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Remineralization of deminrealized dentin by amelogenin peptide P26

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Remineralization of deminrealized dentin by amelogenin peptide P26

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Content 1

Remineralization of Demineralized Dentin by
Amelogenin Peptide P26

by
Gayathri Visakan

A thesis presented to the faculty of
the Ostrow School of Dentistry
University of Southern California
In fulfillment of the requirements for the degree
Master of Science,
Craniofacial Biology
May 2019

2

Acknowledgements

I would first like to convey my heartfelt thanks to my mentor, and supervisor Dr. Janet
Moradian-Oldak for being instrumental in guiding me during my Masters. She is my
source of inspiration, and support and has always had her doors open for help. I would
like to thank Dr. Michael Paine for helping me with the academic requirements of the
degree, and his inputs as my thesis committee member. I thank Dr. Jin-Ho Phark for
having provided unrestrained access to his laboratory facilities where some of the
experiments were carried out, and for his valuable input with experiment design. I would
also like to extend my gratitude to Dr. Malcolm Snead who was kind enough to agree to
be a part of the thesis committee under short notice. His experience and feedback are of
great value to this study.
The members of the Oldak Lab: Dr. Jingtan Su, Dr. Natalie Kegulian, Kaushik Mukherjee,
and Rucha Bapat have helped me learn basic research techniques, and their continued
involvement through active feedback, and comments have helped better myself and the
study.
I am grateful for having an excellent support system comprising of friends and family. My
parents (Brinda Visakan, & Visakan Srinivasan), and my grandmothers (Girija Srinivasan,
and K.R. Saraswathi) have always believed in me and this in turn brings out the best in
me. My close friends (Madhusudhan Krishnamachari, Ramkumar Venugopal, & Farhana
Firdous) are my source of light at the end of any tunnel.
Finally, I thank the Center for Craniofacial Molecular Biology for providing the necessary
facilities that enabled me to successfully carry out this study.

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Table of Contents

ACKNOWLEDGEMENTS 2
LIST OF FIGURES 5
ABBREVIATIONS 6
INTRODUCTION 8
ABSTRACT 8
BACKGROUND 10
A. Non-Carious Cervical Lesions 10
B. Dentin 12
C. Dentin Tensile Strength 14
D. Evaluating mineral density by μ-CT 15
E. Amelogenin, and P26 16
OBJECTIVES, STRATEGY, AND SCOPE 21
CHAPTER 1- TO DETERMINE THE EFFECT OF P26 TREATMENT
ON TENSILE STRENGTH OF PARTIALLY DEMINERALIZED
DENTIN. 23
MATERIALS AND METHODS 23
A. Dentin sample preparation 23
B. Tensile strength testing 25
RESULTS 27
A. Dentin tensile strength 27
CHAPTER 2 - TO TEST THE EFFECT OF P26 TREATMENT ON
MINERAL DENSITY OF PARTIALLY DEMINERALIZED DENTIN. 31
MATERIALS AND METHODS 31
A. Dentin sample preparation 31
B. µ-CT scanning 33
C. Reconstruction and viewing 34
D. Quantitative mineral density measurement 35

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RESULTS 36
A. Reconstructed images 36
B. Quantitative mineral density 39
CHAPTER 3- TO CHARACTERIZE THE MORPHOLOGY OF PARTIALLY
DEMINERALIZED DENTIN AFTER TREATMENT WITH P26 PEPTIDE. 45
MATERIALS AND METHODS 45
A. Dentin sample preparation 45
B. Scanning electron microscopy& EDAX 47

RESULTS 48
A. SEM images 48
B. EDX data 51

DISCUSSION 54
CONCLUSION 59
REFERENCES 60

5

List of Figures

Figure 1. Cervical toothbrush abrasion.……………………………………………………...10
Figure 2. Abfraction lesion.………………...………………………………………………….11
Figure 3. Palatal erosion……………………..……………….………………………….……11
Figure 4. SEM image of dentin structure ………………..……………………….................13
Figure 5. P26 peptide sequence ………………………………………………………...…...19
Figure 6. Etched enamel surface treated with P26 peptide………………………………...20
Figure 7. Sample for UTS testing and sample holder……………………………………….24
Figure 8. Instron 5965 setup…………………………………………………………………..26
Figure 9. Bar graph of mean tensile strength…………………………..……………………28
Figure 10. Boxplot of dentin tensile strength distribution………………………................30
Figure 11. Sample preparation for µ-CT scanning………………………………………….32
Figure 12. µ-CT scanner setup……………………………………………………………….34
Figure 13. Baseline reconstructed image……………………………………………………36
Figure 14. Representative sample P26 group before and …………………………………37
after remineralization.
Figure 15. Representative sample Control group before and……………………………...38
after remineralization
Figure 16. Bar graph representing mean dentin BMD- P26 group………………………...39
Figure 17. BMD change in P26 group sample wise…………………………………………40
Figure 18. Bar graph representing dentin BMD- Control group….. ……………………….41
Figure 19. BMD change in the Control group sample wise ……………………………….42
Figure 20. Boxplot of dentin BMD distribution in P26, and Control…………………….…44
Figure 21. Sample preparation for SEM and EDAX analysis……………………………..46
Figure 22. Normal dentin SEM………………………………………………………………..48
Figure 23. SEM image of demineralized, P26 0.2 mg/mL, and control.…….……………49
Figure 24. SEM image of demineralized, P26 0.5 mg/mL, and control……………………50
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Figure 25. SEM image of P26 treated sample- surface view……………………………….52
Figure 26. SEM of collagen architecture in demineralized, P26 and control dentin…….53

Abbreviations

AC- Attenuation Coefficient
AS- Artificial Saliva
BMD- Bone Mineral Density
CEJ- Cementoenamel Junction
CS-AMEL- Chitosan Based Amelogenin Hydrogel
CT- Computed Tomography
DEJ- Dentinoenamel Junction
EDTA- Ethylene Diamine Tetracetic Acid
EDAX/EDX- Energy Dispersive X-Ray Analyzer
GERD- Gastroesophageal Reflux Disease
GIC- Glass Ionomer Cement
GS- Greyscale
HAP- Hydroxyapatite
HEMA- Hydroxyethymethacrylate
HMDS- Hexamethyldisilazane
LRAP- Leucine-Rich Amelogenin Peptide
LRAP-CS- Leucine-Rich Amelogenin Peptide Incorporated in Chitosan
MID- Minimally Invasive Dentistry
MPa- Mega Pascal
NCCL- Non-Carious Cervical Lesion
PBS- Phosphate Buffered Saline
rP148- C-Terminally Truncated Recombinant Porcine Amelogenin Containing 148 aa
rP172- Full-Length (172-aa) Recombinant Porcine Amelogenin
RT- Room Temperature
7

SD- Standard Deviation
SEM- Scanning Electron Microscopy
TRAP- Tyrosine-Rich Amelogenin Peptide
TMR- Transverse Micro Radiography
μ-CT- Micro-Computed Tomography
UTS- Ultimate Tensile Strength
XRD- X-Ray Diffraction

8

Introduction
Abstract
Dentin is a hydrated biological composite that forms the bulk of the tooth structure. It is
mesenchymal in origin and is embryologically, compositionally, and structurally distinct
from the overlying enamel. Odontoblasts derived from cranial neural crest cells
continually deposit dentin at the expense of the pulp. Dentin is composed of 70%
inorganic material, 20% organic material (Type I collagen), and 10% water (Linde, Bhown
et al. 1980); (Marshall Jr, Marshall et al. 1997). Tooth enamel is the most highly
mineralized tissue in vertebrates. Mature enamel is composed of 95-97% mineral and
less than 1% organic material (Ruan and Moradian-Oldak 2015).
Diseases and disorders of enamel and dentin could be microbial (carious), non-carious,
or of genetic causes. Dental caries leads to dissolution of the inorganic components and
destruction of the organic components of teeth. It warrants the removal of infected tissue
and subsequent restoration. Attrition, abrasion, abfraction and erosion result from
mechanical or chemical loss of the protective overlying enamel and exposure of dentin.
This manifests clinically as dentin hypersensitivity. Despite advancements in dental
material sciences, achieving a seamless interface between the restorative material and
tooth structure remains difficult, failure of which leads to microleakage and secondary
carious lesions beneath the restoration. Dentin poses a restorative challenge owing to its
heterogenous composition.
In treating non-carious cervical lesions (NCCL) the location of the defect poses additional
biomechanical hurdles. The cervical region of the tooth is the point of concentration of
centric and eccentric forces arising from functional and parafunctional mandibular
movements. Centric forces exert compressive stress, eccentric forces tensile (Beresescu
and Brezeanu 2011). Materials that are used as restoratives for NCCL hence have a
higher chance of fracture.
In recent years, there has been a shift towards minimally invasive dentistry (MID). The
most superficial zone of infected dentin is characterized by bacteria filling the dentinal
tubules and granular material in the intertubular space. Lying beneath this layer is the
9

