University of Southern California). Any remnants of tissue were
removed from the teeth with a scalpel. The teeth were washed in de-
ionized water and sonicated for 5 mins to clean them.
b. Red boxing wax was wrapped around the roots of each tooth. Small
blocks measuring about 1cmx1cmx1cm were made from dental stone and
the teeth with wax were mounted into the block when the stone was semi-
set. UHU tabs measuring 5mmX3mmX2mm were placed on the buccal or
lingual surface of each tooth to designate future experimental window. The
vacuform sheets mold around these tabs to form reservoir pouches.
c. About 4 dental stone molds with mounted teeth were placed in the
vacuform machine with approximately one finger space between the
molds (Fig. 14). A flexible vacuform sheet was molded around the teeth.
The sheet was cut to form flexible crowns around each tooth. The crowns
with a reservoir pouch to hold the experimental solution/group in, is peeled
off and the whole tooth was painted with clear nail varnish leaving a
window measuring 5mmX3mmX2mm.
35

Fig. 14: Whole tooth samples mounted on dental stone blocks and dispersed in a
vacuform machine.

2. Whole tooth sample demineralization and remineralization with P26-CS
a. Each tooth sample was immersed in 100ml of demineralizing solution for
14 days. The demineralizing solution was changed every 7 days within 14
days. After 14 days, the teeth were washed with de-ionized water and
sonicated for 5-10mins to clear any debris. The windows were dried and
QLF images made. The visual and numerical results obtained should be
tabulated for future analysis. Clear varnish can be repainted on the tooth
surface if required.
b. 100µL of the experimental solution or hydrogel will be placed in the
reservoir pouch and allowed to stand for 2-3 mins. The thermoplastic
crown will be replaced onto the tooth and the system placed in 100ml of
artificial saliva.
36

c. The system will be in artificial saliva in a water bath at 37
0
C for 1 month.
The artificial saliva will be changed every day and the experimental
solution/hydrogel will be re-applied once a week for 4 weeks.
After the remineralization cycle is complete, the samples should be washed with de-
ionized water and sonicated for approximately 5-10mins to remove any foreign debris.
After being cleaned, the tooth samples will be dried and imaged for QLF analysis.
Comparative visual and numerical QLF analysis post demineralization and post
remineralization will be made to determine effectiveness of the experimental groups.

Fig. 15: Schematic representation of the methodology used to make the whole tooth
samples with reservoir pouches.

Thermoplastic reservoir
crowns prepared on 25
extracted molars
Baseline QLF images
Teeth subjected to 2
weeks of
demineralization
Post demin QLF images
All 25 teeth segregated
into 5 groups (AS, CS,
P26CS, Curodont)
Remin cycle for 4
weeks. Reapplication
once every week.
Post remin QLF images
to be made
Comparisons between
images and ΔF of post
demin and post remin
to be done
37

RESULTS:
Aim 1: To calibrate the QLF system and to optimize the ideal settings for the QLF
to measure mineral loss and mineral gain on tooth sections.

Optimization of the QLF system:
Three different settings of the QLF system were used to capture images of both
sections and whole teeth samples. These settings were
A) ISO 1600, Aperture 18, Shutter Speed 1/125s, Blue light
B) ISO 1600, Aperture 5.6, Shutter Speed 1/30s, Blue light
C) ISO 1600, Aperture 8, Shutter Speed 1/125s, White light.
Comparative visual analysis and quantitative analysis (ΔF: Percentage fluorescence
loss with respect to the fluorescence of sound tooth tissue. Related to lesion depth (%))
of demineralized and remineralized samples using all 3 light sources was done.
Analysis under blue light with ISO 1600, Aperture 5.6, Shutter Speed 1/30s was found
the most reliable. Kruskal-Wallis test was performed to assess if there was any
statistically significant difference in ΔF values at the baseline between the lesions
assigned to the three groups (Artificial saliva, P32-CS hydrogel and P26-CS hydrogel).
No statistically significant (p>0.05) difference was found between P32-CS hydrogel and
P26-CS hydrogel. However, the remineralization potential of artificial saliva was
significantly lower than the other groups.

