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

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

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Chitosan - Amelogenin Hydrogel- Saliva Interactions;
Towards Optimization of a Protocol for Enamel Repair

by
Rucha Arun Bapat

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 2016

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. Her expert guidance has been the most valuable for my academic
progress. I would also like to thank my committee members- Dr. Janet Moradian-Oldak, Dr.
Michael Paine and Dr. Jin-Ho Phark.
I would like to take this opportunity to thank Dr. Michael Paine. He has always been very
supportive and helpful during all my academic endeavors and has helped me time and again
with regards to my career development.
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. Qichao
Ruan, Dr. Parichita Mujumder, Dr. Karthik Chandrababu, Dr. Jingtan Su, Dr. Dongni Ren, Saumya
Prajapati, Kaushik Mukherjee and also the volunteers who worked alongside me- David
Liberman, Esteban Chidez, Gabriel Garcia, Payam Shaaf, Dhwani Shah.
Last but not the least, I thank my parents back home in India, without their unwavering support
this would not have been possible.

3

Table of Contents
ACKNOWLEDGEMENTS 2
LIST OF FIGURES 4
ABBREVIATIONS 5
INTRODUCTION 6
ABSTRACT 6
BACKGROUND 7
A. Enamel and dental caries 7
B. CS-AMEL hydrogel 8
C. Saliva 9
D. pH cycling system 9
OBJECTIVES AND SCOPE 11
CHAPTER 1
TO TEST THE IN VITRO EFFICACY OF CS-AMEL HYDROGEL IN CONDITIONS SIMILAR TO THE ORAL
CAVITY 12
MATERIALS AND METHODS 12
A. Efficacy of CS-AMEL hydrogel in human saliva 12
B. pH cycling 14
RESULTS 17
A. Efficacy of CS-AMEL hydrogel in human saliva 17
B. pH cycling 19
CHAPTER 2
TO EXAMINE THE EFFECT OF HUMAN SALIVA ON AMELOGENIN 22
MATERIALS AND METHODS 22
A. Amelogenin-saliva incubation 22
B. High Performance Liquid Chromatography 22
C. Polyacrylamide Gel Electrophoresis 22
D. Immunochemistry (Western Blot) 22
RESULTS 24
A. High Performance Liquid Chromatography 24
B. Polyacrylamide Gel Electrophoresis 28
C. Immunochemistry (Western Blot) 29
DISCUSSION 30
CONCLUSION 31
BIBLIOGRAPHY 32

4

List of Figures

Figure 1. Fluorescent and micro-CT images of incipient caries like lesions ........................... 15
Figure 2. pH cycling system ...................................................................................................... 16
Figure 3. SEM image showing newly grown hydroxyapatite crystals on enamel ................... 17
Figure 4. SEM images of samples in artificial saliva + human saliva........................................ 18
Figure 5. Fluorescent images of early caries-like lesions with and without Tx Cycle I ............ 19
Figure 6. XRD spectrum of incipient caries-like lesion treated with CS-AMEL ....................... 19
Figure 7. Fluorescent images of early caries like lesions with and without Tx Cycle II ........... 20
Figure 8. SEM images and XRD spectra of early caries like lesions with and without Tx ....... 21
Figure 9. HPLC chromatograph of saliva and amelogenin controls ........................................ 24
Figure 10. HPLC chromatograph of amelogenin and saliva incubated for 1 h ........................ 25
Figure 11. HPLC chromatograph of amelogenin and saliva incubated for 3, 6 h .................... 26
Figure 12. HPLC chromatograph of amelogenin and saliva incubated for 8, 15, 24 h ............ 27
Figure 13. SDS-PAGE gel for amelogenin and saliva incubated for various time intervals ..... 28
Figure 14. Western blot for amelogenin and saliva incubated for various time intervals ...... 29

5

Abbreviations

AS- Artificial Saliva
CS-AMEL- Chitosan Amelogenin
HPLC- High Performance Liquid Chromatography
HS- Human Saliva
KLK- Kallikrein
MMP- Matrix Metalloproteinase
PBS- Phosphate Buffered Saline
rP172- Recombinant Porcine Full Length Amelogenin
SDS-PAGE- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
SEM- Scanning Electron Microscope
TFA- Trifluoroacetic Acid
XRD- X-Ray Diffraction

6

Introduction

Abstract

Enamel, the outermost layer of teeth, is unique in many ways. Mature enamel is completely
acellular, hence incapable of repair; it is the hardest tissue in the human body, almost 99%
mineralized; and unlike other dental tissues (dentin, cementum or pulp) it is ectodermal in
origin (Moradian-Oldak & Paine, 2008). Although unique in so many ways, it is not immune to
diseases or wear. Dental caries is one the most common infectious diseases of mankind.
Attrition, erosion, abrasion, abfraction and fracture are other problems faced by a majority of
the population. The usual approach of conservative dental treatment is excavating infected or
weak enamel and filling the space with artificial materials like dental cements, ceramics or
composite resins. The two main drawbacks of such filling materials are (i) they do not match
the strength of natural enamel, and (ii) the boundary between natural enamel and artificial
materials allows micro-leakage of saliva and bacteria, eventually leading to secondary caries
and failure of the restoration.
To solve these problems dentists and material scientists have been interested in developing
biomimetic materials that would mimic natural enamel tissue. Ruan et al. (2013) developed a
novel chitosan-amelogenin (CS-AMEL) hydrogel to regrow a new layer of enamel-like crystals on
the surface of teeth. The hydrogel is capable of building needle-like, well-oriented
hydroxyapatite crystals with a robust interface on the surface of natural enamel. The final goal
is to treat deeper enamel lesions with a non-invasive approach using CS-AMEL hydrogel.
In order to develop CS-AMEL hydrogel as a potential biomimetic dental material, rigorous
clinical trials need to be conducted after thorough in vitro studies. One of the facets of the
clinical trials will be the mode and frequency of application of CS-AMEL hydrogel in patients. To
determine the application protocol of the hydrogel, in vitro tests in conditions similar to the
oral environment are necessary. This study looked at two of those conditions: pH changes in
oral cavity and interaction of amelogenin with saliva. The pH changes occurring in the oral
cavity throughout the day and night were simulated using a pH cycling model (Buzalaf, et al.,
2010; Ruan, et al., 2015). Interactions with saliva were tested by using the hydrogel in human
saliva as well as by studying the direct interaction between amelogenin and saliva.
The prospective protocol developed following these experiments was to apply CS-AMEL
hydrogel once daily before bedtime in a custom-made tray to prevent its direct interaction with
saliva and to allow maximal contact time between the hydrogel and enamel lesions. This study
is also the first to observe the interaction between saliva and amelogenin to the best of our
knowledge.
7

Background

A. Enamel and dental caries

The formation of enamel is a complex process involving a delicate balance between secretion of
various proteins and proteinases, and deposition of hydroxyapatite crystals. The process of
enamel formation lacks a collagen matrix unlike other mineralized tissues (Moradian-Oldak &
Paine, 2008). Instead, a protein matrix outlines the structure that is eventually replaced by
calcium phosphate crystals (Moradian-Oldak, 2012).
Amelogenin is the smallest but most abundant (almost 90% of total volume) enamel matrix
protein. It plays a vital role in crystal organization, morphology, maintaining length and width of
crystals, and enamel thickness (Moradian-Oldak, 2012). Ameloblastin is the second most
abundant protein, though much lower in volume than amelogenin (10% of total), and has
medium molecular weight, about ~65 kDA (Moradian-Oldak, 2012). It is essential for nucleation
of hydroxyapatite crystals and enamel formation ceases in the absence of ameloblastin (Nanci,
2013). Enamelin is the largest enamel matrix protein (~186 kDA) and is present in the smallest
quantity. Similar to ameloblastin, an enamel layer does not form in the absence of enamelin
(Hu, et al., 2008; Moradian-Oldak, 2012).
Dental caries is one of the most common, irreversible diseases of dental hard tissue. Frank
cavitated caries starts as an early opaque lesion called a white spot lesion or incipient caries.
Early caries can be broadly divided into surface and sub-surface mineral loss (Øgaard, et al.,
1988). Clinically they appear as white spots, sometimes seen only upon drying the teeth, and
may be fully invisible on moist teeth surfaces. The characteristic white appearance of the
lesions comes from the demineralized subsurface enamel losing its translucency (Øgaard, et al.,
1988), thereby becoming unable to reflect the color of underlying dentin. Incipient carious
lesions have always been a cause of concern among dentists because of the difficulty in treating
subsurface mineral loss (Kidd & Fejerskov, 2004). It is one of the major risks in orthodontic
treatment as the areas under and around orthodontic brackets are highly susceptible to
incipient caries (Øgaard, et al., 1988). Once the brackets are in place, there is very little a
dentist can do to prevent the development of white spot lesions and the outcome depends
heavily on the oral hygiene regimen of the patient (Travess, et al., 2004). Early carious lesions, if
not dealt with in time, can progress to cavitated caries which need complete excavation and
restoration (Featherstone, 2008). As white spot lesions usually appear on the labial surfaces of
anterior teeth, they are esthetically unpleasant as well (Bishara & Ostby, 2008). Although
topical fluoride application via toothpastes, fluoride varnishes and fluoride mouth-rinses can
help remineralize white spot lesions, termination of orthodontic treatment has been known to
happen in severe cases (Travess, et al., 2004). Hence treatment of white spot lesions is an
important problem in clinical dentistry.
Apart from caries caused by bacteria, enamel is also lost due to physical and chemical injury.
Non-carious enamel lesions can be classified by their causes: attrition, abrasion, erosion (or
corrosion) and abfraction (Grippo, et al., 2004). These lesions involve loss of dental hard tissue
8

without bacterial infection. Attrition occurs due to friction between adjacent teeth (proximal)
or opposing teeth (occlusal). It typically presents as smooth yellow to brown concavity on
contacting surfaces (Grippo, et al., 2004). Abrasion is caused by friction between teeth and
other objects such as hard tooth brushes. Such trauma is intensified by wrong brushing
techniques. It occurs usually in the cervical portion of a tooth, appearing as a sharp triangular
lesion at the cemento-enamel junction or even on the root surface. Erosion or corrosion is loss
of dental hard tissue by chemical injury, usually from extrinsic or intrinsic acids such as acidic
foods or acids from gastro-esophageal regurgitation (GERD) (Grippo, et al., 2004). This appears
as a smooth concave cervical lesion and is common in GERD patients. Initial stages of these
three lesions start as superficial enamel loss that can be possibly treated more effectively with a
biomimetic layer of enamel, like that developed with CS-AMEL hydrogel, rather than preparing
cavities for placement of traditional restorative materials.
Effective, conservative and non-invasive treatment of white spot lesions as well as non-carious
lesions necessitates the development of novel remineralization techniques, such as our
chitosan-amelogenin (CS-AMEL) hydrogel.

B. CS-AMEL hydrogel

Chitosan-amelogenin (CS-AMEL) hydrogel has been shown to treat artificial erosion-like lesions
(Ruan, et al., 2013) and artificial early caries-like lesions by promoting remineralization of the
lesion from within (Ruan, et al., 2015). CS-AMEL hydrogel can form a new layer of enamel-like
crystals on the etched surface of extracted human third molars in the presence of artificial
saliva (Ruan, et al., 2013). Studies have shown that the new layer is more organized than the
deposition occurring in the presence of fluoride, and the interface between native enamel and
the newly formed crystals is very robust (Fan, et al., 2009; Ruan, et al., 2013). Moreover, the
new layer has hardness comparable to that of native enamel (Mukherjee et al., in preparation).
Further, the hydrogel is stable and active over a range of pH values varying from 4.6 to 7.0
(Ruan, et al., 2015).
Amelogenins are a group of low molecular weight proteins secreted by ameloblasts. They are
rich in proline, histidine and glutamine (Glimcher & Levine, 1966). In vivo, amelogenin is
responsible for the length and width of hydroxyapatite crystals, formation of organized rod
structures, and overall hierarchical structure of enamel (Yang, et al., 2011). In the absence of
amelogenin a thin enamel layer is still formed without distinct enamel architecture (Nanci,
2013). Amelogenin gets degraded by two types of proteinases – matrix metalloproteinase
(MMP)-20 and Kallikrein (KLK)-4 – into multiple smaller fragments of unknown function before
the crystals mature (Yang, et al., 2011).
Chitosan is a biopolymer derived from chitin, found in sea crustaceans. Studies of interaction
between chitosan and hydroxyapatite surfaces have shown that chitosan has antimicrobial and
mucoadhesive properties and it also has the potential to protect enamel surfaces from acid
attack (Arnaud, et al., 2010; Lee, et al., 2012). In the CS-AMEL hydrogel system, it acts as a
9

carrier for the amelogenin protein and provides a means for amelogenin to stay on the tooth at
the site of a white spot lesion because of its mucoadhesive properties (Ruan, et al., 2013).
Chitosan also prevents the growth of human salivary bacteria in LB media as compared to LB-
only and amelogenin-LB broths (Ruan, et al., 2013).
Previous studies of CS-AMEL hydrogel have been conducted in artificial saliva solution (Ruan, et
al., 2013) at a stable pH of 7.0. In order to develop the hydrogel as a potential non-invasive
treatment for white spot lesions, it is necessary to test its efficacy in human saliva.

C. Saliva

Whole saliva is the fluid present in the oral cavity. It is a mix of secretions from major and minor
salivary glands as well as secretions/lysis products of oral microorganisms. The major salivary
glands include the parotid, submandibular and sublingual. Minor salivary glands are distributed
all over the tongue, palate, labial, and buccal mucosa. Saliva secretions can be classified into
serous, mucous and mixed type of secretions. Saliva contains about 99% water, with the
remainder comprised of electrolytes, glycosaminoglycans (GAGs), proteins and glycoproteins
(Fábián, et al., 2008; Humphrey & Williamson, 2001). Mucin is the most abundant glycoprotein.
Common proteins in the saliva are salivary amylase, serum albumin, IgA, lysozyme, histatin,
cystatins, statherins (Fábián, et al., 2008). Functions of saliva including its buffering action,
chemical and mechanical protection of oral tissues, hard and soft tissue repair, maintenance of
tooth integrity, and others are made possible by the presence of these proteins and
glycoproteins (Nanci, 2013). The total number of microbial and human proteins in saliva is as
high as 3000 (Fábián, et al., 2008; Griffin, 2014).The physiological pH of saliva can vary from
5.75 to 7.05 although after consuming carbohydrates or acidic drinks it can go as low as 3.8
(Meurman, et al., 1987). Saliva has various types of buffering systems like phosphate buffers
(HPO 4
-
), carbonate buffers (HCO 3
-
), and protein buffers (García-Godoy & Hicks, 2008) that help
bring the pH back to its physiologic value. However, while it is acidic the tooth enamel starts to
demineralize. The pH below which the enamel starts to demineralize in an acid attack is called
the critical pH and is around 5.5 (Dawes, 2003). Below the critical pH, enamel loses minerals
rapidly and sustained acidic pH leads to development of white spot lesions. Apart from
consumption of food, the pH of saliva also varies depending upon the volume of secretion. The
average flow rate of saliva is ~0.3 ml/min when it is not stimulated by food or speech, but
during sleep it is almost zero; when stimulated, it can increase up to 3.7 ml/min (Fábián, et al.,
2008). As saliva is such a dynamic fluid, it is important to understand the possible interactions
between saliva and CS-AMEL hydrogel.

D. pH cycling system

The pH cycling system is an established system to test the effect of changing pH on the action of
various dental products like dentifrices (Buzalaf, et al., 2010). The pH of the oral environment
10

changes under two main conditions: 1) after consumption of food and 2) during sleep. After
consumption of acidic drinks like sodas or fruit juices, the overall pH of the mouth becomes
acidic. Apart from that, the local pH around the enamel also decreases upon eating because the
carbohydrates from food are digested by oral flora to form acids. Once the local pH reaches its
critical value (Dawes, 2003), demineralization of enamel begins – calcium and phosphate from
enamel start dissolving in saliva – and continues until the pH is buffered to near neutral. During
sleep the overall rate of flow of saliva is very low (Schulz, et al., 2013) which reduces the pH of
the mouth to about 6.5 (Ruan, et al., 2015).
In this project, the pH cycling system was used to subject CS-AMEL hydrogel to the above pH
changes for durations similar to those occurring in the mouth. In this way, a more realistic
assessment of the efficacy of the hydrogel was obtained.

11

Objectives and Scope

The long term objective of the project is to develop Chitosan-Amelogenin (CS-AMEL) hydrogel
as a potential non-invasive treatment for non-carious enamel lesions.
This study was conducted to determine the ability of CS-AMEL hydrogel to grow well-organized
hydroxyapatite crystals on the surface of enamel in conditions similar to those of the oral
cavity.
The experiments were conducted in two parts:
1. To test the in vitro efficacy of CS-AMEL hydrogel in the presence of human saliva and
over a varying pH range
2. To examine the effect of human saliva on amelogenin stability
To test the in vitro efficacy of CS-AMEL hydrogel, it was applied on slices of human third molars
and incubated at 37°C for 7 days in the presence of human saliva. The newly grown layer of
enamel was examined by scanning electron microscopy to assess its organization and
orientation. To determine the effects of change in salivary pH on CS-AMEL hydrogel, pH cycling
was used. Further, the effects of human salivary enzymes on amelogenin in the hydrogel were
studied by incubating amelogenin directly in human saliva. The degradation of amelogenin was
analyzed using HPLC, SDS-PAGE and Western Blot. The scope of this work was limited to
amelogenin only and contents of the saliva were not investigated.

12

Chapter 1
To test the in vitro efficacy of CS-AMEL hydrogel in conditions similar to the oral cavity

Materials and Methods

A. Efficacy of CS-AMEL hydrogel in human saliva

i. Preparation of CS-AMEL hydrogel
CS-AMEL hydrogel was prepared as previously described by Ruan et al. (2013). Chitosan stock
solution was made by dissolving 2% (w/v) chitosan (medium molecular weight, 75-85%
deacetylated by Sigma-Aldrich, MO) in 2% (v/v) acetic acid solution followed by stirring at 80°C
overnight. After cooling the solution to room temperature, it was filtered through a 0.45 μm
filter (Millex-HV 0.45 µm, PVDF, 33 mm, sterile). The pH was adjusted to 5.0 with 1 M NaOH. To
make 1 ml CS-AMEL hydrogel, 960 μl of this chitosan solution was mixed with previously
aliquoted 200 μg recombinant porcine full length amelogenin (rP172), made by Dr Karthik
Chandrababu according to the protocol described by Ryu et al. (1999), 25 μl 0.1 M CaCl 2, and 15
μl 0.1 M Na 2HPO 4 and vortexed until the solution was clear. The pH was adjusted to 6.5 with 1
M NaOH.

ii. Tooth slice preparation
Extracted human third 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 molars were cut longitudinally
into 1-2 mm thick slices using a water-cooled slow speed (45-65 RPM) diamond saw. Each slice
was etched for 30 seconds in 30% phosphoric acid (H 3PO 4), washed with deionized water, and
sonicated for 2 minutes to remove any debris or acid from the surface (Ruan & Moradian-
Oldak, 2014). Clear nail varnish was applied to the slices to make a 2x2 mm window of etched
enamel, covering rest of the surface completely. Two coats of nail varnish were applied, with
the second coat being applied 15 min after the first, and then slices were air dried for at least 2
hours.

iii. Application of hydrogel
About 20 μL of CS-AMEL hydrogel was applied to the windows of enamel using a syringe (Ruan,
et al., 2013). The slices were placed in a desiccator for 15 min to partially dry the hydrogel
before placing them in the saliva solution.

13

iv. Preparation of saliva solution
The teeth slices were placed in a solution containing human saliva (Pooled Normal Human
Saliva, Screened for HIV, Hepatitis B and C, Syphilis, Catalog No.: IR100044P, Innovative
Research, MI). After collection, the saliva was centrifuged at 12,000 g for 20 min at 4°C and
then stored at -20°C before further use (Amado, et al., 2005). The solution used to incubate the
samples contained 1 ml human saliva (HS) and 4 ml artificial saliva (AS- 0.2mM MgCl 2.6H 2O,
16mM KCl, 0.72mM K 2HPO 4, 50mM HEPES, 4.5mM NH 4Cl, 1.2mM CaCl 2.2H 2O in ~800-900ml of
water, pH adjusted to 7.0 with 1M KOH and volume fixed to 1000ml) (Ruan, et al., 2013). To
inhibit the growth of commensal microorganisms from saliva, 0.02% sodium azide (NaN 3) was
added to the solution. To keep the fluoride levels close to those found in the human saliva
(Ingle, et al., 2014) 0.45 ppm of fluoride was added to 5 ml HS-AS solution.

v. Sample preparation for enamel remineralization
After application of CS-AMEL hydrogel, each tooth slice was placed in a scintillation vial
containing 5 ml HS-AS solution and incubated at 37°C for 7 days. The solution was changed and
hydrogel was reapplied every day (once every 24 h) to simulate once-daily application of
hydrogel by patients. The following control and test samples were made:
Sample HS AS Hydrogel
A Control - 5ml CS-AMEL
B Test 1 1ml 4ml CS-AMEL
D Test 3 1ml 4ml Untreated
E Test 4 - 5ml Untreated

Concentrations of sodium azide and fluoride were kept constant throughout.

vi. Characterization of tooth slices
The samples were sputter coated with platinum and observed using a scanning electron
microscope (JEOL JSM-7001F, accelerating voltage 15 kV) to visualize the crystal growth.

14

B. pH cycling

To test whether CS-AMEL hydrogel was effective over a varying pH range on different enamel
lesions, the enamel blocks were subjected to a pH cycling model (Ruan, et al., 2015).

i. Preparation of CS-AMEL hydrogel
CS-AMEL hydrogel was prepared as described in experiment A above.

ii. Tooth preparation to make artificial lesions
Two types of lesions were prepared for the pH cycling experiments:
a. Incipient caries-like lesions: Extracted human 3
rd
molars without any pre-existing caries,
demineralization or restorations were collected from Ostrow School of Dentistry of USC. The
crowns of the molars were cut into approximate quarters (using a water-cooled slow speed
diamond saw, 45-65 RPM) to obtain 4-5 pieces per crown. The enamel surfaces of these pieces
were polished with polishing paper sequentially starting from coarser to finer grit. A window of
enamel was made on the surface of pieces as in part A, using 2 coats of nail varnish. The pieces
were submerged into a demineralization solution (1.5 mM CaCl 2, 0.9 mM NaH 2PO 4, 50 mM
Acetate Buffer, pH 4.6) at 37°C for 7 days to create incipient caries-like lesions. At the end of
this period, the pieces were examined by fluorescent microscopy to verify subsurface
demineralization mimicking white spot lesions (Figure 1) (Buzalaf, et al., 2010).
b. Erosion-like lesions: Extracted human 3
rd
molars were cut into 4-5 pieces and polished as
above (Ruan & Moradian-Oldak, 2014). Two coats of clear nail varnish were applied to the
pieces, leaving a 2 x 2mm window as described above. The pieces were etched for 30 seconds
in 30% phosphoric acid (H 3PO 4), washed with deionized water, and sonicated for 2 minutes to
remove any debris or acid from the surface. After etching with 30% H 3PO 4 for 30 seconds, the
enamel crystals became discontinuous and broken, resembling erosion (Figure 8A).

15

Figure 1. A- Fluorescent and B- micro-CT images of cross-sections of demineralized enamel blocks,
showing an artificial carious lesion (white arrows). Inset: photograph of a demineralized enamel block
showing a white spot lesion (white opaque square) on the tooth sample (Ruan, et al., 2015).

iii. Application of hydrogel
CS-AMEL hydrogel was applied (20μL) on the windows of early caries-like lesions as well as
erosion-like lesions and dried in a desiccator for 15 min.

iv. Preparation of pH cycling solutions
a. The demineralization solution described above was used in Cycle I to replicate the drop in
oral pH after consumption of food.
b. A modified demineralization solution was used in Cycle II to replicate the drop in pH during at
night due to reduced salivary flow. It contained 1.5 mM Ca
2+
, 0.9 mM H 2PO 4
-
, 130 mM KCl, 20
mM Tris, and 0. 45 ppm fluoride at pH 6.5.
c. The remineralization solution simulated physiological salivary pH and contained 1.5 mM Ca
2+
,
0.9 mM H 2PO 4
-
, 130 mM KCl, 20 mM HEPES at pH 7.0.

v. Sample preparation for enamel remineralization (or repair of lesions) (Figure 2)
Cycle I- Simulating changes in pH after consumption of food:
After application of the hydrogel, the enamel pieces were placed in the demineralization (DE)
solution for 6 h, followed by remineralization (RE) solution for 18 h, at 37°C for 5-7 days. The
hydrogel was reapplied before and after each solution change (twice daily). Untreated lesions
were used as a control.

Cycle II- Simulating changes in pH at night:
16

For the modified pH cycle, the pieces were submerged in modified remineralization solution
(mRE) for 8 h followed by remineralization solution (RE) for 16 h at 37°C for 7 days. Similar to
Cycle I, 20 µL CS-AMEL hydrogel was applied before and after each solution change. Untreated
lesions were used as a control.

Figure 2. pH cycling systems representing cycle I and cycle II (Ruan, et al., 2015)

vi. Characterization of newly grown layers
After pH cycling, the enamel pieces were washed, dried and characterized by scanning electron
microscopy (JEOL JSM-7001F, Platinum sputter coating, accelerating voltage 15 kV) for
visualizing the crystal growth, using XRD (Rigaku Diffractometer, Cu Kr radiation, λ = 1.542 Å, 70
kV, 50mA, step size 0.02°, scanning rate 0.02°/s, in a 2θ range from 10°-60°) to determine the
orientation of hydroxyapatite crystals, and by fluorescent microscopy to observe the incipient
caries-like lesions.

17

Results

A. Efficacy of CS-AMEL hydrogel in human saliva

A new layer of apatite crystals was formed on the surface of etched enamel in the presence of
CS-AMEL hydrogel and artificial saliva, similar to that shown by Ruan et al (2013). The layer
showed well-oriented needle-like apatite crystals evenly covering the enamel surface (Figure 3).

Figure 3. SEM image showing newly grown hydroxyapatite crystals on the surface of enamel after 7 days
of treatment with CS-AMEL hydrogel in the presence of artificial saliva. Inset: 30,000 X resolution of the
crystals
The samples in artificial saliva alone (Figure 1) served as a positive control for the next
experiment.
SEM images of samples incubated in a mixture of human saliva (HS) and artificial saliva (AS) are
shown in Figure 4. When samples treated with CS-AMEL hydrogel were subjected to 7 days of
incubation in the HS-AS solution at 37° C there was no discernable layer of newly grown crystals
on the surface of the teeth (Figure 4 A and B). However, the negative control of untreated tooth
slices in HS-AS solution yielded a haphazard accumulation of calcium phosphate crystals on the
surface (Figure 4 B and C).
18

The lack of an organized mineralized layer in the sample with saliva provided a stronger
rationale for testing the effects of human saliva on amelogenin stability using HPLC and SDS-
PAGE.

Figure 4. A and B- SEM images of samples in artificial saliva + human saliva after 7 days of treatment
with CS-AMEL hydrogel; C and D- SEM images of untreated control samples in artificial saliva + human
saliva.

19

B. pH cycling

i. Incipient caries-like lesions - Cycle I
After 7 days of Cycle I (simulating pH changes after meals) in untreated samples, the depth of
caries-like lesions increased from ~100μm to ~150μm (compare Figure 1A with Figure 5A). After
treatment with CS-AMEL hydrogel, the depth of the lesions decreased from ~100µm to ~50µm
(Figure 5B).

Figure 5. Fluorescent image of a cross-section of enamel blocks with early caries-like lesions after 7 days
of Cycle I A- untreated, and B- with CS-AMEL hydrogel treatment.

The XRD spectrum of repaired enamel (Figure 6) (Ruan, et al., 2015) showed a high intensity
ratio of (002) and (211) peaks corresponding to hydroxyapatite (2.323), indicating that
hydroxyapatite crystals grown using CS-AMEL hydrogel were well oriented.

Figure 6. XRD spectrum of incipient
caries-like lesion treated with CS-AMEL
hydrogel for 7 days in Cycle I (Ruan, et al.,
2015)
20

ii. Incipient caries-like lesions - Cycle II
For Cycle II (simulating nightly changes in salivary pH), half of each white spot lesion was
covered with 2 coats of nail varnish and the other half was treated with CS-AMEL hydrogel. The
nail varnish-covered area of the lesion served as its own control, hence reducing the bias from
different levels of mineralization in different pieces of teeth. After 7 days of Cycle II at 37°C, the
depth of early caries-like lesions decreased from ~100µm to ~30µm in the part treated with CS-
AMEL hydrogel (Figure 7) (Ruan, et al., 2015).

Figure 7. A- Fluorescent images of cross-section of enamel blocks with incipient caries like lesions after 7
days of Cycle II – untreated control (top) and CS-AMEL hydrogel treated (bottom). B- Higher resolution
of the portion straddling the dotted line in A. (Ruan, et al., 2015)

21

iii. Erosion-like lesions - Cycle I
After 5 days of Cycle I at 37°C without CS-AMEL treatment, only irregular crystals were
observed on the enamel surface (Figure 8B) (Ruan, et al., 2015). On samples with CS-AMEL
treatment, a layer composed of oriented apatite crystals formed on the surface of the erosion-
like lesion (Figure 8C). In comparison to the sample without CS-AMEL treatment, the XRD
spectrum of enamel repaired by CS-AMEL hydrogel showed a higher ratio of (002) and (211)
peaks, indicating better orientation of crystals achieved by applying the CS-AMEL hydrogel on
the enamel (Figure 8D).

Figure 8. A- SEM image of erosion-like lesion. B- SEM image of erosion-like lesion after 5 days of Cycle I,
untreated. C- SEM image of erosion-like lesion after 5 days of Cycle I and CS-AMEL hydrogel treatment.
Insets show the images at higher magnification. D- XRD spectra of etched enamel and lesions after 5
days of Cycle I, with and without CS-AMEL hydrogel treatment. (Ruan, et al., 2015)

The results of these pH cycling experiments were published in Journal of Biomedical
Engineering and Informatics as ‘Efficacy of amelogenin-chitosan hydrogel in biomimetic repair
of human enamel in pH cycling systems’ (Ruan, et al., 2015).
22

Chapter 2
To examine the effect of human saliva on amelogenin

Materials and methods

A. Amelogenin-saliva incubation

Two hundred µg amelogenin was incubated directly in 1 ml human saliva at 37°C, and its
degradation was followed over time to determine its probable stability in the oral cavity. The
first method used was High Performance Liquid Chromatography (HPLC). The findings were
confirmed using SDS-PAGE and Western Blot. Samples for HPLC and SDS-PAGE were collected
at 1 h, 3 h, 6 h, 8 h, 15 h, and 24 h time points.

B. High Performance Liquid Chromatography

For preliminary determination of how stable amelogenin is in human saliva, the samples were
subjected to reversed phase HPLC. Twenty µL of sample solution containing 0.4 µg amelogenin
(rP172) collected at each time point was injected into 0.1% Trifluoroacetic Acid or (TFA) and
analyzed using a reverse phase C-8 column (Vydak).
Controls of human saliva and rP172 alone were first analyzed (in 0.1% TFA) to confirm the
absence of overlapping peaks (Figure 9). Results showed that the rP172 peak was at 53.051
minutes and there was no coinciding peak in the saliva control. Further, 200 µg rP172 was
incubated in 1 ml saliva, and samples were collected and analyzed at 1 h, 3 h, 6 h, 8 h, 15 h, and
24 h.

C. Polyacrylamide Gel Electrophoresis

The saliva-amelogenin incubated samples were analyzed with SDS-PAGE (16% polyacrylamide
gels, 90 V for 15 min followed by 150 V until completion). Positive control of recombinant full
length porcine amelogenin (rP172) and negative control of human saliva (diluted 4 times) were
used. The gel was stained with Coomassie blue and silver stain.

D. Immunochemistry (Western Blot)

Presence of amelogenin was confirmed in saliva-amelogenin samples incubated for various
time intervals using Western Blot. After electrophoresis as above, the gel was transferred to
PVDF membrane (0.2 µm, Immun-Blot™ PVDF membrane for protein blotting, BioRad) at 120 V
for 60 min on ice. After the transfer, the gel was stained with Coomassie blue and silver stain to
ensure complete transfer had occurred. The membrane was blocked for 2 h in 5% non-fat milk
23

in PBST (0.1% (v/v) Tween 20 in 1x Phosphate Buffered Saline or PBS), then washed and
incubated with anti-full length mouse amelogenin primary antibody (rM179, 1:1000, rabbit-
polyclonal, in 5% non-fat milk in PBST) at 4°C overnight (~12-14 h). The membrane was rinsed
(PBST, 3 times, 15 min each) and incubated for 4 h in secondary antibody (1:4000, HRP
conjugated goat anti-rabbit, in 5% non-fat milk in PBST, BioRad). The results were visualized
using chemiluminescence (Amersham™ ECL™ Western Blotting detection reagents, GE
Healthcare).

24

Results

A. High Performance Liquid Chromatography

To determine whether saliva and amelogenin incubated together can be analyzed using
reversed phase HPLC, first individual controls of each were tested. Figure 9 shows the HPLC
spectra of amelogenin and saliva controls. The peak for rP172 was obtained at 53.051 minutes
(Figure 9 B) and there was no coinciding peak in saliva at the same time (Figure 9 A).
After incubation for 1 hour, rP172 was observed to be stable in saliva, giving a strong peak in
the HPLC chromatogram. Figure 10 compares the 1 h amelogenin-saliva sample (Figure 10 A)
with the rP172 control (Figure 10 B) and saliva control (Figure 10 C).

Figure 9. Reversed phase HPLC spectra of: A- Saliva and B- amelogenin controls to determine absence of
coinciding peaks
25

Figure 10. Reversed phase HPLC spectrum of amelogenin and saliva incubated together for 1 h shows a
strong amelogenin peak at 53.038 min (A). B- Saliva control. C- Amelogenin control.

The peak obtained at the end of 3 h incubation of amelogenin in saliva was comparable to both
that of the rP172 control and the 1 h peak (Figure 11 A and B). The first significant reduction in
peak height was present after 6 h (Figure 11 C).
From 6 h onwards the amelogenin peaks started depleting gradually, as could be seen in the 8,
15 and 24 h samples. By the end of the 24 h, all the amelogenin was lost (Figure 12 A, B, and C
respectively).
It can also be seen that salivary proteins themselves also degrade by the end of 24 h at 37°C
(Figure 12 C).

26

Figure 11. Reversed phase HPLC spectra show presence of amelogenin after 3 h (53.167 min peak in B)
and 6 h (53.589 min peak in C). A- Amelogenin and saliva incubated for 1 h for comparison.
27

Figure 12. Reversed phase HPLC spectra showing amelogenin completely degraded at the end of A- 8 h,
B- 15 h, and C- 24 h. C also shows degradation of saliva components.

28

B. Polyacrylamide Gel Electrophoresis

Figure 13 shows an SDS-PAGE gel after silver staining. Lane 2 and 3 are the rP172 and saliva
controls, respectively. Saliva and amelogenin incubated for 1 h, 3 h, 6 h, 8 h, 15 h, and 24 h are
in lane 4 onwards. The amelogenin-saliva incubated samples showed a band similar in
molecular weight to that of full-length amelogenin which was absent in the saliva control.

Figure 13. Silver-stained polyacrylamide
gel (16%) showing comparison between
rP172 (lane 2, red arrow), fresh saliva
(lane 3), and saliva with amelogenin
incubated for various time intervals
(lanes 4-9)

29

C. Immunochemistry (Western Blot)
There was a clear band in the saliva-amelogenin incubated for 1 h but only a faint band in the
sample incubated for 3 h (Figure 14), which confirmed that amelogenin is stable in saliva up to
3 h and starts degrading between 3-6 h.

Figure 14. Western blot
showing that rP172 is
stable in saliva incubated
for 1 h and 3 h (red
arrows)

30

Discussion
Treating etched enamel with CS-AMEL hydrogel in the presence of saliva showed that
interaction of CS-AMEL hydrogel with saliva does not create a favorable environment in which
well-oriented hydroxyapatite crystals can grow. One possible reason there were no crystals
deposited on the samples in saliva may be that salivary amylase and lysozyme digested chitosan
(Tomihata & Ikada, 1997) and left some degradation products on the surface of the teeth which
prevented or inhibited the deposition of calcium-phosphate crystals. The degradation of
amelogenin that we observed after 3 h of incubation in human saliva may be due to Kallikreins
and matrix metalloproteinases present in saliva, which cleave amelogenin (Chaussain-Miller, et
al., 2006; Buzalaf, et al., 2015).
Based on these results, use of custom-made trays for patients will achieve a two-fold purpose:
1) they will help to hold the hydrogel in place on the white spot or non-carious lesions being
treated and 2) they will prevent a direct interaction between saliva and amelogenin, thereby
allowing the protein to act on the developing crystals. We have shown that CS-AMEL hydrogel is
stable and active at an acidic pH (~4.6); therefore, it will maintain its activity at night (pH ~6.5).
Thus, by wearing the hydrogel-containing trays at night, the hydrogel will act on teeth for about
6-8 h during sleep.
The ability of the hydrogel to remineralize white spot lesions from within, as well as to
remineralize erosion-like lesions, provides a wide array of applications as a dental product.
Apart from treating superficial lesions, there might be possible uses of the hydrogel as a
treatment for larger cavities. Upon treating dentin with CS-AMEL hydrogel, the dentinal tubules
were completely blocked with hydroxyapatite crystals and well-oriented crystals were
deposited on dentin surface as well (Mukherjee et al., in preparation). This suggests an
opportunity for the hydrogel to be used to treat dentinal sensitivity. Instead of superficially
occluding the tubules like many sensitivity treatments, the hydrogel would penetrate deep
within tubules and seal them, while simultaneously creating an enamel-like layer on the surface
of the exposed dentin.
One drawback of this study was that salivary proteins, being such a vast topic in themselves,
were not analyzed. As interaction of amelogenin and saliva does not occur in the oral cavity
naturally, which salivary enzymes degrade amelogenin is not known. During development of
teeth, amelogenin is degraded first by MMP-20 and then by KLK-4 into various fragments that
may fulfill different functions (Bartlett & Simmer, 1999). It would be fascinating to know if
salivary proteinases cut amelogenin in similar places, thereby making small peptides that could
have similar functions to those present during the development of teeth.
Enamel biomimetics is a rapidly developing area of dentistry as well as material science.
Products containing fluoride or casein phosphopeptide-amorphous calcium phosphate (CPP-
ACP) have been successful to some extent in remineralizing enamel. Most notably, building a
new layer of enamel-like hydroxyapatite crystals which are tightly bound to the underlying
surface is a unique characteristic of CS-AMEL hydrogel treatment.

31

Conclusion
The results of the experiments presented here show that CS-AMEL hydrogel is stable and active
over a wide range of pH values from 4.6 to 7.0. The hydrogel can grow enamel like
hydroxyapatite crystals in this pH range on the surface of teeth and also can remineralize
incipient caries-like lesions from within. This ability of the hydrogel shows that it can penetrate
the superficial, more porous layer of enamel and develop crystals from within. Separate studies
have been conducted to compare the hardness of newly grown enamel crystals with natural
enamel and the results have been very encouraging (Mukherjee et al., in preparation).
Although the chitosan-amelogenin hydrogel did not act efficiently in the presence of human
saliva, amelogenin itself was stable in saliva for up to 6 hours. Therefore, it is necessary to
prevent contact between saliva and CS-AMEL hydrogel as much as possible. As our final aim is
to develop an at-home treatment for white spot lesions, isolation of teeth and application in
dental offices was not considered. Instead, application of hydrogel using custom-made arch-
fitting flexible trays may be used to achieve the desired isolation from saliva and prolonged
contact with the tooth surface.
The trays would be similar to those provided with tooth whitening systems and the hydrogel
would be placed in a reservoir for the tooth/teeth to be treated. Patients would be asked to
insert the tray with the hydrogel at night, before bed time, after brushing the teeth and rinsing
with a fluoridated mouthwash. The treatment would continue for 5-7 days.
This system would benefit from the low saliva flow at night as well as create a physical barrier
between the saliva and CS-AMEL hydrogel. The only oral fluid the hydrogel would encounter
would be a small amount of gingival crevicular fluid. Wearing the tray while sleeping would
provide conditions very similar to the experimental conditions tested here and allow the gel to
be in contact with enamel continuously for 6-8 h.
Before moving on into further clinical trials, more studies need to be conducted to elucidate the
interaction between the hydrogel and soft tissues, such as the gingiva and oral mucosa, with
which the hydrogel may come into contact. Chitosan is derived from crustacean cells, so the
possibility of allergic reactions (like shellfish allergy) needs to be taken into consideration. CS-
AMEL hydrogel provides a stepping stone towards rebuilding natural hard tissues in the human
body.

32

Bibliography

Amado, F. M. L. et al., 2005. Analysis of the human saliva proteome. Expert Review of Proteomics, 2(4),
pp. 521-539.
Arnaud, T. M. S., de Barros Neto, B. & Diniz, F. B., 2010. Chitosan effect on dental enamel de-
remineralization: An in vitro evaluation.. Journal of dentistry, 38(11), pp. 848-852.
Bartlett, J. & Simmer, J., 1999. Proteinases in developing dental enamel. Critical Reviews in Oral Biology
& Medicine, 10(4), pp. 425-441.
Bishara, S. E. & Ostby, A. W., 2008. White Spot Lesions: Formation, Prevention, and Treatment. Seminars
in Orthodontics, 14(3), pp. 174-182.
Buzalaf, A. M., C. S. & L, T., 2015. Role of host-derived proteinases in dentine caries and erosion. Caries
Research, 49(1), pp. 30-37.
Buzalaf, M. A. R. et al., 2010. pH-cycling models for in vitro evaluation of the efficacy of fluoridated
dentifrices for caries control: strengths and limitations. Journal of Applied Oral Sciences, 18(4), pp. 316-
334.
Chaussain-Miller, C., Fioretti, F., Goldberg, M. & Menashi, S., 2006. The Role of Matrix
Metalloproteinases (MMPs) in Human Caries. Journal of Dental Research, 85(1), pp. 22-32.
Chaussain-Miller, C., Fioretti, F., Goldberg, M. & Menashi, S., 2006. The Role of Matrix
Metalloproteinases (MMPs) in Human Caries. Journal of Dental Research, 85(1), pp. 22-32.
Dawes, C., 2003. What is the critical pH and why does a tooth dissolve in acid?. Journal-Canadian Dental
Association, 69(11), pp. 722-725.
Du, M. et al., 2012. Randomized controlled trial on fluoride varnish application for treatment of white
spot lesion after fixed orthodontic treatment. Clinical oral investigations , 16(2), pp. 463-468.
Fábián, T. K., Fejérdy, P. & Csermely, P., 2008. Saliva in Health and Disease, Chemical Biology of.. Wiley
Encyclopedia of Chemical Biology.
Fan, D. et al., 2009. In vitro study on the interaction between the 32kDa enamelin and amelogenin.
Journal of Structural Biology, 166(1), pp. 88-94.
Fan, Y., Sun, Z. & Moradian-Oldak, J., 2009. Controlled remineralization of enamel in the presence of
amelogenin and fluoride. Biomaterials, February, 30(4), pp. 478-483.
García-Godoy, F. & Hicks, J. M., 2008. Maintaining the integrity of the enamel surface: The role of dental
biofilm, saliva and preventive agents in enamel demineralization and remineralization. Journal of
American Dental Association, 139(suppl 2), pp. 25S-34S.
Griffin, T. J., 2014. Human saliva proteome: an overview. Proc. SPIE 9112, Sensing Technologies for
Global Health, Military Medicine, and Environmental Monitoring, 5 June.4(91120B).
33

Grippo, J. O., Simring, M. & Schreiner, S., 2004. Attrition, abrasion, corrosion and abfraction revisited: A
new perspective on tooth surface lesions. The Journal of the American Dental Association, August,
135(8), pp. 1109-1118.
Humphrey, S. P. & Williamson, R. T., 2001. A review of saliva: Normal composition, flow, and function.
The Journal of Prosthetic Dentistry, 85(2), pp. 162-169.
Ingle, N. A., Sirohi, R., Kaur, N. & Siwach, A., 2014. Salivary fluoride levels after toothbrushing with
dentifrices containing different concentrations of fluoride. Journal of International Society of Preventive
and Community Dentistry, 4(2), pp. 129-132.
Kidd, E. A. M. & Fejerskov, O., 2004. What Constitutes Dental Caries? Histopathology of Carious Enamel
and Dentin Related to the Action of Cariogenic Biofilms. Journal of Dental Research, Volume 83, pp. C35-
C38.
Lee, H.-S.et al., 2012. Chitosan adsorption on hydroxyapatite and its role in preventing acid erosion.
Journal of Colloid and Interface Science, November, 385(1), pp. 235-243.
Lee, H.-S.et al., 2012. Chitosan adsorption on hydroxyapatite and its role in preventing acid erosion.
Journal of Colloid and Interface Science, November, 385(1), pp. 235-243.
Meurman, J. H. et al., 1987. Salivary pH and Glucose after Consuming Various Beverages, Including
Sugar-Containing Drinks. Caries Research, 21(1).
Moradian-Oldak, J., 2012. Protein- mediated enamel mineralization. Frontiers in Bioscience, Volume 17,
pp. 1996-2023.
Moradian-Oldak, J., 2012. Protein- mediated enamel mineralization. Frontiers in bioscience : a journal
and virtual library, June, Volume 17, pp. 1996-2023.
Moradian-Oldak, J. & Paine, M. L., 2008. Mammalian Enamel Formation. In: A. Sigel, H. Sigel & R. K. O.
Sigel, eds. Metal Ions in Life Science. s.l.:John Wiley & Sons, Ltd, pp. 507-546.
Nanci, A., 2013. Ten Cate's Oral Histology: Development, Structure and Function. 8th ed. St. Loise:
Elsevier Mosby.
Øgaard, B., 1989. Prevalence of white spot lesions in 19-near-olds: A study on untreated and
orthodontically treated persons 5 years after treatment. American Journal of Orthodontics and
Dentofacial Orthopedics, November, 96(5), pp. 423-427.
Øgaard, B., Rølla, G. & & Arends, J., 1988. Orthodontic appliances and enamel demineralization: Part 1.
Lesion development. American Journal of Orthodontics and Dentofacial Orthopedics, 94(1), pp. 68-73.
Ruan, Q. et al., 2015. Efficacy of amelogenin-chitosan hydrogel in biomimetic repair of human enamel in
pH-cycling systems. Journal of Biomedical Engineering and Informatics, 2(1), pp. 119-128.
Ruan, Q. & Moradian-Oldak, J., 2014. Development of amelogenin-chitosan hydrogel for in vitro enamel
regrowth with a dense interface.. Journal of visualized experiments: JoVE, Volume 89.
Ruan, Q. & Moradian-Oldak, J., 2015. Amelogenin and enamel biomimetics. Journal of Materials
Chemistry, B 3(16), pp. 3112-3129.
34

Ruan, Q. et al., 2013. An amelogenin–chitosan matrix promotes assembly of an enamel-like layer with a
dense interface. Acta Biomaterialia, July, 9(7), pp. 7289-7297.
Ruan, Q. et al., 2013. An Amelogenin–Chitosan Matrix Promotes Assembly of an Enamel-Like Layer with
a Dense Interface. Acta Biomaterialia, 9(7), p. 7289–7297.
Ryu, O. et al., 1999. Characterization of Recombinant Pig Enamelysin Activity and Cleavage of
Recombinant Pig and Mouse Amelogenins. Journal of Dental Research, Volume 78, pp. 743-750.
Schulz, B. L., Cooper-White, J. & & Punyadeera, C. K., 2013. Saliva proteome research: current status and
future outlook. Critical Reviews in Biotechnology,, 33(3), pp. 256-259.
Tomihata, K. & Ikada, Y., 1997. In vitro and in vivo degradation of films of chitin and its deacetylated
derivatives. Biomaterials, 18(7), pp. 567-575.
Travess, H., Roberts-Harry, D. & & Sandy, J., 2004. Orthodontics. Part 6: Risks in orthodontic treatment.
British Dental Journal, 196(2), pp. 71-77.
Yang, X. et al., 2011. Amelogenin “nanorods” formation during proteolysis by Mmp-20. Journal of
structural biology, 176(2), pp. 220-228.

Abstract (if available)

Abstract Enamel, the outermost layer of teeth, is unique in many ways. Mature enamel is completely acellular, hence incapable of repair

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Asset Metadata

Creator Bapat, Rucha Arun (author)

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

School School of Dentistry

Degree Master of Science

Degree Program Craniofacial Biology

Publication Date 02/17/2016

Defense Date 01/11/2016

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

Tag amelogenin,biomimetic,chitosan,enamel,enamel regrowth,OAI-PMH Harvest

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Advisor Moradian-Oldak, Janet (committee chair), Paine, Michael L. (committee member), Phark, Jin-Ho (committee member)

Creator Email ruchabap@usc.edu,ruchbapat@gmail.com

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