zone of affected dentin with demineralized inter- and peritubular dentin regions without
the presence of bacteria. The principle behind MID is to retain the affected dentin
(demineralized but devoid of bacteria) and treat it to receive a restoration (Ritter 2017).
To overcome the shortcomings of clinical restoration, several techniques have been
developed that rely on biomimetic strategies involving matrix proteins and their
derivatives. A structural integrity between enamel and dentin exists at the dentinoenamel
junction (DEJ). Amelogenesis and dentinogenesis are closely linked events. The
interaction between the matrix-forming elements of enamel (amelogenin) and dentin
(collagen) has been of significant research interest. (Deshpande, Fang et al. 2010)
demonstrated that collagen fibrils guide the assembly of amelogenin into filamentous
structures along the long axis of the fibril. Furthermore, they demonstrated that
amelogenin-collagen interaction regulates calcium phosphate mineralization leading to
deposition of amorphous mineral particles aligned along the long axis of the collagen
fibrils.
Previous work in our laboratory has demonstrated the ability of amelogenin inspired
synthetic peptides ‘P26’ and ‘P32’ in promoting apatite nucleation in vitro and the re-
growth of aprismatic enamel-like layer on etched enamel surfaces (Mukherjee, Ruan et
al. 2018).
Based on the above knowledge, a research question was formulated: Can amelogenin-
based peptides be used to repair partially demineralized dentin lesions?
I therefore hypothesize that treating partially demineralized dentin with P26 peptide
results in recovery of physical properties and gain in mineral density. Tensile strength and
mineral density were selected as parameters for evaluating the functionality of repaired
dentin. Such physical properties are relevant to addressing the challenges faced in
restoring NCCL in terms of biomechanics and bonding.

10

Background
A. Non-Carious Cervical Lesions
A non-carious cervical lesion (NCCL) is the loss of hard dental tissue on the neck of the
tooth, most frequently located on the vestibular plane (Borcic, Anic et al. 2004). NCCL is
an age-dependent condition. The incidence and severity of NCCL increase with age
(Valena and Young 2002). Abrasion, erosion, and abfraction all lead to NCCL. Abrasion
often results from mechanical wear from overzealous tooth brushing (Fig. 1). These
lesions characteristically occur on the contralateral side of the dominant hand of the
individual (Osborne ‐Smith, Burke et al. 1999). (Borcic, Anic et al. 2004) have
demonstrated that tooth wear index was highest in the premolars, followed by the lateral
and central incisors. These lesions hence have esthetic implications apart from the
functional impairment of the enamel.

Figure 1.
Toothbrush abrasion on mandibular bicuspids and first molar.

Grippo coined the term abfraction. It is the condition in which occlusal loading causes
deformation and flexure at the cervical region leading to disruption of enamel crystals
(Fig. 2). Dental compression syndrome is tooth deformation related to malocclusion and
parafunctional habits (McCoy 1999). (Goel, Khera et al. 1991) developed a 3D linear
elastic finite element stress model of a maxillary first premolar. They found that the shape
of the DEJ differed between functional and non-functional cusps. Tensile stresses were
elevated at the cervical enamel and the authors suggested that the cervical region is more
susceptible to cracking as the interlocking between enamel and dentin is weakest in this
11

region (Wood, Jawad et al. 2008). The periodontal ligament and surrounding alveolar
bone play an important role in the development of these lesions.

Figure 2.
Abfraction lesion on maxillary bicuspids and first molar.

Erosion (Fig. 3) results from chemical insult to the enamel without any microbial
involvement. Habits such as sucking on lemons, which exposes the enamel to harsh
acidic pH conditions, are causative of tooth erosion. Lingual/palatal erosions often result
from repeated vomiting or gastric acid reflux placing patients with eating disorders and
those with gastro-esophageal reflux syndrome (GERD) at heightened risk. In dentin,
erosion leads to dissolution of the peritubular dentin and proceeds laterally on intertubular
dentin from the open tubule (Levitch, Bader et al. 1994). Salivary flow impacts the
occurrence of erosion as the buffering action of saliva helps neutralize the acidic pH.

Figure 3.
Palatal erosion on maxillary teeth.

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NCCL are multifactorial in origin (Osborne ‐Smith, Burke et al. 1999). They have been
described as stress corrosion (Grippo and Simring 1995), a multifactorial physiochemical
degradation of the CEJ area. ‘Bio-dental engineering factors’ (Grippo and Masi 1991)
have been defined as the effect of piezoelectricity at the cervical area (Bartlett and Shah
2006).

The treatment of NCCL begins with elimination of etiology and evaluation of dentinal
sensitivity. Copal varnishes, potassium oxalate, and other desensitizing materials are
used (Francisconi, Scaffa et al. 2009). NCCL pose a restorative challenge. (Ichim,
Schmidlin et al. 2007) investigated the mechanical integration of current cervical
restorative materials in wedge shaped lesions and reported that the current materials are
unsuitable in terms of resistance to fracture. They propose the need for a material
exhibiting increased flexibility and elasticity. Furthermore, published experimental data
have shown that glass ionomer materials (which are commonly employed as NCCL
restoratives) exhibit mixed modes of failure (adhesive and cohesive) both in shear stress
(Sidhu, Sherriff et al. 1999) and in tension (Tanumiharja, Burrow et al. 2000).

B. Dentin
Dentin in the coronal and radicular regions confers the recognizable shape of a tooth.
Mantle dentin is the outermost dentin layer present in most mammals and is 15-30 mm in
thickness. It covers the periphery of the coronal dentin and is atubular in structure
(Goldberg, Kulkarni et al. 2011). Mantle dentin is less mineralized than circum-pulpal
dentin and is believed to provide a cushioning effect to dissipate the forces arising from
the overlying enamel. It is formed from matrix vesicles arising from polarized
odontoblasts, a process akin to bone and cartilage genesis.
Circum-pulpal dentin, which constitutes the bulk of dentin structure, is composed of a
system of tubules surrounded by a cuff of peritubular dentin (Fig. 4). Intertubular dentin
is present between the dentinal tubules. Dentinal tubules converge at the pulp chamber,
so the number and density of the tubules varies depending on their location on the tooth
13

(Marshall Jr, Marshall et al. 1997). The dentinal tubular diameter decreases from 2.5 µm
near the pulp to less than 1 µm near the DEJ (Arola, Ivancik et al. 2009). Since the tubule
diameter and number vary from the DEJ to the pulp, the corresponding dentinal regions—
superficial, middle, and deep—have differing physical properties.

Figure 4.
SEM image of dentin structure

Peritubular dentin is predominantly composed of minerals. Type I collagen, which is found
in abundance in intertubular dentin, is absent from peritubular dentin (Goldberg, Kulkarni
et al. 2011). Peritubular dentin formation is not fully understood. Several mechanisms for
the formation of a more mineralized compartment within circum-pulpal dentin have been
proposed (Marshall Jr, Marshall et al. 1997); (Weiner, Veis et al. 1999); (Goldberg,
Kulkarni et al. 2011). Intertubular dentin can be described using a compartment model
that is often used with reference to bone. It consists of three layers: the cellular stratum
located at the periphery of the pulp, predentin, and mineralized dentin, which reaches up
to the mantel dentin (Goldberg, Kulkarni et al. 2011). It arises consequent to matrix-
controlled processes involving type I collagen.
Depending on when it is formed, dentin is further classified into primary, secondary, and
reactionary/tertiary dentin. Primary dentin is formed by odontoblasts until the erupted
tooth contacts the opposing tooth following which secondary dentin formation is initiated
and continues throughout the lifetime of the individual. Dentinogenesis occurs at the
Dentinal tubule
Peritubular dentin
Intertubular dentin
14

expense of the pulp chamber. Secondary dentin forms the bulk of the dentin. The tubular
structure of secondary dentin is for the most part continuous with that of primary dentin
(Nanci 2012). The S-shaped curvature of dentinal tubules is more accentuated in
secondary dentin (Goldberg, Kulkarni et al. 2011). Reactionary dentin is formed either by
odontoblasts or from the cells in the Höehl’s layer. Reparative dentin, formed in response
to irritants, is formed by pulp precursors and is closer in structure to bone than dentin.

C. Dentin Tensile Strength
Tensile strength is the ability of a material to withstand forces that tend to elongate. Dentin
tensile strength is conferred by the collagen fibers that form the scaffold upon which
minerals are deposited. Dentin tensile strength has been extensively studied since the
mid-20
th
century. Researchers have worked with both human and bovine dentin (Sano,
Ciucchi et al. 1994).
UTS (ultimate tensile strength) of dentin is greatest when tested perpendicular to the
direction of the tubules as samples are elongated along the length of the collagen fibers.
Hence, for fracture to result, the collagen fibers need to disintegrate (Inoue, Pereira et al.
2003). (Miguez, Pereira et al. 2004) reported that collagen alone contributes to 11-12%
of UTS in coronal dentin. The structural anisotropy of dentin gives rise to a tensile
strength gradient from the DEJ to the pulp (Watanabe, Marshall Jr et al. 1996); (Konishi,
Watanabe et al. 2002); (Inoue, Pereira et al. 2003) (Giannini, Soares et al. 2004).
Another aspect that is of interest in dentin tensile strength is the resultant cross-sectional
area at the test site. It has been shown that there exists an inverse relationship between
the cross-sectional area and UTS (Carvalho, Fernandes et al. 2001). Commonly
employed cross-sectional test site areas are 0.25, 0.5, and 1 mm
2
.
Samples for UTS testing can be prepared following one of two models: the beam model
(Konishi, Watanabe et al. 2002) and the hourglass model (Sano, Ciucchi et al. 1994). In
the hourglass model, the stress concentration is minimized. Stress concentration tends
to lower UTS (Sano, Ciucchi et al. 1994).
15

Upon demineralization, the UTS of dentin decreases substantially. Two models of
demineralization—acid-mediated and ethylene diamine tetra-acetic acid (EDTA)-
mediated—have been used in the past with comparable results (Fuentes, Ceballos et al.
2004); (Bedran ‐Russo, Pereira et al. 2007).