38

GROUPS DEMINERALIZED ENAMEL
REMINERALIZED
ENAMEL
P32-CS
HYDROGEL

ΔF=-6.5

ΔF=-5.6
P26-CS
HYDROGEL

ΔF=-8.0

ΔF=-5.7
ARTIFICIAL
SALIVA

ΔF=-8.5

ΔF=-8.7

Fig 16: Visual (A, B, C, D, E, F) QLF images and numerical (ΔF) representation
of the enamel sections after demineralization and after remineralization

A
B
C
D

39

ΔΔF AVERAGE STD DEV STD
ERROR
P26-CS
HYDROGEL
3.23 2.23 1.0
P32-CS
HYDROGEL
3.86 4.94 2.20
ARTIFICIAL
SALIVA
1.24 2.33 1.04

Table 3: Average ΔΔF (ΔF remin – ΔF demin) across 15 sections with standard
deviation and standard error across each group.

Fig. 17: Bar chart representing average mineral loss or mineral gain across the 3
groups. (p>0.05, between P26-CS and P32-CS) (p<0.05, between AS and P26-
CS, AS and P32-CS)

According to the QLF analysis, the ΔF of sound enamel was 0, but the ΔF of artificial
caries lesion baseline appeared to be approximately -12.6 to -6.5 (24 hrs of demin. at
pH 4.6, 37
0
C) in the 3 groups. After the application of the gel, the lesion
depth decreased for both P32-CS (avg Δ ΔF=3.86 ±2.2) and P26-CS (avg
ΔΔF=3.225 ±0.99), indicative of gel-mediated remineralization of carious lesions. The
40

control (treated in artificial saliva only) seemed to have no improvement in the
remineralization process. The values plotted on the graph (Fig. 17) are images taken at
baseline (post-demin) and after treatment (post-remin.) at the end of 1 week of remin
cycle.
Since there was no significant difference between the remineralizing potential between
P26-CS hydrogel and P32-CS hydrogel, it was decided to proceed with P26-CS
hydrogel as it was a smaller peptide and previous studies have shown its efficacy in
solution (Mukherjee et al., 2018).

Aim 2: To compare the remineralization potential between Curodont
TM
, P26-CS
hydrogel, Chitosan (negative control) and Artificial saliva using QLF analysis.

For Aim 2, visual and numerical QLF analysis of remineralizing potential across 40
sections (n=10) was performed for all 4 groups. The blue light settings were the same
as used for Aim 1. Kruskal-Wallis test was performed to assess if there was any
statistically significant difference in ΔF values at the baseline between the lesions
assigned to the four groups. No statistically significant (p>0.05) difference was found
between Curodont
TM
, Chitosan hydrogel and P26-CS hydrogel. However, the
remineralization potential of artificial saliva was significantly lower than the other
groups (Curodont
TM
and AS p=.045, P26-CS hydrogel and AS p=.035, CS and AS
p=.031)

41

GROUPS
DEMINERALIZED
ENAMEL
REMINERALIZED ENAMEL
P26-CS
HYDROGEL

ΔF = -6.6

ΔF = 0.0
CURODONT
TM

ΔF = -9.5

ΔF = 0.0
CHITOSAN
ONLY

ΔF = -6.6

ΔF = -5.4
ARTIFICIAL
SALIVA

ΔF = -7.6

ΔF = -5.5

Fig 18: Visual (A, B, C, D, E, F, G, H) QLF images and numerical (ΔF values)
representation of the enamel sections after demineralization and after
remineralization with P26-CS hydrogel, Curodont
TM
, CS hydrogel and AS.
A
B
C
D
E
F
G
H
42

ΔΔF AVERAGE STD DEV STD ERROR
P26-CS
HYDROGEL
2.33 3.15 1.19
CURODONT
TM
1.57 4.81 1.82
CHITOSAN 1.07 2.24 0.85
ARTIFICIAL
SALIVA
-1.33 1.88 0.71

Table 4: Average ΔΔF (ΔF remin – ΔF demin) across 40 sections with standard
deviation and standard error across each group.

Fig. 19: Bar chart representing average mineral loss or mineral gain across the 4
groups. A) CS hydrogel, B) Curodont
TM
, C) P26-CS hydrogel, D) Artificial Saliva

According to the QLF analysis in this study, the ΔF of sound enamel was 0, but
the ΔF of artificial caries lesion baseline appeared to be approximately -11 to -6.3
1.071428571 1.571428571
2.328571429
-1.33
-4
-2
0
2
4
6
8
1 2 3 4
AVERAGE
A
B C D
ΔΔF (Mineral gain/loss)
43

(24 hrs of demin. at pH 4.6, 37
0
C) in the 3 groups. After the application of gel, the
lesion depth decreased for both P26-CS hydrogel (avg ΔΔF=2.33 ±3.15) and
Curodont
TM
(avg ΔΔF=1.57 ±1.82), indicative of gel-mediated remineralization
of carious lesions. The control (treated in artificial saliva only) seemed to have
very minimal improvement in the remineralization process as did chitosan
alone. The values plotted on the graph (Fig. 19) are images taken at baseline
(post-demin) and after treatment (post-remin.).

Aim 3: To characterize the newly formed remineralized layer after treatment
with P26-CS using SEM imaging & XRD.

GROUPS P26-CS HYDROGEL CURODONT
TM

X2000

A
B
44

X5000

X30000

Fig 20: SEM images of enamel windows treated with P26-CS hydrogel (A, C, E) and
Curodont
TM
(B, D, F) after 7 days at X2000 (A, B), X5000 (C, D) & X30000 (E, F)
magnification.

Following remineralization in artificial saliva and in the presence of P26-CS the
demineralized enamel windows (artificial lesions) were uniformly coated with apatite
containing material (Fig. 20A and B). At higher magnifications, rod-like crystals were
observed in both P26-CS and Curodont
TM
samples and at 30000 magnification, uniform
plate-like crystals were observed (Fig 20C and D). The crystals growing in the P26-Cs
samples were more uniform than in the Curodont
TM
samples (Fig. 20E and F).
C
D
E F
45

Fig. 21: XRD spectra of Curodont
TM
, P26-CS hydrogel, Artificial Saliva and Demin
enamel
Fig.21 is the preliminary XRD spectrum of newly deposited mineral crystals showing the
presence of hydroxyapatite peaks for both Curodont
TM
and P26-CS hydrogel while the
presence of other calcium phosphate mineral phases cannot be ruled out. Further
analysis is needed to better characterize the mineral components in the newly formed
mineralized layer.

10 20 30 40 50 60
Theta(deg)
0
250
500
750
1000
1250
Intensity(Counts)
[deminenamel.raw] demin enamelhb2
[AS only_7d.raw] AS only_7d
[P26 CS_1A.raw] P26 CS_1A
[CURODONT.raw] CURODONT
211
300
Curodont
P26-CS
Artificial Saliva
Demin enamel
46

AIM 4: To establish a model that uses whole tooth sample for future
comparative remineralization of WSLs.

Designing the Whole Tooth Model for WSL
Though, I used enamel sections to optimize the QLF system for comparatively study
between commercially available Curodont
TM
and P26-CS hydrogel, sections are not an
entirely accurate depiction of the clinical environment. Whole teeth, with intact enamel
are more resistant to both carious onslaught and attempts at remineralization. To
simulate the clinical environment in vitro, in this aim I propose to use whole tooth
samples. These samples, when demineralized, will provide a more accurate
representation of WSLs as seen clinically.

Fig. 22: A) Whole tooth samples with UHU
TM
tabs to form reservoirs in thermoplastic
crowns. B) Thermoplastic crowns with reservoir pouches.
A
B
47

The crowns formed from vacuform sheets were made with reservoir pouches to hold the
peptide hydrogel. The reservoir pouches were made such that they reciprocate the
WSLs on the tooth surface. It is surmised that the pouches would allow peptide
hydrogel to remain in contact with the WSL for longer period. An essential part of using
these thermoplastic crowns is that they are not completely constricted at the cervix of
the tooth, thus allowing exchange of saliva while the crown is in place.
A clinical extrapolation of this model would be to make custom-made trays with similar
reservoir pouches for patients with WSLs or retainers with strategically placed pouches
to hold the gel in place. A similar system has been used earlier to test the efficacy of
using reservoirs for extended application of carbamide peroxide. It would involve the
waxing up of teeth in question before forming the bleaching tray or retainer (Fig. 23, 24)
(Jahaveri & Janis, 2000).