D. Evaluating mineral density by μ-CT
X-rays were discovered by William Roentgen in 1895. In computed tomography (CT) a
series of 2-D x-ray images are recorded of the object of interest from several angles and
are then 3-dimensionally reconstructed (Du Plessis, Broeckhoven et al. 2017). CT
systems can be classified into synchrotron or commercial systems. Both systems employ
attenuation mechanisms (photoelectric absorption and Compton scattering) to produce a
shadow image of the object of interest (Zou, Hunter et al. 2011). Combined with
appropriate software, mineral density measurements can be recorded from the
reconstructed images. Synchrotron systems, due to their monochromatic beam, lead to
more accurate mineral density measurements but are not cost effective. Additionally, they
require high speed electrons in a particle accelerator.
µ-CT has been extensively employed in dental research due to its non-destructive nature.
Enamel, bone, and dentin are some tissues that have been studied extensively using µ-
CT. (Weatherell, Robinson et al. 1974) reported that there exists a mineral density
gradient from the cusp towards the cervical region in outer enamel. (Huang, Jones et al.
2007) used pure-HAP phantoms to quantify mineral density of white spot lesions. (Yu,
Mei et al. 2017) used a commercial µ-CT system to study the effect of fluoride on two
modes of enamel demineralization, using either acetate or lactate buffer. (Shahmoradi
and Swain 2017) demonstrated that naturally arrested brown spot lesions have different
mineral density and surface layer thickness from those of white spot lesions.
Using µ-CT as a tool to measure response of enamel and dentin to a treatment system
has been carried out either by measuring the difference in the lesion depth before and
after treatment (Lo, Zhi et al. 2010, Wang, Gao et al. 2017), greyscale intensity analysis
(Ribeiro, Costa et al. 2015, Wang, Gao et al. 2017), or by quantitative measurement of
16

mineral density. Quantitative mineral density is determined by measuring linear
attenuation coefficient and/or bone mineral density (BMD) (Zhi, Lo et al. 2013).
BMD measurements made using µ-CT systems can be compared using either physical
measurements made by ashing the sample (Magne 2007) or more accurate methods like
transverse microradiography (TMR). (Lo, Zhi et al. 2010) comparatively analyzed the
remineralization of artificial carious lesions using polarized microscopy, TMR, and µ-CT.

E. Amelogenin, and P26
a. Functionality of amelogenin
Amelogenin is the most abundant enamel matrix protein. Mouse amelogenin c-DNA was
discovered (Snead et al., 1983, 1985) and sequenced (Snead, Zeichner-David et al.
1983). Amelogenin sequences, especially the N- and C–termini, are highly conserved
among species (Toyosawa, O’hUigin et al. 1998). Amelogenin size as determined by
laser desorption and mass spectrometry is 20.16 kDa and it migrates in the 25-26 kDa
range on sodium dodecyl sulphate gels (Simmer, Lau et al. 1994).

An important function of amelogenin is to form supramolecular structures which are
believed to guide enamel mineralization (Fincham, Moradian-Oldak et al. 1995);
(Tompkins, Alvares et al. 2005); (Du, Falini et al. 2005). Amelogenin nanospheres were
observed adjacent to hydroxyapatite (HAP) crystallites during in vivo enamel formation
(Robinson, Fuchs et al. 1981); (Fincham and Moradian-Oldak 1995). Using Y2H assay
(Paine and Snead 1997) demonstrated two amelogenin self-binding domains: ‘domain A,’
corresponding to amino acid residues +1 through 42, and ‘domain B,’ closely bordering
the highly conserved hydrophilic C-terminal 10 residues and defined by 17 residues.
Domain A is responsible for amelogenin-amelogenin interactions and has binding ability
in isolation (Paine and Snead 1997).

The hydrophilic carboxy-terminus of amelogenin binds HAP as demonstrated in vitro, and
this is believed to aid in the initial orientation of amelogenin towards the developing
17

enamel crystallites (Aoba, Moreno et al. 1989, Lijima, Moriwaki et al. 2002); (Maycock,
Wood et al. 2002). (Moradian-Oldak, Bouropoulos et al. 2002) demonstrated that both
native and recombinant amelogenin lacking C-terminus had significantly lower apatite-
binding affinity than full-length amelogenin. The affinity of the C-terminus of amelogenin
to apatite crystals is necessary during early stages of amelogenesis to prevent premature
crystal-crystal fusion (Moradian-Oldak, Bouropoulos et al. 2002).

A single phosphorylated serine is present at position 16, which falls within the domain A
and plays a role in enamel biomineralization (Fincham, Moradian-Oldak et al. 1994);
(Torres-Quintana, Lecolle et al. 2000). (Le Norcy, Kwak et al. 2011) demonstrated that
the phosphorylated serine is necessary to stabilize amorphous calcium phosphate and
prevent its transformation to HAP. Phosphorylation might play an important role in crystal
formation in vivo. Furthermore, (Lu, Xu et al. 2013) demonstrated that the structure and
conformation of certain amino acid residues changed as a function of the phosphorylation.
It was found that a lysine residue at position 24 adopted a configuration that consisted
only of 25-30% alpha helix, down from an original 40-50% helical configuration that was
uncovered upon dephosphorylation. The authors propose that phosphorylation is
essential for controlling the structure of the N-terminus and its interaction with HAP.

Self-assembly of amelogenin guides elongated hydroxyapatite growth during enamel
formation by virtue of the formation of supramolecular structures (Fan, Sun et al. 2009).
The C-terminus of amelogenin is essential for amelogenin self-assembly. (Paine, Wang
et al. 2003) identified deletions within the C-terminus occurring at and beyond position
169 (proline residue) that impacted self-assembly. Proline-169 is essential for the stability
of amelogenin nanospheres. The organization of amelogenin nanospheres into
nanochain structures that further guide elongated crystal growth by acting as a template
is dependent on the C-terminus (Fan, Sun et al. 2007). The authors demonstrated that C-
terminally truncated amelogenin (rP148) could not form chains of nanospheres, unlike
full-length amelogenin (rP172). In the presence of calcium phosphate, rP172 formed
bundles of rod-like structures, whereas rP148 could only form globular structures. This
was believed to be due to the difference in self-assembly. The newly grown crystal layer
18

in the presence of rP172 and calcium phosphate had improved mechanical properties
(hardness and elastic modulus). Similar effects have been observed in LRAP, an
alternatively spliced variant of amelogenin. LRAP lacking C-terminus could not form
nanochain assemblies even in the presence of calcium phosphate (Le Norcy, Kwak et al.
2011).

b. Amelogenin, its peptides, and tooth repair
Full-length amelogenin and peptides derived from it have been used for biomimetic tooth
repair. Using phage display, peptides were identified by (Gungormus, Oren et al. 2012)
within full-length amelogenin that had sequence similarity to a group of HAP-binding
peptides. The authors demonstrated that peptides ADP5 and ADP7 controlled
mineralization kinetics and phase transformation of calcium phosphate to HAP. Upon
testing the peptides in terms of their ability to repair artificially created lesions on acellular
cementum, they found that ADP5 was able to promote the regrowth of plate-like apatite
crystals arising from the surface of dentin to form a cementomimetic layer on the root
surface. ADP5 was later demonstrated (Dogan, Fong et al. 2018) to aid in enamel repair
for artificially created white spot lesions. ADP5 promoted remineralization by forming a
10-µm-thick dense mineral layer containing HAP on the surface integrating with
underlying enamel.

Ruan from our laboratory used chitosan as a mode of delivery of amelogenin to
demonstrate superficial enamel reconstruction. Chitosan was chosen due to its
antimicrobial properties. Amelogenin stabilized calcium phosphate in the CS-AMEL
hydrogel and guided its arrangement into linear chains. The newly grown layer bonded
well with the existing enamel and increased the hardness and elastic modulus of enamel
by nine and four times, respectively. The authors further used peptides with focused
regions of interest. They first examined the ability of leucine-rich amelogenin peptide
(LRAP) incorporated in chitosan (LRAP-CS) to repair enamel. (Mukherjee, Ruan et al.
2016) demonstrated that, after 3 days of remineralization in artificial saliva following
treatment with LRAP-CS, a 10-µm layer of well-organized, closely packed, fine needle-
like crystals were detected on the surface of the previously demineralized enamel. It was
19

observed that LRAP-CS promoted faster crystal nucleation and growth compared to
rP172-CS.

Using knowledge of the significance role in mineralization played by the N- and C-termini
of full-length amelogenin, a synthetic bioinspired 26-amino-acid-long peptide P26 was
designed (Fig. 5).