Fig. 23: Wax up of potential white spot lesions on dental stone cast of patient.
48

Fig.24: Bleaching tray for maxillary arch with reservoir pouches.

Fig. 25: QLF image post-remin with P26-CS of whole tooth sample. The blue marked
region is the region of analysis. The grey patches in the thumbnail represent WSLs.

Fig. 25 represents the QLF result of a pilot sample of experimentation with whole tooth
model after remineralization with P26-CS. The above sample was subjected to the
protocol of demineralization and remineralization as mentioned in the methodology
49

above. Hyper-fluorescence was observed in most of the QLF images made, which may
interfere with the readings of the QLF system. This phenomenon could be explained by
the presence of remnant peptide on the tooth surface. The natural curvature of the tooth
may also pose a technical difficulty as the QLF system is sensitive to the position of the
tooth. It becomes imperative to have the window on a flat surface for the experiment.
The selected surface of the tooth must be polished on fine grit paper to ensure the
surface is relatively flat. It is also important to ensure the images are made with the
same orientation each time. The video capture option of the QLF system will help in this
as it recaptures images in the same orientation as the previous capture. A very
important fact in the selection of the window is to have adjacent intact enamel that has
been covered in varnish to, which the experimental window can be compared for QLF
analysis.

50

DISCUSSION:
In the clinic, the QLF system is an integral diagnostic tool for incipient lesions. However,
it is extremely technique sensitive and has an associated learning curve. The most
important factor in using a QLF system is to ensure comparative images are made
under the same settings. It is equally important to ensure the lesions are captured at
approximately the same position each time, which is enabled by the video capture
option in the system. To study an image, it is essential to include sound enamel along
with the lesion window as QLF measures the difference in enamel crystal structure
between sound and demineralized/remineralized lesion. The blue light setting of ISO
1600, Aperture 5.6 and Shutter speed 1/30s was found to be the most consistent light
settings for optimal analysis. P26-CS hydrogel and Curodont
TM
were successful in
remineralizing the demineralized lesions but the difference in remineralization wasn’t
statistically significant. SEM images of both lesions showed a uniform coating on the
artificial lesion, not seen on the adjacent sound enamel. Rod-like apatite crystals
resembling enamel crystals were observed. This image was different from the needle-
like crystals seen in previous studies (Mukherjee et al., 2018). This could be explained
because peptides in chitosan hydrogel were used instead of peptides in solution. The
uniform layer formed on the sections was not removed by sonication and would
contribute to the nature of QLF light reflected to the system and hence the quantitative
as well as qualitative results. The finding that differences in remineralization between
P26 hydrogel and Curodont
TM
was not significant could be attributed to the sample size
of the study. My findings indicate that the P26-CS hydrogel is as effective as the
commercially available Curodont
TM
with a much lower peptide concentration. The
51

concentration of Curodont
TM
is much greater (10mg/ml) than P26 (0.2mg/ml) for
approximately the same effect.
The whole tooth models I developed in Aim 4 are highly representative of a clinical
situation. The thermoplastic vacuform crowns with reservoir pouches could be
translated in to a clinical model through bleaching trays with reservoir pouches
corresponding to white spot lesions in a patient’s mouth.
Corelating the QLF images and ΔF values with SEM images and XRD analysis at all
stages during the experiment, including post-demineralization and post-remineralization
would help to better understand the validity of the analysis. Comparative studies
between P26-CS hydrogel and more commercially available remineralizing agents will
help to study the efficacy of hydrogel system.

CONCLUSIONS:
QLF is an excellent clinical tool for detecting white spot lesions and assessing the ability
of different remineralizing agents available in the market. For comparative study, the
QLF system needs to be used under identical setting, each time in order to ensure
accurate and reliable results. The amelogenin derived peptide P26-CS hydrogel was
found to be as effective as the commercially available Curodont
TM
in remineralizing
artificial enamel lesions. Amelogenin peptide-based hydrogel systems have a great
potential for future remineralization of incipient lesions.

52

BIBLOGRAPHY
1. Pandya and Diekwisch. Enamel biomimetics-fiction or future of dentistry.
International Journal of Oral Science. 2019; 11:8.
2. Ruan Q, Moradian-Oldak J. Amelogenin and Enamel Biomimetics. J Mater Chem B.
2015; 3:3112-3129.
3. Bansal AK, Shetty DC, Bindal R, Pathak A. Amelogenin: A novel protein with diverse
applications in genetic and molecular profiling. J Oral Maxillofac Pathol. 2012
Sep;16(3):395-9.
4. Pretty IA, Ekstrand KR. Detection and monitoring of early caries lesions: a review.
Eur Arch Paediatr Dent. 2016 Feb;17(1):13-25.
5. Buzalaf MA, Pessan JP, Honório HM, Ten Cate JM. Mechanisms of action of
fluoride for caries control. Monogr Oral Sci. 2011; 22:97-114.
6. Kidd EA, Fejerskov O. What constitutes dental caries? Histopathology of carious
enamel and dentin related to the action of cariogenic biofilms. J Dent Res. 2004;83
Spec No C:C35-8.
7. Selwitz RH, Ismail AI, Pitts NB. Dental Caries. The Lancet. 2007 Jan; 369(9555):51-
59.
8. Featherstone JD. Dental caries: a dynamic disease process. Aust Dent J. 2008
Sep;53(3):286-91.
9. Denis M, Atlan A, Vennat E, Tirlet G, Attal J-P. White defects on enamel: diagnosis
and anatomopathology: two essential factors for proper treatment (part 1).
International Orthodontics. 2013;11(2): 139-65.
53

10. Neuhaus KW, Longbottom C, Ellwood R, Lussi A. Novel lesion detection aids.
Monogr Oral Sci. 2009; 21:52-62.
11. Waller E, Van Daelen CJ, Van der Veen MH. Application of QLF™ for Diagnosis
and Quality Assessment in Clinical Practice. 2012.
12. Committee, International Caries Detection and Assessment System Coordinating,
"Rationale and Evidence for the International Caries Detection and Assessment
System (ICDAS II)," 2005.
13. Sudjalim TR, Woods MG, Manton DJ. Prevention of white spot lesions in
orthodontic practice: a contemporary review. Aust Dent J. 2006 Dec;51(4):284-9;
quiz 347.
14. Pretty IA, Hall AF, Smith PW, Edgar WM, Higham SM. The intra- and inter-
examiner reliability of quantitative light-induced fluorescence (QLF) analyses. Br
Dent J. 2002 Jul 27;193(2):105-9.
15. Pliska BT, Warner GA, Tantbirojn D, Larson BE. Treatment of white spot lesion with
ACP paste and microabrasion. Angle Orthod. 2012 Sep;82(5):765-9.
16. Kugel G, Arsenault P, Papas A. Treatment modalities for caries management,
including a new resin infiltration system. Compend Contin Educ Dent. 2009 Oct;30
Spec No 3:1-10; quiz 11-2.
17. Aoba T. Solubility properties of human tooth mineral and pathogenesis of dental
caries. Oral Dis. 2004 Sep;10(5):249-57.
18. Tao, S., Zhu, Y., Yuan, H., Tao, S., Cheng, Y., Li, J., & He, L. Efficacy of fluorides
and CPP-ACP vs fluorides monotherapy on early caries lesions: A systematic
review and meta-analysis. PloS one, 2018. 13(4), e0196660.
54

19. Borges AB, Caneppele TM, Masterson D, Maia LC. Is resin infiltration an effective
esthetic treatment for enamel development defects and white spot lesions? A
systematic review. J Dent. 2017 Jan; 56:11-18.
20. Paine ML, Luo W, Zhu DH, Bringas P Jr, Snead ML. Functional domains for
amelogenin revealed by compound genetic defects. J Bone Miner Res. 2003
Mar;18(3):466-72.
21. Paine ML, Snead ML. Tooth developmental biology: disruptions to enamel-matrix
assembly and its impact on biomineralization. Orthod Craniofac Res. 2005
Nov;8(4):239-51.
22. Simmer JP, and Snead ML. Molecular biology of the amelogenin gene. In Dental
enamel formation to destruction. CRC Press 2017. pp. 59-84.
23. Simmer JP, Hu JC. Dental enamel formation and its impact on clinical dentistry. J
Dent Educ. 2001 Sep;65(9):896-905.
24. Gibson CW, Yuan ZA, Hall B, Longenecker G, Chen E, Thyagarajan T, Sreenath T,
Wright JT, Decker S, Piddington R, Harrison G, Kulkarni AB. Amelogenin-deficient
mice display an amelogenesis imperfecta phenotype. J Biol Chem. 2001 Aug
24;276(34):31871-5.
25. Wright JT. The molecular etiologies and associated phenotypes of amelogenesis
imperfecta. Am J Med Genet A. 2006 Dec 1;140(23):2547-55.
26. Ruan Q, Zhang Y, Yang X, Nutt S, Moradian-Oldak J. An amelogenin-chitosan
matrix promotes assembly of an enamel-like layer with a dense interface. Acta
Biomater. 2013 Jul;9(7):7289-97.
55