MPLPSYEVLTPLKWPSTDKTKREEVD
Figure 5.
Amelogenin-derived P26 peptide sequence. N- and C-termini are shown in red and blue,
respectively, and blue arrow points to the serine residue corresponding to the phosphorylated
S16 in full-length amelogenin.

Presented in red are the amino acids from the N-terminus, including the phosphorylated
serine at position 16, and shown in blue are the C-terminal residues. The N-terminal
residues were chosen due to the role they play in amelogenin-amelogenin interaction and
control of mineralization kinetics, the C-terminal residues for their significance in apatite
binding (as previously described in detail). Upon analyzing the effects of P26 on calcium
phosphate mineralization in vitro using transmission electron microscopy, (Mukherjee,
Ruan et al. 2018) observed that P26 accelerated crystal nucleation as evidenced by the
formation of dense amorphous lamellar structures after 25 min of aging. Presence of P26
led to the formation of thin plate-like hydroxyapatite crystals 24 hours after aging. After 2
days of treating artificially created lesions on human tooth enamel sections with P26, clear
diffraction peaks were observed on XRD that resembled those expected for HAP. Seven
days after application, a clear preference for c-axial growth perpendicular to the enamel
surface was reported. SEM observation revealed presence of needle-like crystals on the
demineralized enamel surface 2 days after treatment with P26. Upon repeated peptide
application, a dense multilayer apatite of approximately 30 µm in thickness growing
preferentially along the c-axis of the apatite crystals was observed at the end of 7 days.
20

Figure 6.
Acid etched human enamel section after repeated P26 applications.

21

Objectives, Strategy, and Scope

The long-term objective of this project is to develop bioinspired, amelogenin-derived
synthetic peptides as viable candidates for repairing non-carious cervical lesions
extending into dentin. The proposed mechanism of repair is by promoting growth of a
hyper-mineralized layer over the exposed dentinal surface and creating a suitable
substrate for bonding and further restorative rehabilitation.
The experiments were carried out in three parts:
1. To measure the micro-tensile strength as a functional attribute after P26 treatment of
partially demineralized dentin.
2. To assess the change in gross calcium content and density of partially demineralized
dentin after P26 treatment using bone mineral density (BMD) calculations from µ-CT
scans.
3. To characterize the structure and composition of partially demineralized dentin after
P26 treatment using scanning electron microscopy (SEM) and energy dispersive x-ray
analyzer (EDX).
Transverse dentin sections prepared from healthy human third molars were used for
micro-tensile strength testing following the hour-glass model of specimen preparation. For
µ-CT scanning, a 2x2-mm window was created on the transverse dentin section to have
a focused area of interest. µ-CT system calibration was done prior to BMD measurements
using commercial manufactured hydroxyapatite phantoms. Tooth quarters were used for
SEM and EDX analysis. Samples were analyzed at different points in the experiment to
obtain data pertaining to normal, demineralized, P26-treated and remineralized and
control remineralized dentin. Demineralization and remineralization cycles were carried
out by incubation in 37˚C. The concentration of P26 was 0.2 mg/mL for all three aims and
for SEM imaging; 0.5 mg/mL P26 solution was also used to study the effect of a higher
concentration and duration of exposure. The scope of this work was limited to the use of
tooth sections arising from one region of dentin. To address the heterogeneity present in
22

dentin with varying distances from the cementoenamel junction and dentinoenamel
junction was outside the scope of this study.

23

Chapter 1
To determine the effect of P26 treatment on tensile strength of partially
demineralized dentin.
Strategy
The high organic content in dentin, consisting mainly of type I collagen, confers a
considerable amount of tensile strength, or resistance to forces that tend to pull/elongate.
Measurement of dentin tensile strength is therefore partly a measurement of the strength
of its collagen fibers. Weakening the dentinal collagen (i.e. after demineralization) hence
causes tensile strength to diminish, and a gain in tensile strength following
remineralization could be indicative of changes pertaining to the collagen fibers. To test
dentin tensile strength, micro-tensile method was used and the hour-glass model of dentin
sample preparation for tensile strength testing was chosen.
Materials and Methods
A. Dentin Sample Preparation
a. P26 solution preparation
The peptide used in the study (Fig. 5) was synthesized commercially by CHEMPEPTIDE
Limited (Shanghai, China) and was prepared as per the protocol defined by (Mukherjee,
Ruan et al. 2018). 200 μg peptide was dissolved in 960 μL ultra-pure water, 25 μL CaCl2,
and 15 μL Na2HPO4 and the final pH was adjusted to 6.50. Final concentration of P26
solution was 0.2 mg/mL. Ruan et al. (2014) demonstrated that the optimal concentration
of amelogenin required to produce organized enamel-like crystals in a CS-AMEL hydrogel
is 200 μg/mL. Following this, (Mukherjee, Ruan et al. 2018) demonstrated that the same
concentration of P26 solution produced multilayer aprismatic enamel on an etched
enamel surface. Hence the same concentration of P26 solution was continued for the
present dentin experiments. Peptide solution was stored at 4˚C.
24

b. Tooth sectioning
Caries-free human third molars extracted using standard procedures (Herman Ostrow
School of Dentistry, USC) were used in this study. Remnant soft tissues were removed
using a scalpel and the teeth were rinsed in 70% ethanol to get rid of gross debris. They
were then sonicated for 20 min in a water bath. Cleaned teeth were placed in a freshly
prepared tooth storage media (PBS with 0.002% sodium azide) and stored at 4˚C until
use. Transverse dentin sections (0.7 +/- 0.2 mm thickness, and 1.4 mm above the CEJ)
were obtained using a low speed diamond saw under constant water cooling (BUEHLER
IsoMet 1000). Transverse sections were further prepared into hour-glass shaped test
specimens using a dental handpiece ensuring copious irrigation (Fig. 7a). For each
sample, an effective test site area of 1 mm
2
was chosen. Tensile strength test samples
were then divided into 4 groups: normal dentin, demineralized dentin, P26-treated
demineralized dentin, and control. Control samples were treated in artificial saliva only
without P26.

Figure 7a. b.
Fig. 7a. A representative image of a sample following the hour-glass model specimen preparation.
b. Sample mounted on the two detachable arms of the tensile strength grips.

25

c. Demineralization cycle to create partially demineralized dentin
The test samples from all groups excluding normal dentin were coated with two coats of
acid-resistant nail varnish. The second coat of varnish was applied 10 minutes following
the first application. Test site was left exposed (Fig. 7a). Samples from the three groups
were then immersed in 30 mL demineralization buffer (2 mM CaCl2·2H2O, 2 mM KH2PO4,
50 mM sodium acetate, and 0.05M acetic acid) (Xu, Neoh et al. 2011) pH 4.6 for 3 days
at 37 °C. After 3 days of demineralization, the samples from P26 treatment and control
groups were further taken through the remineralization cycle.
d. Remineralization cycle
Demineralized dentin sections were dried and placed in a clean glass vial. Samples in
the P26 treatment group were immersed in 20 µL of P26 solution and were left
undisturbed at RT for 15 min. All the samples were then placed in 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) at pH 7.0 and incubated in a water bath
at 37˚C for 10 days. Artificial saliva solution was changed every 24 hours to prevent the
chance of potential contamination and to replenish the ions in the solution. Peptide re-
application was done on the samples from P26 group as previously described on Day 5
of the remineralization cycle.

B. Tensile Strength Testing
Prior to tensile strength testing, varnish coating on the samples was carefully
mechanically removed and the samples were immobilized on the grips of a Ciucchi’s jig
(Fig. 8b) using cyanoacrylate glue (Scotch, USA). Samples were then tested under
tension using Instron 5965 (operating at 0.5 mm/min) until fracture (Fig. 8.a, b). The
maximum load calculated by the machine (in Newton) was then converted to tensile
strength in Mega Pascal (MPa) by calculating the force/ unit cross-sectional area of the
sample. Data were gathered only from samples that fractured in the desired test site (Fig.
26

8c). Statistical tests were carried out either in Microsoft Excel 2018 (parametric tests) or
using online calculators non- parametric tests).

Figure 8a. b. c.
Fig. 8a. Instron 5965 machine in operation with assembled parts. Note the sample holder mounted
between the two hydraulic pull arms of the device. Samples were tested in air and were hence
not hydrated during testing. b. A sample mounted on its holder showing how the bridge was placed
accurately at the point of separation of the two detachable ends of the sample holder. c. A
magnified image of the same sample after fracture in the desired bridge area. Note the change in
the color (chalky white) of the bridge in comparison to the surrounding normal dentin.

27

Results
Dentin Tensile Strength
Table 1.
A numerical representation of tensile strength data from different groups. Sample attrition
occurred in the P26 and control groups.
Sample type Mean Tensile
strength (MPa)
Standard
Deviation
Minimum and
Maximum UTS
values (MPa)
Normal dentin
(n = 15)
64.61 13.26 45.48-90.73
Demineralized
dentin (n = 10)
15.50 6.90 6.28-29.21
P26 treated
(n = 10)
34.40 19.94 7.6-69.47
Control (n = 10) 18.74 13.77 4.23-50.96

28

Figure 9.
Mean tensile strength of dentin in each group. MPa= Mega Pascal.