27. Mukherjee, K.; Ruan, Q.; Liberman, D.; White, S. N.; Moradian-Oldak, J. Repairing
human tooth enamel with leucine-rich amelogenin peptide-chitosan hydrogel J.
Mater. Res. 2016; 31:556– 563.
28. Dogan S, Fong H, Yucesoy DT, Cousin T, Gresswell C, Dag S, Huang G, Sarikaya
M. Biomimetic Tooth Repair: Amelogenin-Derived Peptide Enables in-Vitro
Remineralization of Human Enamel. ACS Biomaterials Science & Engineering.
2018; 4(5):1788-1796.
29. Kamal D, Hassanein H, Elkassas D, Hamza H. Comparative evaluation of
remineralizing efficacy of biomimetic self-assembling peptide on artificially induced
enamel lesions: An in vitro study. J Conserv Dent. 2018 Sep-Oct; 21(5): 536–541.
30. Mukherjee K, Ruan Q, Nutt S, Tao J, Yoreo JJD, Moradian-Oldak J. Peptide-Based
Bioinspired Approach to Regrowing Multilayered Aprismatic Enamel. ACS Omega.
2018 Mar 31; 3(3): 2546–2557.
31. Habibi S. 2018. Characterizing the appearance of ex vivo remineralized white spot
lesions with a novel peptide.
https://digital.lib.washington.edu/researchworks/handle/1773/41763 (Accessed 24
March 2019)
32. Gungormus M, Oren EE, Horst JA, Fong H, Hnilova M, Somerman MJ, Snead ML,
Samudrala R, Tamerler C, Sarikaya M. Cementomimetics-constructing a
cementum-like biomineralized microlayer via amelogenin-derived peptides. Int J
Oral Sci. 2012 Jun;4(2):69-77.
56

33. Ceci M, Mirando M, Beltrami R, Chiesa M, Colombo M, Poggio C. Effect of self-
assembling peptide P11-4 on enamel erosion: AFM and SEM studies. Scanning.
2015; 38:344–351.
34. Kind L, Stevanovic S, Wuttig S, Wimberger S, Hofer J, Müller B, Pieles U.
Biomimetic Remineralization of Carious Lesions by Self-Assembling Peptide. Dent
Res. 2017 Jul;96(7):790-797.
35. Costa EM, Silva S, Tavaria FK, Pintado MM. Study of the effects of chitosan upon
Streptococcus mutans adherence and biofilm formation. Anaerobe. 2013 Apr;
20:27-31.
36. Javaheri DS, Janis JN. The efficacy of reservoirs in bleaching trays. Oper Dent.
2000 May-Jun;25(3):149-51.

Abstract (if available)