The mean tensile strength of normal dentin was 64.61 MPa (n = 15) (Table 1). The tensile
strength of dentin declined sharply following 3 days of acid demineralization to 15.50 MPa
(n = 10). This is a decrease of 76% which is highly significant as described in detail in the
statistical analysis section.
Following 10 days of remineralization in artificial saliva and in the presence of P26, a
partial recovery of tensile strength: 34.40 MPa (n = 10) was observed (Fig. 9). The control
samples (n = 10), which were remineralized in AS in the absence of P26, did not show
any statistically significant change in tensile strength when compared to demineralized
dentin (n = 10).
Note that even if sample variability and sample attrition were high, statistical analysis
shows that the difference between demineralized and P26-treated dentin samples was
significant.

29

Statistical analysis
a. Data were tested for normality using three separate online calculators using Shapiro
Wilk, and Kolmogorov Smirnoff tests.
The data were normally distributed in all groups (p = 0.12754). Non-parametric statistical
tests were used due to the difference in sample size between the groups. The alpha level
for statistical significance was set at 0.05.
b. Upon testing the 4 groups for statistical significance in tensile strength values using
Kruskal Wallis test, it was found that the difference in tensile strength between the groups
(shown in Fig. 9) was statistically very highly significant (p < 0.001).
c. Student t-test run between the demineralized dentin and P26 treatment groups
revealed that the difference in tensile strength between demineralized dentin and P26-
treated dentin was statistically significant (p = 0.0163).
d. Student t- test between the control and demineralized dentin groups revealed that there
was no statistically significant difference in tensile strength between the two groups (p =
0.5179).
e. Furthermore, box-plots were used to understand the data distribution within and
between groups (Fig. 10).
In less than 25% of P26-treated samples, the magnitude of tensile strength recovery was
less than that of 50% of the tensile strength values of demineralized and control samples.
30

Figure 10.
Tensile strength distribution within and between the 4 groups in Figure 3. Note that the data
distribution is wider in the P26 group compared to others, indicating that not all samples recorded
a high magnitude of tensile strength recovery.

The tensile strength measurements collectively showed that there was indeed an
increase in tensile strength resulting from P26 treatment of partially demineralized dentin.
The significance of our findings will be discussed separately in the discussion section.
Following these measurements, it was essential that this functional enhancement upon
remineralization in the presence of P26 be characterized more specifically in terms of
composition and structure. To achieve this, gross overall calcium density (Chapter 2) was
measured using BMD as a first step, and then structural resolution and elemental
compositional analysis (Chapter 3) were carried out using SEM and EDAX to characterize
the remineralized dentin in greater detail.

31

Chapter 2
To test the effect of P26 treatment on mineral density of partially
demineralized dentin.

Strategy
In order to examine whether improvement in tensile strength of remineralized dentin is
correlated to its mineral density, we quantitatively determined the bone mineral density
(BMD) parameter following P26 treatment of partially demineralized dentin. Dentin unlike
enamel is composed of a collagenous framework upon which hydroxyapatite (carbonated
HAP) is deposited to confer its hierarchical structure. It is compositionally similar to bone
(Weiner and Wagner 1998).
To measure the BMD of dentin, as a pre-requisite the μ-CT system needs to be
appropriately calibrated. The dentin sections were scanned at different stages of the
treatment cycle under similar conditions to draw meaningful comparisons. As the
measurements were concerning dentin, samples need to be hydrated at all times. A target
area of 2x2 mm in dimension was created to focus the area of interest to a confined
region. Quantitative measurements were made within the confines of the window to
minimize error from surrounding normal dentin and enamel. Multiple measurements were
recorded within different regions to get BMD values representative of the entire window.

Materials and Methods
A. Dentin sample preparation
a. P26 solution preparation
0.2 mg/mL P26 solution (pH 6.50) was prepared as described in Chapter 1A.a.
b. Tooth sectioning
32

The teeth were obtained and cleaned as described in Chapter 1A.b. Subsequently, the
teeth were sectioned transversely using a low speed diamond saw (MTI Corporation SYJ-
160, USA) to obtain cross sections from middle dentin. Each section was 1.2 +/- 0.2 mm
in thickness and was obtained 1.5 mm above the CEJ. Samples were prepared carefully
to ensure that there was no interference from the pulp or the overlying enamel. The
sections were then randomly divided into two groups: P26 treatment and control. A dentin
window of 2x2 mm in dimension was prepared at the center of each section (Fig. 11) by
painting the rest of the section with 2 coats of acid resistant nail varnish. Unlike in Chapter
1, the same sample from the P26, and control group was analyzed at different time points
of the experiment.

Figure 11.
A representative sample showing the 2x2 mm window within which the changes observed were
measured. Surrounding the window is the acid resistant nail varnish (green). Sample thickness
was 1.2 +/- 0.2 mm.

c. Demineralization cycle to create partially demineralized dentin
33

All the samples were immersed in demineralization buffer (2 mM CaCl2·2H2O, 2 mM
KH2PO4, 50 mM sodium acetate, and 0.05M acetic acid) at pH 4.6 for 3 days at 37 °C
following which they were subjected to μ-CT scanning.
d. Remineralization cycle
Samples in the P26 group were placed in a clean glass vial and 20 µL of P26 solution
was applied on the dentin window. Samples were left undisturbed at RT for 15 min. All
the samples were then immersed in 5 mL 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) at pH 7.0, and incubated in a water bath at 37˚C for 10 days. Artificial
saliva solution was changed every 24 hours to reduce the chance of potential
contamination, and to replenish the ions in the solution. Peptide was re-applied as
previously described on Day 5 of the remineralization cycle. Samples were then subjected
to μ-CT scanning.

B. μ-CT scanning
Prior to scanning, a custom holder was prepared using a polystyrene centrifuge tube
and modeling wax to ensure that the samples remained hydrated during the duration of
the scan (Fig. 12). A SkyScan 1174 μ -CT (Bruker) scanner operating at an accelerating
voltage of 52 kV and tube current of 790 mA was used. A 0.5-mm aluminum beam filter
was used to selectively remove the low energy x-rays. Samples were rotated through
360° to allow for maximum detail capture. An optimum voxel size resolution of 9.6 μm
was chosen with an averaging of four frames, and 0.70° rotational step.
μ-CT calibration was carried out using commercially manufactured 250-g/cm
3
and 750-
g/cm
3
hydroxyapatite phantoms. BMD was calibrated against the attenuation coefficient
(AC) of the phantoms. The equation relating the two is as follows:
BMD = AC – 0.0084005/0.065438 g/cm
3
(Equation 1)
34

Equation 1 was used to calculate the density of the calibration phantoms, this way
ensuring that the calibration was accurate. Additionally, the hydroxyapatite phantoms
were scanned using the pre-prepared polystyrene tubing filled with water to ensure
identical scan conditions as that of the test samples.
Samples were mounted on the water filled sample holder with their long axes
perpendicular to the stage. Shadow images were obtained at the end of the
demineralization and remineralization cycle and were 3-dimensionally reconstructed
using NRecon (software, version 1.6.9.8; SkyScan Bruker) software to generate a
projection dataset. A 100% beam hardening correction was used.

Figure 12.
The scan set-up. A 0.5-mm aluminum beam filter is in place. Sample is mounted on wax within
the polystyrene tubing to ensure hydration. Sample immobilized on the turntable using wax. Same
holder was used for all the samples.

C. Reconstruction and viewing
The generated projection dataset was then viewed using Amira 6.5.0 (Thermo Fisher
Scientific). Representative samples were selected to obtain images of normal,
35

demineralized, P26-treated and control dentin. 3D reconstructed images were sectioned
axially and viewed using greyscale, heatmap, and glow settings.
D. Quantitative mineral density measurement
The projection dataset was then further analyzed using CTAn (Software Version 1.6.9.8
SkyScan, Bruker) to record quantitative measurements of BMD. Five lines were randomly
selected within the 2000-μm-long window and within each line a region of interest (ROI)
was drawn surrounding the demineralized region. To minimize error, each ROI was drawn
5 times and the mean BMD was recorded. Between each measurement and the next, the
software was closed and re-opened in order to simulate maximum error. Hence, 25
measurements in total were obtained per sample and the mean of those was used to
represent the BMD of that sample at that stage. While calculating the BMD following the
remineralization cycle, the lines that were chosen were closely matched with those at the
end of the demineralization cycle. Data obtained were tabulated for further statistical
analysis. Paired sample t-tests were carried out in Microsoft Excel between demineralized
dentin BMD, and remineralized dentin BMD in both the P26, and control groups.

36

Results
A. Reconstructed Images
Normal Dentin (baseline)

Figure 13.
Representative baseline reconstructed images of normal dentin. Fig. 13a shows a representative
image in ‘glow ’view, b in greyscale, and c in heatmap.
a.
b. c.
37

Fig. 13.a represents a ‘glow’ view of the sample. In this setting, the colors are arbitrary.
Enamel is represented by blue and dentin by orange. Glow setting was chosen to aid the
visualization of demineralized samples and to monitor changes upon remineralization. In
the greyscale image (Fig. 13.b), enamel appears white and dentin appears grey due to
differences in mineral densities between the two tissues. Fig. 13.c represents a heatmap
view where tissues with a high mineral density appear orange/red (hotter) and the tissues
with lower mineral density in blue/green(cooler) The upper and lower ends of the sample
are intact and have been isosurface volume rendered.

P26-treated Dentin
Figure 14.
Representative images from a sample from the P26 group. Fig. 14. (a-c): 3 days demineralized.
(d-f):10 days remineralized.
Figure 14. represents axial sections of sample P1 after reconstruction. The wall of the
demineralized window is seen on isosurface volume rendering (shown by an arrow in Fig.
14.a and d). In Fig. 14a-c, the window wall is clearly visible. After 10 days of
remineralization in AS in the presence of P26, the visibility of the demineralized window
a.

b.

c.

d.

e.

f.

38

wall was reduced (Fig. 14d-f). The vertical height of the sample beneath the window as
shown by a blue arrow was greater after P26 treatment (Fig. 14d) The region below the
lower border of the window appears to be less dense than the remaining dentin as it
appears darker in images b and e (arrows).
Control

Figure 15.
Representative images of a sample from the control group. Fig15. (a-c): 3 days demineralized.
(d-f): 10 days remineralized.

In the representative control sample, the extent to which the demineralized window wall
was visible at the end of 10 days of remineralization (Fig. 15d-f) was the same at that
observed after demineralization (Fig. 15a-c). No visually appreciable change was
detected in the images recorded after remineralization in AS in the absence of P26.

a.

d.

b.

c.

e.

f.

39

B. Quantitative Mineral Density
Although image reconstruction provided a visual representation of the change following
P26 treatment, the interpretation of this technique is subjective. BMD measurement is
needed to quantify the change caused by P26. It also helps with understanding how
effective the treatment has been across the samples tested thereby confirming that the
change observed is consistent and reproducible. Such quantitative measurements are
the prelude for future efficacy studies for the concerned material(s).

Figure 16.
Bar graph representing mean BMD values at different points of the experiment in the P26 group
(n = 10).
Table 2
A numerical representation of data from Fig. 16. The variability in BMD values across samples
within any group was low. There was no sample attrition in this group. n = 10. S.D. = Standard
Deviation.
40

The mean BMD (n = 10) of the samples before initiation of demineralization (Normal) was
1.61 g/cm
3
(Fig. 16, Table 2). Following 3 days of acid-mediated demineralization
(Demineralized), the BMD declined to 1.06 g/cm
3
. This could correspond to loss of
superficial mineral content leading to exposure of underlying collagen. However, following
10 days of remineralization in artificial saliva in the presence of P26, the BMD increased
by 10.74% to 1.16 g/cm
3
(P26 treated). This is a direct indicator of increased gross
calcium content from that of demineralized dentin.

Figure 17.
Graph representing BMD change in each sample in the P26 group during different stages of the
experiment (n = 10).
Sample Type Mean BMD
(g/cm
3
)

S. D. Minimum and Maximum
BMD values (g/cm
3
)
Normal (n = 10) 1.52 0.06 1.45 - 1.65
Demineralized (n = 10) 1.05 0.13 0.87 - 1.26
P26 Treated (n = 10) 1.16 0.13 0.95 – 1.40
41

BMD gain after P26 mediated remineralization was observed in all the samples (Fig. 17).
The mean BMD gain of 10.74% was not consequent to isolated changes occurring only
in a subset of samples. BMD gain ranged between 6.83% to 14.43%. The 10 samples
which were taken through the treatment cycle exhibited a very low data variability for
BMD. Even those samples that exhibited > 35% decline in BMD following
demineralization (Samples P1-P3), recorded a substantial increase following P26
treatment (7.69-12.9%). The BMD gain pattern was hence consistent and reproducible
across all samples. Such changes hold potential for further exploitation in clinical dentistry
as production of a mineral dense layer over dentin could be advantageous in terms of
bonding to dentin.

Figure 18.
Bar graph representing mean BMD values at every stage of the experiment in the Control group
(n = 7).
Table 3
Numerical representation of Fig. 18. Note the low sample variance in this group as well. n = 7.
S.D. = Standard Deviation.
42

The mean BMD in the control group at the beginning of the experiment (Normal) was 1.65
g/cm
3
, identical to the P26 treatment group. Following 3 days of acid-mediated
demineralization, identical to the P26 treatment group, BMD declined to 1.08 g/cm
3
.
However, the difference in the BMD after 10 days of remineralization in artificial saliva
was within the error limit (Fig. 18, Table 3) indicating no change in the BMD in the control
group. Sample size was 7 in this group as some samples had interference from enamel
that warranted their removal from the experiment.

Figure 19.
Graph representing BMD change in each sample in the control group during different stages of
the experiment (n = 7).
Data variability was low in the control group (Fig. 19). None of the samples exhibited a
gain in BMD that was outside the error range of 1-4%.
Sample Type Mean BMD
(g/cm
3
)
S. D. Minimum and Maximum
BMD values (g/cm
3
)

Normal (n = 7) 1.65 0.11 1.50 - 1.79
Demineralized (n = 7) 1.08 0.18 0.82 - 1.33
Control AS only (n = 7) 1.06 0.18 0.83 - 1.32
43

Table 4
Summary of ∆BMD.
After 10 days of P26 mediated remineralization, the mean BMD of partially demineralized
dentin increased by a magnitude of 0.105 g/cm
3
(Table 4).

Statistical Analysis
a. The data were first tested for normality using the same online calculators as those
enumerated in Chapter 1.
The data were normally distributed in the P26 (p = 0.47137), and control groups (p =
0.48476). The alpha was set at 0.05.
b. Paired sample t-test run between demineralized and P26-treated samples revealed
that the difference in bone mineral density of demineralized samples following P26
treatment was statistically significant (p < 0.001).
c. In contrast, paired t-test run between demineralized and control samples revealed that
there was no statistically significant difference in BMD values from demineralized dentin
in the control (p > 0.05).
d. Box-plots were plotted for both groups to analyze data distribution (Fig. 20).

Sample
Type
Mean Demin
BMD
Mean ∆BMD (BMD
Remin-BMD
Demin)
p value
P26
1.05 g/cm
3
0.105 g/cm
3

p= 0.0000008838
Control
1.08 g/cm
3
-0.01429 g/cm
3

p=0.1010755
44

Figure 20.
Box-plot to analyze the data distribution within each stage and how it compares with the other
stages. Fig. 20 a. P26 group (n = 10), b. Control group (n = 7).
From the box-plot it was observed that following P26 treatment, more than 50% of the
samples exhibited a gain in BMD which exceeded the BMD of more than 75% of the
samples following demineralization (Fig. 20). Furthermore, the BMD of some of the P26-
treated samples approached that of normal dentin.
However, in the control sample, the BMD distribution within the control group following
demineralization and remineralization was identical indicating that there was no change
in BMD in this group (Fig. 20b). All the control samples recorded BMD values that were
lower than the least observed BMD value of the samples prior to initiation of the
experiment (normal dentin).

a. b.
45

Chapter 3
To characterize the morphology of partially demineralized dentin after
treatment with P26 peptide.
Strategy
In the previous chapters, it was demonstrated that P26 treatment of partially
demineralized dentin leads to an increase in tensile strength and mineral density.
However, in order to understand how these physical properties were changed as the
result of P26 addition, it is pivotal to determine the structure and composition of the
remineralized dentin. Scanning electron microscopy (SEM) in conjunction with elemental
dispersive x-ray analyzer (EDAX/EDS) were used for this purpose. Teeth were prepared
into quarters to facilitate SEM viewing. The effect of different P26 concentrations, and
application methods were analyzed.
Materials and Methods
A. Dentin Sample Preparation
a. Tooth Sectioning
Human third molars were obtained and cleaned as described in Chapter 1. Transverse
dentin sections were prepared from 1.5 mm above the CEJ measuring 1.2 mm+/- 0.2mm
in thickness. Each cross-section was further sectioned to obtain 3-4 usable quarters per
sample. Quarters were used instead of whole sections to aid SEM viewing. They were
stored in tooth storage media in 4˚C until use. A 1x1-mm window was prepared on each
quarter by covering the surrounding regions with two coats of acid resistant nail varnish
(Fig. 21).
46

Figure 21.
A tooth quarter after varnish application.
All the quarters were then placed in demineralization buffer to create partially
demineralized dentin lesions as previously described. Two quarters from each section
were then taken through the remineralization cycle. One quarter from each group was
used as control and the other as P26 treatment.
b. P26 treatment
Two concentrations of P26 were used and their methods of application were different.
P26 solution at a concentration of 0.2 mg/mL (pH 6.50) was prepared by dissolving 200
µg P26 powder in 960 µL of ultra-pure water, 25 µL of 0.1 M CaCl2, and 15 µL of 0.1 M
Na2HPO4.
P26 solution at 0.5 mg/mL concentration was prepared by dissolving 500 µg P26 powder
in the same quantities of the remainder of the ingredients.
For the 0.2 mg/mL concentration, 20 µL P26 was applied on the demineralized window
and was left undisturbed at RT for 30 mins. The samples were then immersed in 5 mL
AS and incubated at 37˚C for 10 days with AS replacement every 24 hours. Control
samples were immersed in AS without prior treatment with P26.
47

In the 0.5 mg/mL group, samples were immersed in 40 µL P26 solution and were left
undisturbed overnight at RT. They were then placed in AS for 10 days with the AS solution
being replaced daily.
c. Dehydration and sample drying
Prior to SEM, samples from all groups were dehydrated in ascending ethanol series (10-
100%) by immersing in solvent at ethanol concentration for 10 minutes (Tay and Pashley
2008). Then samples were chemically dried as per protocols defined by the Utah State
University for biological sample preparation for SEM. The quarters were immersed for 15
minutes each in the following:
● 2:1- 100% ETOH: HMDS
● 1:1- 100% ETOH: HMDS
● 2:1- HMDS: 100% ETOH
● HMDS alone- 2 times, 15 mins each
● HMDS alone- left to evaporate overnight in a fume hood.
Following chemical drying, samples were mounted on an aluminum stub using carbon
tape and sputter-coated with Au for 30 seconds.
B. SEM imaging and EDAX analysis
All the samples were imaged using Field emission SEM (JEOL JSM-7001F, JEOL Ltd.,
Tokyo, Japan) at the Center for Nanoimaging, University of Southern California. Surface
views of the samples were captured under low and high magnification to visualize both
structure and the extent of change. Samples were imaged at an accelerating voltage of
10kV. Elemental compositional analysis was carried out using an EDAX detector coupled
to the SEM (JEOL 7001SEM-EDX).

48

Results
A. SEM images
Normal Dentin

Figure 22.
SEM image of normal dentin. PT, peritubular dentin; IT, intertubular dentin.
Normal dentin has tubular structure surrounded by a cuff of hyper-mineralized peritubular
dentin (Fig. 22). Intertubular dentin, being less mineral-dense appears dull in comparison.
Note that the average diameter of the tubule is 1 µm and less in certain regions. Tissue
fixation was not carried out prior to SEM viewing. Therefore the collagen is not visible.

PT
IT
49

Figure 23.
Representative SEM images. Panel 1: 3-day demineralized dentin. Panel 2: P26 0.2mg/mL 10-
day remineralized. Panel 3: Control 10-day remineralized (AS alone).
Demineralized P26 0.2 mg/mL Remineralized Control Remineralized
Panel 1 Panel 2 Panel 3
50

Figure 24.
Representative SEM images. Panel 1: 3-day demineralized dentin. Panel 2: P26 0.5 mg/mL 10-
day remineralized. Panel 3: Control 10-day remineralized (AS alone).
Demineralized P26 0.5 mg/mL Remineralized Control Remineralized
Panel 1 Panel 2 Panel 3
51

Partially demineralized dentin samples displayed a lack of distinction between intertubular
and peritubular dentin (Fig. 23 and 24, Panel 1). Widening of dentin tubular diameter at
the expense of peri- and intertubular dentin was observed. Following 10 days of P26-
mediated remineralization in AS, samples treated with 0.2 mg/mL P26 displayed
narrowing of dentin tubular lumen by an electron dense deposit (Fig. 23, Panel 2). This
narrowing of tubular lumen was observed over a wide region, as seen under 2000
magnification (Fig. 23, Panel 2, image 1). The control samples in the 0.2 mg/mL
concentration group (Fig. 23, Panel 3) displayed widely open tubules with loss of
distinction between intertubular and peritubular dentin identical to images in Fig. 23, Panel
1. Following graded ethanol dehydration and chemical fixation, the collagen network in
the intertubular dentin was apparent (Fig. 24, Panel 1). Exposed collagen fibrils were seen
in the intertubular dentin matrix in demineralized and control samples (Fig. 24. Panels 1
& 3). 0.5 mg/mL P26 treatment led to the deposition of an electron dense layer occluding
the dentinal tubules and presenting in the intertubular dentin matrix over the collagen
fibrils (Fig. 24, Panel 2). Multiple regions were identified where the electron-dense
deposits were observed on the collagen fibrils (Fig. 24, Panel 2, images 3, and 4) as
shown by arrows. Collagen architecture and tubule diameter in the control samples are
identical to those of demineralized dentin (Fig. 24, Panels 1&3).
B. Elemental dispersive x-ray analyzer(EDAX/EDX) confirmed that the electron dense
deposits were composed of Ca and PO4.
Table 5
The ratio of weight % of calcium and
phosphate in normal dentin was 1.90 (n = 3).
Following acid- mediated demineralization,
the ratio of weight% decreased to 1.57 (n =
3). P26 0.2 mg/ml group recorded a ratio of 1.86 (n = 3) and 0.5 mg/ml group recorded
1.88 (n = 3).
52

Figure 25.
SEM image of surface view of a P26
treated sample.
The electron dense deposits were
composed of thin elongated
crystals that were deposited on the
surface of dentinal tubules. Low-
magnification imaging (Fig. 25.a)
revealed that the crystals were
spread over certain regions of the
intertubular dentin and dentinal
tubules. High-magnification
imaging (Fig. 25.c) revealed that in
some regions the thin crystals were
aligned to form bundle-like
structures (arrow).
a.
b.
c.
53

Figure.26
A comparative image of the collagen
architecture in the a. Demineralized
b. P26-remineralized, and c. Control
groups.
Exposed collagen fibrils were
observed in the intertubular
dentin matrix in the
demineralized and control
groups. (Fig. 26 a, c). When
remineralization was carried out
in the presence of 0.5 mg/mL
P26, electron-dense deposits
were observed on the surface of
the collagen fibrils resulting in a
beaded appearance of the
collagen. This pattern was
observed in a widespread
manner. The dull grey
appearance of the collagen fibrils
in the demineralized and control
groups was replaced by a bright
electron dense appearance after
P26-mediated remineralization in
AS.
a.
b.
c.
54

Discussion
The mechanical interlocking between enamel and dentin is weakest at the cervical region
(Goel, Khera et al. 1991). Under excessive and abnormal occlusal loading, the enamel in
the cervical region is susceptible to fracture, resulting in an abfraction lesion. NCCL, apart
from causing functional impairment of enamel and dentin, cause pain and sensitivity and
pose an esthetic problem. The location of these lesions makes restoration challenging.
Treatment modalities that focus on symptomatic relief employ desensitizing materials like
potassium salts of nitrates. Topical fluoride applications constitute an important part of
caries prevention strategy. The efficacy of fluorides to effectively remineralize dentin
lesions is controversial. Fluoride application leads to the formation of a barrier layer on
the surface of the lesion when used to remineralize enamel (Lynch, Churchley et al.
2011), and dentin (Wang, Gao et al. 2017). This barrier layer is important for the anti-
demineralization effect of fluoride, but it prevents calcium and phosphate ions from
penetrating into the deeper layers of the lesion. Furthermore, the remineralizing ability of
fluoride in dentin lesions is dependent on the presence of residual hydroxyapatite crystals.
(Zhang, Neoh et al. 2012) demonstrated that in regions of dentin where HAP was
depleted, fluoride could not effectively remineralize the lesion.
Strategies that employ molecules involved in biomineralization of these tissues can exert
control over events in the mineralization process like formation of a supersaturated
solution, mineralization kinetics, apatite binding, etc. Biomimetic strategies can hence
prove to be superior in promoting remineralization and aiding repair of damaged tissues.
In this study we used a synthetic peptide derived from amelogenin (P26), containing the
functional domains of amelogenin responsible for protein-protein and protein-mineral
interactions, and applied mineralization kinetics to demonstrate remineralization of
partially demineralized dentin.
A) Presence of P26 in the remineralization solution increases UTS
It was observed that treatment of partially demineralized dentin with P26 had a positive
effect. Partial demineralization means removal of superficial mineral content in dentin to
expose underlying collagen framework (Habelitz, Balooch et al. 2002). The tensile
55

strength value recorded for sound dentin 64.6 +/- 13.26 MPa and demineralized dentin
15.50 +/- 6.90 MPa were in concurrence with those obtained by (Giannini, Soares et al.
2004) and (Nishitani, Yoshiyama et al. 2005).
Following P26 treatment and 10 days remineralization in AS, a 2.2-fold increase in tensile
strength of partially demineralized dentin was observed. Magnitude of tensile strength
increased from 15.5 +/- 6.9 MPa to 34.4 +/- 19.94 MPa. The mechanism of tensile
strength enhancement after P26 treatment was analyzed using SEM. In a study by
(Pashley, Agee et al. 2003), it was demonstrated that the hydrogen bonding ability of a
solvent as measured by Hansen’s solubility parameter δh exhibited an inverse relationship
with the ultimate tensile strength (UTS). They treated demineralized dentin with several
polar solvents like acetone, HEMA, methanol, etc., and upon measuring the UTS, the
tensile strength magnitude following HEMA treatment was 31 MPa. The proposed
mechanism of UTS improvement in their study was the low hydrogen bonding ability of
certain solvents which in turn permits interpeptide H bond formation in collagen.
The magnitude of tensile strength in the control sample following 10 days of
remineralization in AS was 18.7 +/- 13.77 MPa, not significantly greater than that of
demineralized dentin (p > 0.05). However, from the box-plot analysis, it was seen that
some control samples exhibited UTS values on the order of 50 MPa. This was probably
consequent to insufficient demineralization. Some of the P26 samples also recorded a
UTS magnitude of 70 MPa, also attributable to insufficient demineralization prior to
initiation of treatment. It is also important to note that even within sound dentin, a spectrum
of tensile strength values was recorded ranging from 45 to 90 MPa. This demonstrates
the inherent heterogeneity and variability of the dentin substrate. However, the magnitude
of tensile strength recovery at the end of 10 days of remineralization in AS with P26 was
statistically significantly higher than that of partially demineralized dentin.
B) Presence of P26 in the remineralization solution increases mineral density
µ-CT was used to analyze mineral density of dentin samples after remineralization with
P26. µ-CT systems are classified broadly into synchrotron and commercial systems. In
this study a commercial µ-CT system SkyScan 1174 was used. Commercial systems
56

unlike synchrotron produce a polychromatic energy beam that needs to be filtered prior
to use. The resultant beam hardening artifacts are corrected for in the
reconstruction. Bone mineral density is a measure of bone density reflecting the strength
of bones as represented by calcium content (MedicineNet). Compositionally dentin is
similar to bone (Weiner and Wagner 1998), making BMD an appropriate candidate for
measurement of change in calcium content following treatment. Additionally, BMD can be
calibrated against attenuation coefficient or Hounsfield units, which renders this more
suitable for calculations and drawing comparisons.
To calibrate the system, 250-g/cm
3
and 750-g/cm
3
commercially manufactured HAP
phantoms were used, as a partial demineralization model was chosen wherein the
organic content was left undisturbed. This way the two-phase dentin calibration system
proposed by (Zou, Gao et al. 2009) using both HAP and liquid K2HPO4 was not followed.
It is also interesting to note that, according to (Kinney, Marshall et al. 1994) and
(Clementino-Luedemann, Ilie et al. 2006), the contributions by water and organic
components were considered negligible and thus did not warrant a two-phase calibration
system.
The BMD for sound dentin obtained in this study was 1.52 g/cm
3
, and 1.65 g/cm
3
in the
P26 and control groups. These values closely approach those obtained by (Clementino-
Luedemann, Ilie et al. 2006) (1.49 g/cm
3
); (Kinney, Marshall et al. 1994) (1.29 g/cm
3
); and
(Willmott, Wong et al. 2007) (1.42 g/cm
3
). It is important to note that there are differences
between these studies and ours. First, in the study by Kinney et al., a synchrotron source
and human canines were used unlike the molars in our study. Molars play a significant
role in mastication. It is possible that due to this functional significance, the mineral density
of molars might be greater than that of canines. (Willmott, Wong et al. 2007) used
deciduous teeth. Furthermore, in the Clementino Luedemann study, the method of
recording BMD was different from our study as an aluminum post was used as reference
material and the BMD was calculated based on an equation relating the theoretical and
experimentally obtained mass attenuation coefficients of materials.
57

Following acid demineralization of sound dentin, in my study the BMD declined to 1.05
g/cm
3
and 1.08 g/ cm
3
in the P26 and control groups. The lowest BMD value recorded in
a carious model was 0.55 g/cm
3
as obtained by (Kinney, Marshall et al. 1994), and the
BMD of demineralized deciduous molar was 0.37 g/cm
3
(Willmott, Wong et al. 2007).
However, to the best of my knowledge, there is insufficient support in the literature for
dentin BMD change following treatment. In one study by (Zhi, Lo et al. 2013), the BMD
following treatment in AgF was found to be 0.950 g/cm
3
.
C) Morphological characterization of remineralized dentin
Acid-mediated demineralization of dentin led to widening of dentin tubular diameter and
loss of distinction between peritubular and intertubular dentin. SEM evaluation of the P26-
treated dentin surface showed that upon application of 0.2 mg/mL of P26 solution,
narrowing of tubular diameter, as well as tubular occlusion in some regions, was evident.
However, immersion in 0.5 mg/mL P26 solution overnight was more effective in promoting
mineral deposition and led to the growth of a synthetic mineral layer on the dentin surface.
The collagen architecture in the P26 remineralized group was distinct from that of the
demineralized and control groups. Remineralization in the presence of 0.5 mg/mL P26
led to mineral-rich deposits on the surface of the collagen resulting in a beaded
appearance of collagen. The ability of P26 to promote nucleation of apatite crystals in
solution in a dose-dependent manner has been reported by (Mukherjee, Ruan et al.
2018). Interactions of P26 with self-assembled collagen is currently under investigation in
our laboratory. Preliminary data suggest that P26 promotes mineral nucleation on
collagen fibrils in vitro (Mukherjee, PhD Thesis). Whether this is due to the formation of
peptide-mineral complexes or other mechanisms of collagen mineralization remains to be
explored.
In a study comparing the tensile strength of mineralized and demineralized healthy and
carious dentin, (Nishitani, Yoshiyama et al. 2005) speculated that the lower bond strength
of adhesive resins to carious dentin was associated with lack of mineral around and within
the collagen fibrils (Kinney, Habelitz et al. 2003). Mineral deposition on collagen fibrils
can hence prove to be highly valuable. Although intrafibrillar collagen mineralization is
pivotal to enhancement of dentin mechanical properties (Kinney, Habelitz et al. 2003), it
58

has been demonstrated that apatite crystallites contribute 66-72% of dentin UTS (Sano,
Ciucchi et al. 1994); (Nishitani, Yoshiyama et al. 2005); (Miguez, Pereira et al. 2004).
Furthermore, around 70% of minerals in collagen are extrafibrillar in distribution (Chien,
Tao et al. 2017). Therefore, gain in dentin BMD and mineral deposition on the collagen
fibrils leading to their encasement in response to P26-mediated remineralization of
partially demineralized dentin could account for the UTS enhancement.
D) Challenges and future studies
The scope of this study was limited in exploring the differences in structure within dentin
and their possible effects on the remineralization potential. Additionally, the teeth used in
this study were 3
rd
molars, sclerotic dentin formation and other age-related changes
present in dentin and the possible effects of age-related changes on P26 treatment were
outside the scope of this study.
Furthermore, artificial saliva was used to remineralize the lesions. Previous work in our
lab has demonstrated that CS-AMEL did not act efficiently in the presence of natural
saliva (Rucha Arun Bapat, MS thesis). The effects of natural saliva on the interactions,
stability, and remineralization potential of P26 peptide need to be explored.
All the experiments were carried out on coronal dentin. Radicular dentin behaves
differently in comparison to coronal dentin. (Miguez, Pereira et al. 2004) demonstrated
that the collagen-crosslinking patterns vary between coronal and radicular dentin and
demineralized radicular dentin UTS is higher than that of coronal dentin. Regarding
structure, (Schilke, Lisson et al. 2000, Camargo, Siviero et al. 2007) previous reports
showed that in both bovine and human dentin the diameter of dentinal tubules was larger
in radicular dentin, but the tubule density was lower. Such structural distinctions can have
implications in terms of mineral density and physical properties and it is of relevance in
addressing NCCL. Often NCCL are accompanied by gingival recession leading to
exposure of root surface. Enamel matrix proteins are commonly employed for periodontal
therapy. Effects of P26 on cementum remain to be explored.

59

Conclusion

Using µ-CT, tensile strength testing and imaging, I demonstrated that amelogenin-derived
P26 peptide is effective in promoting remineralization of partially demineralized dentin.
P26- mediated remineralization of partially demineralized dentin led to a 2.2-fold increase
in tensile strength. A 10.74% gain in dentin BMD was recorded after 10 days of
remineralization in AS in the presence of P26. Finally, P26 treatment of partially
demineralized dentin led to the growth of a synthetic mineral layer covering the etched
dentin, occluding the dentinal tubules and presenting on the collagen in a concentration-
dependent manner.
The tensile strength enhancement and the resultant intrinsic reinforcement of dentin can
prove to be crucial as far as restoring cervical lesions are concerned. The mineral density
gain could be beneficial for bonding of restorative materials. P26 could potentially be used
for NCCL as a liner upon which restorations can be placed. A suitable mode of delivery
of the peptide needs to be developed for future clinical trials.

60

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Abstract (if available)

Abstract Dentin is a hydrated biological composite that forms the bulk of the tooth structure. It is mesenchymal in origin and is embryologically, compositionally, and structurally distinct from the overlying enamel. Odontoblasts derived from cranial neural crest cells continually deposit dentin at the expense of the pulp. Dentin is composed of 70% inorganic material, 20% organic material (Type I collagen), and 10% water (Linde, Bhown et al. 1980)

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Creator Visakan, Gayathri (author)

Core Title Remineralization of deminrealized dentin by amelogenin peptide P26

School School of Dentistry

Degree Master of Science

Degree Program Craniofacial Biology

Publication Date 04/29/2019

Defense Date 03/01/2019

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

Tag dentin,dentin tensile strength,micro-CT,mineral density,non-carious cervical lesions,OAI-PMH Harvest,remineralization

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Moradian-Oldak, Janet (committee chair), Phark, Jin-Ho (committee member), Snead, Malcolm (committee member)

Creator Email gayathri.visakan@gmail.com,visakan@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-158056

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