Abstract Dental Caries is one of the most prevalent diseases in the modern world. Due to its widespread and debilitating nature, it is imperative to detect the carious lesions early and treat them. The process of cavitation begins as sub surface demineralization caused by plaque accumulation on the surface of teeth. This sub surface demineralized enamel appears chalky-white and is called a White Spot Lesion (WSL). ❧ Multiple treatment modalities are currently available to treat WSLs like topical fluoride, MI Paste plus and ICON resin infiltration. However, a newer approach to treat these lesions is through biomimetics. A biomimetic solution emulates the delicate and unique process of nature by inducing the growth of apatite-like crystals on the demineralized surface. One such approach was taken in this study using amelogenin peptides P26 and P32 chitosan hydrogel (P26-CS, P32-CS) to remineralize windows of demineralized enamel sections. Curodont™ was used for comparison as it is currently commercially available. ❧ Quantitative Light-induced Fluorescence (QLF) was used as an analytical tool for the study as it is one of the few clinical detection techniques for WSLs. A protocol was developed to analyze demineralized enamel sections (as a model for WSL) and their consequent remineralization using Curodont™ and P26-CS. Both visual and numerical changes after demineralization and after remineralization were analyzed. A proposed model for whole tooth samples was also developed to mimic WSL and enable the study of the remineralization by the peptides under more clinically relevant scenario. ❧ The QLF system was optimized to make images using Blue light at ISO 1600, Shutter speed 1/30s and Aperture 5.6. In the pilot study to compare demineralized lesions on enamel sections between artificial saliva, P26-CS hydrogel and P32-CS hydrogel, significant results were obtained between artificial saliva and the other groups while the remineralization potential between P26-CS and P32-CS was not significantly different. Visual QLF images and numerical analysis of ΔF values across the groups Artificial Saliva, Curodont™, P26-CS hydrogel and CS hydrogel were conclusive to demonstrate remineralization potential of Curodont™, P26-CS hydrogel and CS hydrogel. However, there was no significant difference between these 3 groups.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Determination of mineral density of remineralized enamel and dentin: a QLF study

PDF

Amelogenin peptides for biomimetic remineralization of enamel and dentin

PDF

Chitosan‐amelogenin hydrogel‐saliva interactions: towards optimization of a protocol for enamel repair

PDF

Remineralization of deminrealized dentin by amelogenin peptide P26

PDF

Dynamics of amelogenin self-assembly during in vitro proteolysis by Mmp-20

PDF

Amelogenin-ameloblastin protein interaction and function in dental enamel formation

PDF

Influence of enamel biomineralization on bonding to minimally invasive CAD/CAM restorations

PDF

Leucine-rich amelogenin peptide induces osteogenesis in mouse embryonic stem cells

PDF

The accuracy of intraoral scans of interproximal spaces with and without saliva: a prospective clinical study

PDF

Efficacy of fibrin-assisted soft-tissue promotion (FASTP) in treatment of multiple gingival recession defects: a retrospective 3-D volumetric analysis

PDF

Maxillary sinus floor and alveolar crest alterations following extraction of maxillary molars and ridge preservation: a retrospective CBCT analysis

PDF

Enamelysin (MMP20) and Kallikrein 4 (KLK4) functions during enamel formation

PDF

3D assessment of virtual bracket removal for modern orthodontic retainers: a prospective clinical study

PDF

Maxillary sinus floor and alveolar crest alterations following extraction of maxillary molars: a retrospective CBCT analysis

PDF

An overview of the dental and skeletal relationships in individuals of Vietnamese ancestry

PDF

Influence of a novel self-etching primer on bond-strength to glass-ceramics and wettability of glass-ceramics

PDF

Cephalometric norms for the Vietnamese: a clinical appraisal

PDF

Patient reported outcomes measures (PROMs) and medication count of the three coronal advancement methods for the treatment of multiple gingival recession defects

PDF

Relationship of buccal bone plate thickness and healing of extraction sockets with or without alveolar ridge preservation: a systematic review

PDF

Bonding accuracy of a novel lingual customized orthodontic appliance (INBRACE™): an in-vivo study

Asset Metadata

Creator Chakraborty, Amrita (author)

Core Title Amelogenin peptide - chitosan for remineralization of artificial enamel lesions: a QLF analysis

School School of Dentistry

Degree Master of Science

Degree Program Craniofacial Biology

Publication Date 04/30/2019

Defense Date 04/30/2019

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag amelogenin peptide,chitosan,OAI-PMH Harvest,QLF,remineralization,white spot lesions

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Oldak, Janet (committee chair)

Creator Email amritach@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-162462

Unique identifier UC11660002

Identifier etd-Chakrabort-7372.pdf (filename),usctheses-c89-162462 (legacy record id)

Legacy Identifier etd-Chakrabort-7372.pdf

Dmrecord 162462

Document Type Thesis

Format application/pdf (imt)

Rights Chakraborty, Amrita

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA