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Role of MMP-20 in preventing protein occlusion in enamel apatite crystals: relevance in enamel biomineralization and biomimetics

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Role of MMP-20 in preventing protein occlusion in enamel apatite crystals: relevance in enamel biomineralization and biomimetics

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Content 1

Role of MMP-20 in preventing protein occlusion in enamel apatite
crystals: Relevance in Enamel Biomineralization and Biomimetics
By
Saumya Prajapati

A dissertation presented to the faculty of the Graduate School University of Southern California
In partial fulfillment of the requirements for the degree
DOCTOR OF PHILOSOPHY
(Craniofacial Biology)
August 2016

2

Table of Contents

Dedication ....................................................................................................................... 4
Acknowledgments ........................................................................................................ 5
List of Tables .................................................................................................................. 6
List of Figures ................................................................................................................ 7
Abbreviations .............................................................................................................. 10
Abstract ......................................................................................................................... 12
Chapter 1: Introduction ........................................................................................... 15
1.1Biomineralization ............................................................................................................................... 15
1.2 Amelogenesis .................................................................................................................................... 16
1.3 Enamel Matrix proteins .................................................................................................................... 18
1.3.1 Amelogenin ................................................................................................................................ 18
1.3.2 Non-amelogenin enamel matrix proteins .................................................................................. 20
1.4 Enamel Proteases .............................................................................................................................. 22
1.4.1 Matrix Metalloproteinase-20 ..................................................................................................... 22
1.4.2 Kallikrein-4 ................................................................................................................................. 23
1.5 Mmp-20 null mouse .......................................................................................................................... 24
1.6 Amelogenesis Imperfecta .................................................................................................................. 26
1.7 Significance, objectives, and scope of the work ............................................................................... 27
Chapter 2: Analysis of proteins trapped inside isolated enamel crystals
.......................................................................................................................................... 30
2.1 Background and objectives: .............................................................................................................. 30
2.2 Results ............................................................................................................................................... 31
3

2.2.1 Phenotypic characteristics of Mmp-20 null mouse ................................................................... 31
2.2.2 Analysis of proteins associated with isolated enamel crystals .................................................. 32
2.2.3 Raman spectroscopy of secretory- and maturation-stage enamel crystals .............................. 37
2.2.4 In situ AFM force spectroscopy .................................................................................................. 38
2.3 Discussion ........................................................................................................................................ 39
2.4 Methods and materials: .................................................................................................................... 43
Chapter 3: Analysis of size and morphology of enamel crystals ................ 48
3.1 Background and objectives ............................................................................................................... 48
3.2 Results ............................................................................................................................................... 49
3.2.1 Morphology of enamel crystals of WT and Mmp-20 null mouse .............................................. 49
3.2.2 Width and thickness of enamel crystals of WT and Mmp-20 null mouse ................................. 50
3.2.3 Analysis of areas of imperfections in the Mmp-20 null enamel crystals ............................. 51
3.2.4 Crystallinity of Mmp-20 null enamel crystals ........................................................................ 53
3.3 Discussion ........................................................................................................................................ 55
3.4 Materials and methods ..................................................................................................................... 57
Chapter 4: In vitro growth of artificial enamel-like layer ............................. 60
4.1 Background and objectives ............................................................................................................... 60
4.2 Results ............................................................................................................................................... 62
4.2.1 Proteolysis of amelogenin rP172 by MMP-20 in the presence of 2% chitosan solution ........... 62
4.2.2 Enamel “re-growth” ................................................................................................................... 63
4.2.3 Characterization of the newly grown layer ................................................................................ 67
4.2.4 Mechanical testing ..................................................................................................................... 69
4.3 Discussion .......................................................................................................................................... 72
4.4 Materials and Methods ..................................................................................................................... 77
Chapter 5: Conclusion and future work .............................................................. 81
References .................................................................................................................... 86

4

Dedication

This dissertation is dedicated to my parents, Late Dr. Kamlesh Kumar and Suman Lata. My mother
became the sole bread earner for my family when my father passed away. She showed me the way to
be strong willed and never give up in the face of difficulties. My father was a kind-hearted and a very
generous doctor. I wish I had the chance to share my experiences and achievements with him. His life
will live on in me.

5

Acknowledgments

I would like to take this opportunity to thank Dr. Janet Moradian-Oldak for her guidance, expert advice
and immense amount of support during my entire doctoral training. I would also like to thank my
committee members; Dr. Michael Paine, Dr. Baruch Frenkel, Dr. Ian Haworth and Dr. Steven Nutt for
their expertise, guidance and advice.
I am immensely grateful to all my colleagues and friends in my laboratory; Dr. Victoria Gallon who
mentored me in the beginning of my doctoral training, Dr. B.C. Karthik and Dr. Parichita Mazumder for
sharing their biochemistry knowledge with me, Dr. Qichao Ruan, Dr. Kaushik M and Dr. Dongni Ren for
helping me with the in vitro experiments and Dr. Rucha Bapat for her support.
I would like to thank our collaborators Dr. James J DeYoreo and Dr. Jinhui Tao form Pacific Northwest
national Laboratory for their expertise in atomic force spectroscopy and Raman spectroscopy and Dr.
Steven Nutt, Dr. Yuzheng Zhang and Jon Lo from Mork Family Department of Chemical Engineering and
Materials Science at University of Southern California for their help with Fourier Transform Infrared
Spectroscopy (FTIR) and mechanical testing.
Special thanks to all the student volunteers Lianna Damargi, David Leiberman, and Esteban Chidez
and Mahsa Tehrani for their help and dedication to my project.

6

List of Tables

Table 1 Known cases of Amelogenesis Imperfecta

26
Table 2 Mass spectrometry results of porcine crystals dissolved
in 1M HCl.

33
Table 3 Ratios of intensities at I (002)/I (211) and I (002)/I (300) to
determine the crystallinity and length to thickness ratio
respectively.

68
Table 4 Ratios of absorbance of the PO 4 peaks at 600 and 1028
cm
-1
with amide III peak.

71

7

List of Figures

Figure 1 Amelogenin sequence for mouse, human and porcine showing the
conserved regions of the protein.
19
Figure 2 Amelogenin sequence showing the 20KDa C-terminally cleaved
P148 peptide, TRAP and the cleaved C-terminus.
19
Figure 3 Comparison of WT and Mmp-20 null mouse enamel. 31
Figure 4 UV-adsorption of proteins extracted from isolated porcine enamel
crystals.
32
Figure 5 SDS-PAGE and Western blot of proteins extracted from porcine
enamel crystals.
33
Figure 6 A set of control experiments (n=3) to show the trend of decrease
in the amount of adsorbed proteins on mouse enamel crystals
with each wash.
34
Figure 7 UV-adsorption of proteins extracted from isolated enamel crystals
of WT, Heterozygous and Mmp-20 null mouse.
35
Figure 8 SDS-PAGE and Western Blot of Wild Type and Mmp-20 null mouse
enamel crystals.
35
Figure 9 UV-adsorption and Western blot of proteins present in dissolved
(1M HCl) enamel crystals of WT and Mmp-20 null mouse from
secretory and maturation stages.
36
Figure 10 Raman Spectra of WT and Mmp-20 null mouse enamel crystals at
secretory and maturation stages.
37
Figure 11 In situ AFM images and force curves of WT and Mmp-20 null 38
8

mouse enamel crystals at secretory and maturation stages.
Figure 12 Scheme showing the methods and steps involved in the isolation
of enamel crystals from WT and Mmp-20 null mouse enamel.
43
Figure 13 Comparison of WT and Mmp-20 mouse enamel morphologies
using SEM and TEM.
50
Figure 14 Width and thickness of enamel crystals of Wild Type and Mmp-20
null mouse.
51
Figure 15 HRTEM of WT and Mmp-20 null mouse. 52
Figure 16 Analysis of areas of imperfections in enamel crystals of Mmp-20
null mouse.
53
Figure 17 Full width of half maximum (FWHM) of Raman peaks of WT and
Mmp-20 null mouse secretory and maturation stage enamel
crystals.
54
Figure 18 Reverse phase chromatography and SDS-PAGE showing the
amelogenin proteolysis products at various time intervals.
63
Figure 19 SEM images showing etched enamel, newly grown HAP crystals in
chitosan hydrogel, amelogenin-chitosan hydrogel and MMP-20
containing amelogenin-chitosan hydrogel.
64
Figure 20 SEM images showing direction of growth in the newly grown HAP
crystals in chitosan hydrogel, amelogenin-chitosan hydrogel and
MMP-20 containing amelogenin-chitosan hydrogel.
65
Figure 21 SEM images showing higher magnification of the newly grown HAP
crystals
66
Figure 22 XRD spectra of newly grown layer grown in chitosan hydrogel,
amelogenin-chitosan hydrogel and MMP-20 containing
67
9

amelogenin-chitosan hydrogel.
Figure 23 Calculation of FWHM using the peak obtained at 2Ɵ = 25.8 (002). 68
Figure 24 Graph showing modulus of elasticity for the newly grown crystals
and their comparison with healthy and etched enamel.
69
Figure 25 Graph showing hardness for the newly grown crystals and their
comparison with healthy and etched enamel.
70
Figure 26 FTIR spectra of the newly grown HAP layer in the chitosan-
hydrogel system.
71
Figure 27 Proposed model to show the effect of the absence of MMP-20 on
enamel HAP crystals in Mmp-20 null mouse
84

10

Abbreviations
25 KDa
amelogenin
Full length native amelogenin
ACP Amorphous calcium phosphate
AFM Atomic force microscopy
AI Amelogenesis Imperfecta
AMELX Amelogenin gene for humans on the X chromosome
C-terminal
amelogenin
Hydrophilic C-terminal tail of amelogenin
ECM Extracellular matrix
EMP Enamel matrix protein
FTIR Fourier transform infrared spectroscopy
FWHM Full width of half maximum
HAP Hydroxyapatite
HRTEM High resolution Transmission electron microscopy
KLK-4 Kallikrein-4
MMP-20 Matrix metalloprotease-20
OCP Octacalcium phosphate
P148 or “20K”
C-terminally cleaved amelogenin, 148 amino acids in
length and 20KDa in molecular weight
rhMMP-20 Recombinant human MMP-20
11

rP172 Recombinant full length pig amelogenin
SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel
electrophoresis
SEM Scanning electron microscopy
TRAP
Tyrosine rich amelogenin peptide, 1-45 of full length
amelogenin
WT Wild type mouse
XRD X-ray diffraction
12

Abstract

Introduction: Defects in enamel formation are an important topic of study in the world of dentistry and
biomineralization. The formation of tooth enamel is a typical example of cell- and matrix- mediated
biomineralization. Enamel is composed of a well-organized hierarchical structure of long apatite crystals
which are arranged in parallel in prisms or rod-like structures. Amelogenesis is a dynamic process
involving hydroxyapatite crystal nucleation and maturation concomitant with formation of an enamel
matrix of proteins like amelogenin, ameloblastin, enamelin, etc. and proteases like matrix
metalloprotease-20(MMP-20) and kallikrein-4 (KLK-4). These proteases degrade the enamel matrix
(EMP) proteins to create space for the hydroxyapatite crystals to grow in width and length. I designed a
series of in vivo experiments to study the effects of the absence of MMP-20 on HAP crystal formation
using Mmp-20 null mouse as my animal model. Using the information that I extracted from in vivo
experiments, I designed in vitro experiments to re-grow artificial enamel-like materials with improved
structural and mechanical properties.
Materials and methods: 6 month old pig third and fourth molars were used for the extraction of enamel
crystals and establishment of a protocol. Enamel crystals were isolated from adult mandibular and
maxillary incisors from wild type and Mmp-20 null mouse. The isolated enamel was washed with a series
of extraction buffers to wash off adsorbed proteins followed by their dissolution in 1M HCl. Qualitative
and quantitative analysis of the proteins associated with isolated enamel crystals of WT and Mmp-20
null mouse was done by using UV-adsorption, immunochemistry, Raman spectroscopy and in situ
Atomic force microscopy (AFM). The morphology and structural changes in the Mmp-20 null mouse as
compared to the WT mouse was studied by Scanning electron microscopy (SEM), high-resolution
Transmission electron microscopy (HRTEM) and Atomic force microscopy.
13

The in vitro experiments were performed on extracted human 3
rd
molar tooth slices. Recombinant
porcine amelogenin rP172 was dissolved in 2% chitosan followed by the addition of Na 2HPO 4 and CaCl 2
solution. The pH of the solution was adjusted to 6.5 with 1M NaOH. Recombinant human MMP-20
(rhMMP-20) was added to this solution at a ratio of 1:1000. The chitosan hydrogel alone was used as a
negative control and the solution containing rP172 only was used as positive control. 30µl of each of
these solutions were carefully applied to exposed enamel windows of the prepared tooth slices and air
dried for 15 minutes. The tooth slices were then immersed in 30 ml artificial saliva solution with a
fluoride (F
-
) concentration of 1ppm at 37°C for 5 days. After the allotted time, the tooth slices were
removed from the solution, sonicated in a water bath for 10 mins and air dried. Characterization of the
newly formed layer was done by SEM, X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy
(FTIR) and mechanical testing.
Results: UV-adsorption and immunochemistry showed that there was a significant difference in the
amount of proteins in the enamel of Mmp-20 null mouse as compared to that of the WT mouse. The
dissolved crystals in the Mmp-20 null mouse showed the presence of full-length amelogenin which
suggests that in the absence of MMP-20; full-length amelogenin gets trapped inside the hydroxyapatite
crystals during the early stages of amelogenesis. SEM and AFM showed an absence in the decussating
pattern of enamel and a decrease in the length of enamel crystals in the Mmp-20 null mouse as
compared to that of the WT mouse. HRTEM showed a significant difference (p<0.05) in the diffraction
pattern of the hydroxyapatite crystals when compared to the WT mouse suggesting the presence of
organic matter (protein) inside the crystal. The in vitro experiments showed the formation of an apatite
layer with long thin needle-like crystals which were well oriented in a direction parallel to the underlying
enamel rods. The newly formed layer was found to be hydroxyapatite (HAP) by XRD. The MMP-20
containing amelogenin-chitosan hydrogel showed a statistically significant improvement in the
14

mechanical properties as compared to the controls. FTIR results showed a decrease in the amount of
proteins associated with the newly formed layer in the MMP-20 containing samples.
Conclusions: The proteolysis of enamel matrix proteins by MMP-20 prevents the occlusion of
amelogenin inside the HAP crystals during the secretory stage of amelogenesis. Presence of full length
amelogenin inside the HAP crystals cause various morphological and structural defects in the crystals as
shown in the enamel crystals of Mmp-20 null mouse. The introduction of MMP-20 in the in vitro
experiment showed the formation of an enamel-like layer with improved orientation, growth and
mechanical properties. We suggest that MMP-20 improves the HAP crystal formation by preventing
occlusion of amelogenin inside the crystals during the early stage of its formation. These strategies can
be used in the future for the formation of enamel-like restorative materials for the treatment of dental
enamel defects.

15

Chapter 1: Introduction
Dental enamel is a unique example of a bioceramic with a complex hierarchical microstructure which
provides remarkable mechanical properties. Enamel matrix proteins and proteases play specific roles in
the process of amelogenesis to form enamel. Biomimetic strategies have been tried previously to
restore enamel structure in vitro [1-5]. Biomimetics requires understanding of biological and chemical
principles involving protein secretion and assembly, proteolytic degradation of enamel matrix proteins,
nucleation and growth of hydroxyapatite (HAP) crystals. Matrix metalloprotease-20 (MMP-20) is a
proteinase which is secreted during the early stages of amelogenesis and is involved in the timely
proteolysis of enamel matrix proteins [6]. The aim of the present study was to understand the
mechanism by which MMP-20 affects HAP crystal formation during amelogenesis and use this
knowledge to develop strategies for “re-growing” a biomimetic enamel layer on human tooth sections.
1.1 Biomineralization

Biological materials such as bone, teeth, and shells are examples of how the process of biomineralization
can form composite materials which have an organic matrix reinforced by an inorganic phase [7]. The
inorganic minerals are highly organized, with uniform crystal size, morphology and crystallographic
orientation. The process of mineralization in biological organisms is believed to be achieved by
modulating the growth of mineral phases [8]. This occurs when macromolecules in solution direct crystal
morphology by adsorbing to specific crystal faces. The process can be inhibited by the attachment of
certain macromolecule additives, which occupy the potential adsorption sites [9, 10]. In an in vitro study
it was observed that a phosphorylated peptide of osteopontin (OPN) inhibits the growth of calcium
oxalate monohydrate, which shows that peptides with higher adsorption energies are more likely to
become incorporated into a growing crystal face and inhibit crystal growth in a certain direction
[11].
16

Biomineralization can be divided into two fundamentally different classes. Biologically induced
mineralization involves precipitation of minerals due to secondary interactions between the metabolic
process of the organism and its environment, such as bacteria which contain minerals like iron and
manganese oxides. In biologically controlled mineralization, the organism has direct control over the
process and has evolved specific strategies to regulate mineral deposition, nucleation, morphology and
growth. Typical examples include bones, shells and teeth. In this study we are focusing solely on tooth
enamel, its formation and biological regulation by enamel matrix proteins and proteases [12].
Dental enamel is the outermost layer of the tooth and the hardest structure in the human body. The
enamel is composed of a well-organized hierarchical structure of long hydroxyapatite (HAP) crystals.
These crystals are arranged in parallel prisms or rod-like structures. Enamel formation involves
secretion, assembly and degradation of enamel matrix proteins to form a hardened structure which is
composed of approximately 95-97% mineral [13, 14]. Amelogenin is an important component of the
enamel matrix and plays a pivotal role in normal enamel formation [6, 15]. In an in vitro study it was
shown that, in the presence of amelogenin, calcium phosphate clusters stabilize and form nanospheres
and nanochains of Amel-Ca-P to form Amel-ACP (amorphous calcium phosphate) nanorods. These
nanorods elongate to form HAP crystals [16]. Amelogenin has been shown to be involved in the early
nucleation process in vitro [17].
1.2 Amelogenesis
Enamel formation is a highly orchestrated process that involves degradation of enamel matrix proteins
and precise cell signaling mechanisms. It can be broken down into 4 stages—the presecretory, secretory,
transitional, and maturation stages—according to the morphology and function of enamel-forming cells
called ameloblasts [15].
17

During the presecretory stage, odontoblasts (dentin-forming cells) lay down predentin just under the
future dentino-enamel junction (DEJ), which is the first area to mineralize. Immediately after the
formation of predentin, pre-ameloblasts differentiate into ameloblasts and begin secreting enamel
matrix proteins to initiate enamel formation [18].
The secretory stage involves secretion of copious amounts of an organic matrix which consists of
proteins including amelogenin, enamelin and ameloblastin along with proteases like matrix
metalloprotease-20 (MMP-20). This stage also involves movement of ameloblasts away from the dentin
surface so that the new enamel layer can thicken [19]. Long, thin mineral ribbons start forming at this
stage. These crystallite ribbons are arranged in parallel and eventually come together to form a rod;
each ameloblast is responsible for creating a single rod. Once the Tomes’ processes are formed by the
ameloblasts, a two-compartment system which helps in the formation of rod and interrod enamel is
formed. MMP-20 is secreted during this stage to degrade the enamel matrix proteins. Protein cleavage
products are either resorbed by the ameloblasts or deposited between the rod and interrod enamel.
The ameloblasts also move in groups so that they slide by one another, which creates the characteristic
decussating (crisscross pattern of rods and interrods in rodent enamel) pattern observed in rodents[20]
or the gnarled prism pattern seen in humans. By the end of the secretory stage, enamel has achieved its
full thickness, but it doesn’t harden until the end of the maturation stage [15].
During the transition stage, which is a short period between the secretory and maturation stages, the
ameloblasts retract their Tomes’ processes and form a final coating of aprismatic enamel.
The maturation stage involves the ameloblasts becoming shorter and broader, and they modulate
between smooth- and ruffle-ended cell types at the enamel surface [21]. Kallikrein-4 (KLK-4), a serine
protease, is actively secreted during this stage, which removes all the previously cleaved proteins and
degraded peptides so the enamel crystallites can now expand in width and thickness.
18

Mature enamel consists primarily of HAP crystals, which constitute about 95-97% of enamel by weight.
The rest is occupied by residual enamel matrix proteins and water. The HAP crystals are organized into
prisms, which are essentially rods 3-6 µm in diameter running from the surface of the enamel to the DEJ
[6]. The interface between the rod and interrod regions is occupied by residual enamel matrix proteins
and water, and is termed the rod sheath. The rods and interrods are arranged such that they form a
decussating pattern in rodents. In humans, these rods and interrods are arranged in a keyhole structure
with interprismatic enamel between them. The mature enamel has excellent mechanical properties with
a hardness of 4.5 GPa and toughness of 2 MPa m
1/21
[22]. In my thesis I will analyze mouse enamel
crystals from both secretory and maturation stages.
1.3 Enamel Matrix proteins
1.3.1 Amelogenin
Amelogenin has emerged as an important protein in the matrix-mediated biomineralization of dental
enamel. Out of the extracellular proteins secreted during the secretory stage, which control crystal
nucleation and organization, 90% are amelogenin. In humans, AMEL sequences are located on the distal
short arm of the X chromosome in the p22.1 → p22.3 region and near the centromere on the Y
chromosome, possibly in the proximal long arm (Yq11) region. However, in the mouse this gene is
located solely on the X chromosome [23].
Amelogenin’s primary structure can be divided into three domains (Figure 1 and 2), each of which plays
a pivotal role in enamel formation [24]. The N-terminal domain is 45 amino acids in length, has high
tyrosine content, and is commonly known as the tyrosine-rich amelogenin peptide (TRAP). This region is
involved in the self-assembly of amelogenin, as shown in in vitro and in vivo studies [25, 26]. It also
contains a lectin binding motif which may be responsible for nanosphere assembly [27, 28]. The second
domain is the central region of the protein and is the largest segment. It is enriched in the amino acids
19

proline, leucine, and glutamine, and is generally hydrophobic [29]. The C-terminal forms the third
domain, which is highly charged and hydrophilic, and may be responsible for calcium binding and
nucleation of HAP [30]. Amelogenin’s primary structure at both the N- and C- termini is highly conserved
among mammals. It is known that during the secretory stage, full-length porcine amelogenin (P173 or
“25K”) is cleaved by MMP-20 at the C-terminus to produce the “20K” amelogenin (P148). This is
followed by cleavage at the N-terminus by KLK-4 during the maturation stage [31]. Studies on
amelogenin knockout mouse showed that the mutants’ enamel is thin, chalky white in appearance, and
lacks the organized prismatic structure of the wild type [32]. Another in vivo study showed that
Figure 1. Amelogenin sequence for mouse, human and porcine showing the conserved regions of
the protein. Purple and red arrows show the cleavage site for MMP-20 at the C and N-terminus
respectively
MPLPPHPGHPGYINFSYEVLTPLKWYQNMIRHPYTSYGYEPMGGWL
HHQIIPVVSQQTPQSHALQPHHHIPMVPAQQPGIPQQPMMPLPGQ
HSMTPTQHHQPNLPLPAQQPFQPQPVQPQPHQPLQPQSPMHPIQP
LLPQPPLPPMFSMQSLLPDLPLEAWPATDKTKREEVD
C-terminus
Figure 2. Porcine amelogenin sequence showing the 20KDa C-terminally cleaved P148 (black
font) peptide and the cleaved C-terminus (red font). The first 45 amino acids from the N-
terminal region constitute TRAP region of amelogenin.
N-terminus, TRAP region
20

amelogenin knockout mouse enamel was rescued by transgenic M180 mouse amelogenin, which
strongly supports the idea that the alteration in crystallite growth is a direct result of amelogenin
protein loss [33].
Evidence suggests that amelogenin facilitates the formation of hydroxyapatite by self-assembly and
formation of nanospheres in the developing dental enamel. One study indicates that amelogenin
controls prenucleation cluster aggregation during the earliest stages of Ca-P mineralization [34].
Hydrophobic portions of amelogenin affect crystal formation by regulating the arrangement of crystals
into parallel arrays [35]. All these studies show that amelogenin is an important matrix protein and plays
a major role in enabling enamel HAP crystals to grow in an organized manner. However, amelogenin
does not seem to be the only factor important for crystal nucleation, because both human and mouse
studies show that enamel formation does occur in the absence of amelogenin [36, 37].
1.3.2 Non-amelogenin enamel matrix proteins
Ameloblastin is the second most abundant enamel matrix protein which is highly expressed during the
secretory stage but not during the maturation stage [38]. It also localizes near the cell surface rather
than deep in the enamel matrix layer [39]. We recently showed that the ameloblastin N-terminal and
the amelogenin N-terminal (TRAP) co-localize and interact around the rods, and that the rod sheath
possibly plays a role in the maintenance of rod-interrod structure (In press J DENT
RES 0022034516645389). Studies of ameloblastin mutant mouse have shown that ameloblastin is
involved in the regulation of adhesion, proliferation and differentiation of ameloblasts during enamel
development [40]. Ameloblastin has also been shown to be involved in mineralization of enamel by
providing Ca-binding sites at its C-terminal. Enamel formation completely shuts down in ameloblastin
mutant mouse. A thin calcified material is secreted instead of enamel [40]. Ameloblastin and
amelogenin have synergistic roles. An in vivo study showed that Amelx
-/-
/Ambn
-/-
mouse have additional
21

enamel defects which are not seen in either Amelx
-/-
or Ambn
-/-
mouse [41]. In another co-localization
study we showed that amelogenin and ameloblastin show strong overlap with each other during the
secretory stage of amelogenesis in postnatal day 1 mouse [42].
Enamelin is another important enamel matrix protein comprising about 1-2% of total enamel proteins. It
is secreted as a 186-kDa acidic phosphorylated glycoprotein, which quickly degrades into short-lived
cleavage products [43, 44]. The 32-kDa cleavage product is the only stable product that is known to
accumulate deep in the enamel and have a specific role in enamel formation [45]. Enamelin is thought
to be crucially involved in the formation of the mineralization front and promotion of extension of
enamel crystallites. Enamelin knockout mouse clearly illustrate the importance of enamelin during
normal enamel development [46]. Enamel is virtually absent in these mice, which also display severe
dentin abrasion. The teeth are covered with a layer of calcified material which is not enamel.
Thus, though enamelin is not very abundant, it is the largest enamel matrix protein and plays a critical
role at the mineralization front during enamel formation.
Odontogenic ameloblast-associated protein (ODAM) is a calcium-binding phosphoprotein expressed by
maturation-stage ameloblasts and gingival cells that form the junctional epithelium (JE). It plays a role in
in the adherence of junctional epithelium to the tooth and maintenance of integrity of the JE. ODAM
knockout mouse showed that its absence has no major effect on enamel. However, older animals
showed signs of periodontal disease. ODAM may affect the local inflammatory activity of the
periodontium of the mutant animal [47].
Amelotin (AMTN) is another enamel matrix protein which is secreted predominantly during the
maturation stage of amelogenesis. The function of this protein is not well understood. Mutations in
AMTN gene have not yet been shown to cause Amelogenesis Imperfecta (AI) in human. However, Amtn
knockout mouse show a hypomineralization/hypomaturation phenotype which is most pronounced at
22

the surface enamel. This suggests that AMTN is mainly related to the dense aprismatic surface enamel,
and may a role in biofilm formation [48]. It may also have some effect on the JE due to its interaction
with ODAM in the basal lamina of mature ameloblats [49]. A recent study showed that AMTN is a potent
promoter of calcium phosphate mineralization and is a key player in the formation of the aprismatic
surface enamel [50].
1.4 Enamel Proteases
Protease function during enamel formation is crucial for amelogenesis. By the end of the maturation
stage, protein content in enamel matrix has been reduced to 1-2% [15, 51]. In porcine enamel, the
protein weight at the maturation stage is only 2% as compared to 30% at the secretory stage. Thus,
intact proteins are cleaved and removed during various stages of amelogenesis, allowing the enamel
layer to achieve a high degree of mineralization. Two main proteases are secreted during amelogenesis:
MMP-20 and KLK-4.
1.4.1 Matrix Metalloproteinase-20
Matrix metalloproteinases (MMPs) are a class of enzymes that are involved in many physiological
processes such as angiogenesis, bone remodeling, and organ morphogenesis, and in disease processes
such as arthritis, atherosclerosis, cardiovascular diseases, and many more [52-54]. These enzymes are
responsible for extracellular matrix degradation.
MMP-20 has a similar structure to the other MMPs. It contains three domains, which are conserved in
all the MMPs: the prodomain, the catalytic domain, and the hemopexin sequence, which is conserved in
all family members [55]. In addition to these domains, it also contains a signal peptide and a hinge
region.
Similar to other MMPs, MMP-20 is secreted as a proenzyme and is activated by the cleavage of its
propeptide. In MMP-20 the active peptide starts at Tyr108 and has a calculated mass of 42.6 kDa. MMP-
23

20 also contains a conserved Met244, like other MMPs. It is not yet clear what role this plays in the
action of MMP-20, but it is speculated that it may be responsible for structural integrity of the zinc
binding site, similar to its role in MMP-1[55, 56].
MMP-20 is a tooth-specific protease that is secreted during the secretory stage of amelogenesis [57]. It
has been suggested that it cleaves the enamel matrix proteins to create space for the forming enamel
crystals to grow in length [15]. Processing of amelogenin is important for its function, as shown by
several studies. MMP-20 cleaves the less-abundant proteins ameloblastin and enamelin to produce
cleavage products that accumulate in the enamel subsurface layer [45, 58]. MMP-20 has a broad
substrate specificity but high selectivity for hydrophobic residue at the P1’ position, and this is true of its
natural substrates, most of which contain leucine at this site. The P3 position also shows selectivity for
proline, which is also present in its natural substrate enamel matrix proteins [52]. MMP-20 is
responsible for cleaving the KLK-4 propeptide to activate the enzyme during the transition and
maturation stages of amelogenesis. However, this cannot be the only process that activates KLK-4
because KLK-4 is active in MMP-20 null mouse. MMP-20 is also expressed in odontoblasts, where it
cleaves dentin sialophosphoprotein along with other MMPs to form dentin.
1.4.2 Kallikrein-4
Kallikrein-4 is a serine protease that is secreted in the dental enamel matrix during the transition and
maturation stages of amelogenesis. KLK-4, like MMP-20, plays specific roles in enamel development,
even though KLK-4 was also shown to be expressed in other soft tissues such as the liver, prostate,
kidneys, submandibular glands, ovaries, testes, vas deferens, and epididymis. However, the expression
of KLK-4 in maturation stage ameloblasts was far stronger than any other tissue that was tested [59].
KLK-4 activation is essential for its own activity because the removal of its propeptide allows the
formation of a salt bridge between the new N-terminus and the side chain of Asp194 [16]. This is
24

believed to be achieved by the action of MMP-20 on KLK-4; however, again, MMP-20 cannot be the sole
activator because KLK-4 is active in MMP-20 null mouse [17]. Another study suggests that dipeptidyl
peptidase I (Cathepsin C, CTSC) activates KLK-4 in vitro and could possibly be one of its activator in vivo
[18]. KLK-4 activity during the transition and maturation stages facilitates exporting of small peptides out
of the enamel to allow apatite crystals to grow and harden. This occurs as the MMP-20 cleavage
products, such as TRAP, are further cleaved into smaller peptides [31] . Several studies indicate the role
of KLK-4 in this final stage of ameloblastin and enamelin cleavage [19]. Thus, KLK-4 cleaves all major
enamel matrix proteins into small peptides and creates further space for the growth of apatite crystals
[60].
1.5 Mmp-20 null mouse
The Mmp-20 null mouse was created to study the effects of Mmp-20 on enamel formation. Exon 5 of
Mmp-20 contains the highly conserved zinc binding site HEXGHXXGXXH, which is responsible for its
catalytic activity. [4]. The Mmp-20 null mouse was engineered by deleting the majority of exon 4 and 5,
which rendered the enzyme inactive. The resulting mouse had enamel which was hypoplastic and
hypomineralized, and had an altered enamel rod pattern; the enamel broke off from the dentin and had
deteriorating morphology. Phenotypically, the Mmp-20 null mouse showed a large number of enamel-
free areas in the maxillary molars when observed under SEM. These enamel-free areas were observed
near the cusps, which come into contact during mastication with food or the opposite molars. Their
enamel also delaminates from the underlying dentinal surface [4].
Unlike what was seen in Klk-4 knockout mouse, the Mmp-20 null mouse enamel fractured at the DEJ.
The Mmp-20 null mouse displayed plate-like mineral projecting upward at the fracture surfaces. These
projections made individual rooms or cells which were not aligned (unlike in Klk-4 knockout mouse) and
were uniform in size. The diameter of the holes observed on the fracture surface was less than 1 µm,
suggesting that they likely represented the ends of dentinal tubules. The overall picture resembled the
25

surface of mantle dentin. These observations confirmed that the fracture point in Mmp-20 null mouse
enamel was at the DEJ [4, 7, and 8].
The above findings for both the knockout mice can be explained based on the sequence in which the
proteases are secreted. The Mmp-20 null mouse showed a decrease in the enamel thickness (70 µm) as
compared to the wild type (120 µm). They also lacked the characteristic decussating pattern and had
uneven rod diameters. This confirmed that the pattern and thickness of enamel is established in the
secretory stage of amelogenesis and that Mmp-20 plays a key role in determining pattern and thickness
of enamel [4, 6]. A study using 1 mm strips of Mmp-20 null and Klk-4 knockout mouse enamel (from
both secretory and maturation stages) yielded the following observations: a) The Mmp-20 null mouse
had an overall 50% decrease in mineral content as compared to the wild type, b) the amount of proteins
and water content in Mmp-20 null mouse increased to almost twice that of the wild type, and c)
kallikrein-4 was not able to compensate for the loss of Mmp-20. It was suggested that the removal of
proteins and degradation products is critical for the crystallites to undergo maturational volumetric
expansion [61].
Mmp-20 null mouse show altered ameloblast morphology, with abnormal extension, retraction and
subsequent re-extension of their Tomes’ processes during enamel development [62]. Mmp-20 has been
shown to play a role in ameloblast cell signaling by cleaving the extracellular domains of cadherin (a part
of the adherens junction complex responsible for ameloblast cell-cell adhesion). This process removes β-
catenin and p120-catenin, which in turn are translocated to the nucleus where they promote cell
migration, invasion and proliferation.
Studies on Mmp-20 null mouse show the importance of proteolytic processing of enamel matrix
proteins and its role in normal enamel development.
26

1.6 Amelogenesis Imperfecta
Amelogenesis Imperfecta (AI) is a genetic disorder that affects the tooth enamel (refer to table 1 for all
the types of AI caused by mutations in major structural proteins and proteinases). It can be genetically
transmitted in autosomal dominant, autosomal recessive or X-linked fashions. AI can be subdivided into
three types: hypoplastic, hypomaturation, and hypocalcified. Hypoplastic AI presents with thin enamel
and defective matrix synthesis. Hypomaturation AI enamel is of normal thickness but it is soft and
typically stained. This is usually associated with a failure to remove proteins from the matrix during
amelogenesis. Hypocalcified AI enamel is soft and rough, and undergoes a lot of attrition. It is the most
severe form of AI and affects both the early and late stages of enamel development [63]. In humans,
seven different mutations in the MMP-20 gene are known to cause hypomaturation or hypoplastic AI.
Gene No of known mutations Type of AI
AMELX 18 X-linked AI[70-73]
ENAM 12 Autosomal recessive [74-79]
MMP-20 7 5- pigmented hypomaturation AI[64-67]
2- Hypoplastic hypomaturation AI[68, 69]
KLK-4 2 Hypoplastic hypomaturation AI[69, 80]
AMBN, AMTN and ODAM None found to date
Table 1. Known cases of Amelogenesis Imperfecta caused by mutations in genes coding for
different enamel matrix proteins and proteases.
27

People with these forms of AI have normal-sized teeth, but the enamel tends to chip away from the
dentin. One of the phenotypes is characterized by enamel roughness and a yellowish-brown
pigmentation when the tooth is erupting in the oral cavity [64-69]. In addition to the forms of AI
associated with mutations in MMP-20, two different mutations in KLK-4 also cause AI in humans. The
latter forms of AI are characterized by yellowish-brown teeth and extreme sensitivity to hot and cold
food. The enamel in these patients has normal thickness but is only slightly different in opacity from the
dentin, which indicates reduced mineral content in the enamel. The enamel also typically shows
fractures at the occlusal surfaces of the molars [69, 70]. Eighteen [71] mutations in the amelogenin
gene (AMELX have been shown to cause AI in humans to date [72, 73]. Additionally, twelve mutations in
the ENAM gene have been shown to cause Amelogenesis Imperfecta in humans [74-78].
Taken together, these diseases demonstrate that both MMP-20 and KLK-4 are important proteases
during normal enamel development. Patients suffering from such disorders usually suffer from attrition,
dental caries, hypersensitivity, discoloration, and other functional and aesthetic problems. Conventional
treatment for such patients includes fillings with amalgam, composites and ceramics; laminates;
bleaching; etc. Even after these treatments, the normal function and aesthetics of the dentition are
sometimes not restored. While there are some alternative biomimetic materials available to repair
enamel defects, treatment still remains a challenge [79, 80].
1.7 Significance, objectives, and scope of the work
For the proper genesis of enamel, enamel matrix proteins such as amelogenin, ameloblastin, and
enamelin along with MMP-20 and KLK-4 play key roles in the proteolytic degradation, assembly and
nucleation of HAP crystals. Thus, understanding the role of proteases during enamel formation is vital to
understanding the mechanism of HAP crystal formation. In this study I focused on MMP-20 and its
indirect effect on HAP crystal formation during the secretory stage of amelogenesis. Mature enamel is a
28

non-living tissue so enamel defects cannot repair themselves. The repair of enamel defects due to
caries, erosion, trauma, hypomineralization and other causes therefore remains a major challenge in
dentistry. There is a need for restorative materials with improved adhesion properties, to avoid the
problem of secondary caries that often arises at restoration sites due to the poor bond between
restorative materials and the natural dental tissues. Artificial enamel can be a solution to such problems.
The present study explores a potential mechanism by which we can build artificial enamel for the
treatment of the above-mentioned enamel defects and diseases. My study is divided into two major
parts: (i) an in vivo analysis of Mmp-20 null mouse enamel to study the effects of the absence of MMP-
20 on enamel crystal formation, and (ii) the use of that knowledge to build artificial enamel in vitro using
a chitosan hydrogel.
We hypothesized that in the absence of MMP-20, full-length amelogenin becomes trapped inside the
HAP crystals during the secretory stage of amelogenesis, which affects the growth of these crystals. If
correct, this would show that MMP-20, along with its other functions, also helps in the formation of
enamel crystals during amelogenesis by preventing occlusion of proteins inside the apatite crystals. Our
hypothesis was based on the results of the systematic analysis by Smith et al. [61] in which the amounts
of proteins and minerals associated with Mmp-20 null mouse were found to differ from those seen in
the wild type. Furthermore, an in vitro study also showed the presence of organic molecules and altered
morphology of calcite crystals when they were grown in the absence of MMP-20 and the presence of
amelogenin, as compared to calcite crystals grown in the presence of both MMP-20 and amelogenin
[81].
To verify the above hypothesis, three specific aims were proposed:
 Specific Aim 1: To perform qualitative and quantitative analyses of proteins trapped inside
enamel crystals isolated from developing incisors during secretory and maturation stages of
29

amelogenesis in: a) wild type and b) Mmp-20 null mouse by immunochemistry (SDS-PAGE
and Western blot), Raman spectroscopy, and in situ atomic force microscopy (AFM).
 Specific Aim 2: To compare the size and morphology of enamel crystals isolated from Mmp-
20 null and wild type mouse incisors using high-resolution transmission electron microscopy
(HRTEM), scanning electron microscopy (SEM), Raman spectroscopy, and atomic force
Microscopy (AFM).
 Specific Aim 3: To grow an artificial enamel-like material by adding MMP-20 to an
established amelogenin-chitosan hydrogel system on isolated human teeth and perform
qualitative analysis of these crystals using SEM, X-ray diffraction (XRD), Fourier transform
infrared spectroscopy (FTIR), and mechanical testing.
I applied various biochemical and biophysical techniques to study the proteins trapped inside the
enamel crystals of Mmp-20 null mouse and compare them with wild type (WT) mouse enamel crystals. I
developed a novel protocol to isolate, clean, and study the enamel crystals. The following two chapters
describe the systematic analysis of isolated enamel crystals. In Chapter 2, the enamel crystals from
Mmp-20 null and WT mouse will be studied to compare the proteins associated with them using
immunochemical and biophysical techniques. This chapter will show how protein processing by MMP-20
is crucial during early enamel formation. In Chapter 3, a systematic comparison of enamel crystal
morphology in both Mmp-20 and WT mouse will be presented, using electron microscopic analysis to
study the effect of Mmp-20 on enamel crystal morphology. Chapter 4 will demonstrate the use of MMP-
20 in the formation of artificial enamel in vitro.

30

Chapter 2: Analysis of proteins trapped inside isolated enamel
crystals
2.1 Background and objectives:
The extracellular matrix (ECM) is essential for the normal development of enamel. Even though the
exact role played by all its components is not yet fully understood, we know amelogenin functions as a
potent regulator of HAP crystallite growth. Amelogenin may also function as a protective envelope for
the early enamel crystallites due to its adherence effect on them. This is attributed to the formation of
nanospheres by amelogenin self-assembly. These tightly packed nanosheres interact with the apatite
crystals and protect them from early fusion [82, 83]. Processing of amelogenin and other proteins by
MMP-20 and KLK-4 regulates crystal growth by controlling mineral deposition on specific crystal faces to
grow either in length (secretory stage) or width and thickness (maturation stage).
It has been shown that the hydrophilic C-terminal of amelogenin can adsorb on certain faces of HAP
crystals in enamel to facilitate growth only along the c-axis [30]. Other ECM proteins are also important
during amelogenesis. Studies on Enamelin knockout mouse showed that enamel formation stops
completely in the absence of enamelin [46]. This suggests that enamelin is a critical component of the
mineralization front which promotes the extension of enamel crystals along the length of the crystal (c-
axis). Enamelin is also essential for ameloblast integrity and initial mineralization during the secretory
stage of amelogenesis [84]. Ameloblastin, in addition to its role in cell adhesion, may have another
function in providing scaffolding to organize enamel matrix proteins (EMPs) for the initiation of enamel
crystal structure, as suggested in a transgenic mouse model [85].
Studies have shown that organic macromolecules can become occluded inside crystals during
biomineralization. One such example is the case of calcium carbonate biominerals in sea urchins, where
31

the organic macromolecules can adsorb on certain faces of the growing crystal or on step edges and
terraces and ultimately get occluded within them [86, 87]. These macromolecules within a crystal can
result in significant changes in the texture and mechanical properties of the biominerals. It has also been
shown that soluble macromolecules accelerate crystal growth at low concentrations but retard crystal
growth at high concentrations by non-specifically binding to the surfaces of the growing crystallites [88].
Even though several studies have been previously performed to show how protein processing is affected
in the Mmp-20 null mouse, there is a gap in the understanding of how Mmp-20 specifically affects HAP
crystal formation. I used Mmp-20 null mouse to demonstrate how Mmp-20 affects the secretory stage
of amelogenesis by preventing occlusion of amelogenin inside the enamel crystals. In this chapter, I will
show complete qualitative and quantitative analyses of the un-processed proteins and their effects on
HAP crystal growth. Amelogenin is the sole focus of this study because it is the most abundant protein in
the ECM.
2.2 Results
2.2.1 Phenotypic characteristics of Mmp-20 null mouse

As previously described [54], only the enamel of Mmp-20 null mouse was affected (Figure 3). The
A
D B
C
Wild Type Mmp-20 null
A
D B
C
Wild Type Mmp-20 null
Figure 3. Comparison of WT and Mmp-20 mouse enamel. A-B Wild Type and C-D Mmp-20 null mouse. The
Mmp-20 null mouse have soft cheesy enamel, absence of a decussating pattern and enamel that delaminates
at the DEJ.
32

incisors and molars displayed a chalky white appearance with an absence of the translucent enamel
seen in WT mouse. The Mmp-20 null enamel had a soft, cheese-like consistency and could also be
scraped off the dentin easily.
When observed under SEM, the enamel lacked the characteristic decussating pattern of enamel found
typically in rodents. The enamel rods were disorganized and appeared to delaminate from the
underlying dentinal surface (Figure 3).
2.2.2 Analysis of proteins associated with isolated enamel crystals
I used porcine tooth enamel crystals to establish a protocol for the isolation of enamel crystals and
quantitative and qualitative analysis of the proteins associated with them.
Porcine model: Un-erupted 3
rd
and 4
th
molars were extracted from the jaws of 6-month-old pigs. These
teeth were used to isolate enamel crystals. Protein analysis by UV- adsorption (Figure 4) and SDS-PAGE
0
1
2
3
4
5
6
W1P W2P W3P W1UT W2UT W3UT W1P' W2P' W3P' W1HCl
Absorbance (220nm)
Supernatants obtained after washings
Figure 4. UV-adsorption of proteins extracted from isolated porcine enamel crystals. W1P-
W3P: 1st Phosphate wash, W1UT-W3UT: Urea and Tris wash, W1P’-W2P’: 2
nd
phosphate
wash and W1HCl: crystals dissolved in 1M HCl
33

showed a decrease in the amount of total protein with each wash (Figure 4, lanes W1HCl-W3P’).
Supernatants W1UT-W3UT show more proteins associated with them due to the presence of 4M urea in
the extraction buffer. The last three supernatants, W1P’-W3P’, show almost negligible readings,
indicating that all the proteins adsorbed on the surface of the isolated crystals were removed. The
crystals were then dissolved in 1M HCl to analyze the proteins inside them. Both UV-adsorption and
SDS-PAGE showed the presence of some proteins in W1HCl. Western blotting confirmed the presence of
amelogenin. Mass spectrometry and Western blot analysis showed that this amelogenin was of length
148 (refer to Figure 2 for P148 sequence), as shown in Figure 5 and Table 2.

Sample
Mass
(Da)
Residue
Porcine
Crystals
16839 1-148
Porcine
Crystals
16919 1-148PO4
37
25
20

15
Figure 5. SDS-PAGE and Western blot of proteins extracted from
porcine enamel crystals. Lanes W1P-W2P’ show a decrease in the
amount of proteins with each buffer wash. W1HCl show a band which
corresponds with P148 in both SDS-PAGE and the Western Blot.
Table 2. Mass spectrometry results of porcine
crystals dissolved in 1M HCl. 1-148 represents the
P148 amelogenin fragment of amelogenin as
shown in figure 2.
34

Mouse model: Similar to the the procedure followed above for the porcine enamel, the isolated mouse
enamel crystals were washed with a series of extraction buffers to extract the adsorbed proteins. Figure
6 represents a set of 3 experiments using WT mouse enamel crystals to show the decrease in proteins
with each buffer wash followed by dissoltuion in 1M HCL and measurement by UV-adsorption. The
results show a similar trend as in the porcine samples. The above experiment was repeated with WT,
Mmp-20 heterozygous and Mmp-20 null mouse. UV-adsorption showed that the amount of total protein
associated with the enamel crystals of Mmp-20 null mouse was more than in either the WT or the
heterozygous mouse, which is a result of the absence of Mmp-20 and the failure to degrade enamel
matrix proteins (Figure 7). It was observed that that amount of protein in W1HCl (dissolved enamel
crystals in 1M HCl) was also almost 4 times that of the WT and heterozygous mouse (Figure 6, W1HCL).
SDS-PAGE (Figure 7A) confirmed that the amount of protein decreased with each buffer wash and that
more protein was present inside the isolated enamel crystals in W1HCl (Figures 7B and 8A) in the Mmp-
20 null mouse. The Mmp-20 null enamel crystals dissolved in 1M HCl showed a band corresponding to
full-
0
1
2
3
4
5
6
W1P W2P W3P W1UT W2UT W3UT W1P' W2P' W3P' W1HCl
Absorbance (280nm)
Supernatants obtained after washings
Figure 6. A set of control experiments (n=3) to show the trend of decrease in the
amount of adsorbed proteins on mouse enamel crystals with each wash. W1P-W3P: 1st
Phosphate wash, W1UT-W3UT: Urea and Tris wash, W1P’-W2P’: 2
nd
phosphate wash
and W1HCl: crystals dissolved in 1M HCl

35

length amelogenin (M 180) (refer to Figure 2 for M180 sequence), which was confirmed by Western blot
(Figure 8B, shown by arrow).
0
5
10
15
20
25
30
35
40
45
50
W1P W2P W3P W1UT W2UT W3UT W1HCl
Absorbance (280nm)
Supernantants obtained after buffer wash
Wild Type Heterozygous Mmp-20 Null
0
0.1
0.2
0.3
0.4
0.5
W1HCl
A B
Figure 7. UV-adsorption of proteins extracted from isolated enamel crystals of WT, Heterozygous and Mmp-20 null
mouse. B shows the proteins dissolved in 1M HCl. Mmp-20 null mouse W1HCl have almost 4 times the proteins as
that of the WT.
W1P-W3P: 1st Phosphate wash, W1UT-W3UT: Urea and Tris wash, W1P’-W2P’: 2
nd
phosphate wash and W1HCl:
crystals dissolved in 1M HCl

75
50
37
25
20

15
WT
Mmp-20
null
Figure 8. A) SDS-PAGE and B) Western Blot of Wild Type and Mmp-20 null mouse enamel crystals. The
Mmp-20 null mouse shows a protein band at 23KDa which corresponds to full length amelogenin M180
(shown by arrows). A decrease in the protein content can also be seen with each wash which is consistent
with the UV-adsorption profile.
WT Mmp-20 null
B
A
36

Analysis of proteins inside the enamel crystals isolated from secretory and maturation stage: Since
Mmp-20 is released in the secretory stage, the enamel crystals from the secretory and maturation
stages were separated and then processed as described in Chapter 2.4. Very similar results were
observed for these sets of samples as well. There was an overall increase in the amount of protein in
the Mmp-20 null enamel. The UV-adsorption showed a similar trend to the porcine and murine crystals
shown above in Figures 4,6 and 7 (data not shown).
As observed in Figure 9, the amounts of proteins in the dissolved enamel crystals were as follows: Mmp-
20 null mouse secretory stage > WT mouse secretory stage > MMP-20 null mouse maturation stage >
WT mouse maturation stage. Lanes 1 and 2 in the Western blot (Figure 9B) represent the samples from
secretory stage from Mmp-20 null and WT mouse, respectively. There is a definite increase in the
amount of protein in the Mmp-20 null mouse, as observed earlier (Figure 7 and 8). Lane 2 (WT) shows
some protein, which is expected because the samples represent an earlier stage of amelogenesis and
some protein retention is expected at this stage. Lanes 5 and 6 (Figure 9B) correspond to the maturation
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
WT Maturation
Stage
WT Secretory
Stage
Mmp-20
Maturation Stage
MMP-20
Secretory Stage
Absorbance (280nm)
W1HCl
Secretory Stage

Maturation Stage

Figure 9. A) UV-adsorption and B) Western blot of proteins present in dissolved (1M HCl) enamel
crystals of WT and Mmp-20 null mouse from secretory and maturation stages. The Mmp-20 null
mouse maturation stage crystals show the presence of amelogenin as compared to none in the WT
(as shown by arrow).
A
B
37

stages of Mmp-20 null and WT mouse, respectively. Mmp-20 null enamel crystals show the presence of
large quantities of protein, particularly full length amelogenin (M180), whereas WT samples show none
,confirming that WT enamel crystals do not retain any protein at the maturation stage.
2.2.3 Raman spectroscopy of secretory- and maturation-stage enamel crystals
Raman spectroscopy (Figure 10) was performed to confirm the presence of organic macromolecules
inside the isolated Mmp-20 null mouse crystals. The results show the presence of a broad peak at 3000
cm
-1
in the secretory- and maturation-stage enamel crystals of Mmp-20 null mouse. This peak
corresponds to the C-H virational ring and is indictive of organic macromolecules in the sample. The WT
enamel crystals show a small broad peak at the secretory stage and no peak at the maturation stage at
3000 cm
-1
. Thus, the organic macromolecules are present in both the secretory and maturation stage
enamel crystals in Mmp-20 null mouse but only during the secretory stage in the WT mouse. The Raman
results are also consistent with UV-adsorption and Western blot findings (Figures 7 and 9B).
Figure 10. Raman Spectra of WT and Mmp-20 null mouse enamel
crystals at secretory and maturation stages.
38

2.2.4 In situ AFM force spectroscopy
Enamel crystals during the isolation process tend to aggregate. In my study, I performed in situ AFM
force spectroscopy to address this problem and confirm the presence of intra-cryastalline proteins in
isolated enamel crystals.This technique measures the adhesive forces between the silicon nitride tip of
the AFM and the surface of the enamel crystal sample before and after surface dissolution of the
crystals with HCl at pH 4.6 (Figure 11). Figure 11A shows an example of the typical curves that were
obtained in this experiment for WT and Mmp-20 null mouse secretory- and maturation-stage enamel
crystals. The graph shows the presence of multiple rupture forces in Mmp-20 maturation-stage (Figure
Figure 11. In situ AFM images and force curves of WT and Mmp-20 null mouse enamel
crystals at secretory and maturation stages. A) Single and multiple rupture curves for
WT and Mmp-20 null samples. B) Probability of the Silicon nitride tip to pick up organic
macromolecules before and after surface dissolution. Images obtained before and after
surface dissolution: C&D) WT maturation, E&F) Mmp-20 null maturation, G&H) WT
secretory and I&J) Mmp-20 secretory stages.
39

11E&F), WT secretory-stage (Figure 11G&H), and Mmp-20 null secretory-stage (Figure 11 I&J) enamel
crystals before and after dissolution with HCl. In contrast, WT maturation-stage (Figure 11 C&D) enamel
crystals show a single rupture curve.
The presence of multiple rupture curves is an indication of the interaction between between the silicon
nitride AFM tip and stretchable organic macromolecules such as proteins or polysaccharides present on
the sample surface [89, 90]. The probability of the silicon nitride tip picking up an organic
macromolecule from the surface of the sample can be measured by calculating the percentage of
multiple rupture curves out of the total number of force curves measured. WT secretory-stage, Mmp-20
secretory-stage, and Mmp-20 maturation-stage samples showed a significant increase in the percentage
of multiple rupture curves after surface dissolution, whereas in WT maturation stage there was no
difference in the percentage of multiple rupture curves before and after surface dissolution with HCl (Fig
11B).
2.3 Discussion
By isolating the enamel crystals from pig mandibular jaws I established a reliable protocol. I then used
mouse incisors to show the amount of proteins adsorbed on the surfaces of the enamel crystals as well
as the proteins occluded inside the Mmp-20 null mouse enamel crystals.
Both UV-adsorption and immunochemical analysis showed that the amount of proteins associated with
isolated enamel crystals of Mmp-20 null mouse is 4 times of that of the WT. In a previous report, it was
shown that the amount of volatiles (water and protein) in Mmp-20 null mouse enamel crystals was
almost twice that of the WT [61]. The difference in our findings could be due to the difference in
techniques used to measure the proteins. We isolated the proteins by using an extraction buffer
whereas an ashing method was used to measure the protein in the prior study [61].
40

In my study, the role of Mmp-20 during the secretory stage of amelogenesis was studied more closely by
separating the secretory stage enamel from the maturation stage. The enamel crystals from the
secretory stage in WT mouse showed an increase in the amount of protein compared to maturation
stage, which is expected because of the active secretion of enamel matrix proteins by the ameloblasts at
this stage. The Mmp-20 null mouse enamel crystals showed an increase in the amount of protein
compared to the maturation stage, at levels much higher than in the WT. This shows that right from the
scretory stage, the amount of protein increase in the Mmp-20 null mouse is due to the absence of the
enzyme. It is already known that EMPs are proteolytically processed by Mmp-20 as soon as they are
secreted into the future enamel space. During the maturation stage, the Mmp-20 null mouse showed
an increased amount of protein compared to the WT. This finding indicates that Klk-4, which is
expresssed during the maturation stage, is not able to compensate fully for the lack of Mmp-20 in these
mouse, as shown by the Klk-4 knockout mouse in which the enamel formation was normal up to the
secretory stage of amelogenesis [91]. We performed Western blots to confirm the presence of
amelogenin in the Mmp-20 null mouse enamel crystals, which show full-length amelogenin even in the
maturation-stage enamel crytals. Based on these findings, we suggest that Klk-4 is unable to degrade the
excess amount of EMPs because they are occluded inside the HAP crystals in the Mmp-20 null mouse.
To further investigate the intracrystalline proteins, I performed Raman spectroscopy and AFM force
spectroscopy. The Mmp-20 null mouse secretory- and maturation-stage enamel crystals both show the
presence of a broad peak at 3000 cm
-1
, which is indicative of the presence of organic macromolecules
within the enamel crystals. No such peak was observed in the WT maturation-stage enamel crystals.
AFM force spectroscopy showed the presence of multiple rupture curves in the Mmp-20 null enamel
crystals, which further confirmed that organic macromolecules are present in the Mmp-20 null enamel
crystals at the maturation stage. This finding confirmed our hypothesis that, in the absence of MMP-20
and subsequent protein degradation, amelogenin gets occluded inside the HAP enamel crystals,
41

affecting their structure and mechanical properties. The change in the structural and mechnical
properties of Mmp-20 null enamel is described in subsequent chapters.
Amelogenins are known to adapt a β-sheet structure when they come in contact with HAP crystals. This
conformational change could faclilitate the occlusion of amelogenin inside the HAP crystals when the
protein is present in excess [92]. Full-length amelogenin has a tendency to adhere to the crystals, thus
facilitating its occlusion [93, 94]. In another in vitro study, Tao et al. showed that during the
transformation of ACP to HAP in the presence of glycine and glutamic acid, these amino acids were
expelled from the insides of the crystals, giving us more evidence that protein proteolysis can facilitate
the movement of small peptides from within the crystal [95]. In the case of Mmp-20 null mouse enamel,
such an event may not occur due to the absence of proteolysis in the secretory stage, which may result
in the occlusion of proteins inside the crystals.
It has already been shown that Mmp-20 null enamel displays inferior mechanical properties compared
to WT enamel. Our results show that the mechanical properties of Mmp-20 null enamel could be
affected by protein occlusion inside the HAP crystals. Several examples of intracrysatilline organic
macromolecules affecting the mechanical properties of the biomineral exist in nature. For example, in
European abalone, the addition of an organic matrix with CaCO 3 enhances the mechanical properties of
the shell [96]. In another study on calcite crystals in sea urchins, the presence of occluded proteins in
the sea urchin skeletal elements was suggested to be responsible for the changes in their fracture
properties. It was also shown that the glycoproteins adsorbed selectively on specific crystal planes and
that continuous growth of the crystal results in the occlusion of glycoproteins within the crystals [86, 87]
and inhibit certain active growth sites within the crystals, which affects the way in which fractures
propagate.
42

Proteins are believed to participate in the nucleation and the growth process of the biomineral. A
number of in vitro studies have been performed to elucidate the role of amelogenins. In an in vitro
study, it was shown that amelogenin can lower the interfacial energy for nucleation, thus making in vivo
nucleation a possibility. It was also shown that the proteins’ ability to form nanospheres forms a
template composed of charged sites to promote the accumulation of calcium and phosphate and favor
nucleation [17]. During the secretory stage of amelogenesis, amelogenin guides the formation of thin
ribbons of apatitic crystals which ultimately form the template for the rod structure of mature enamel
[29]. The hydrophilic C-terminus of amelogenin has also been shown to be associated with proper
enamel formation. Beniash et al. showed in an in vitro study that the charged C-terminal of amelogenin
aids in the arrangement of crystals into parallel arrays and that calcium ions influence amelogenin
assembly by interacting with the charged C-terminus of the protein [35]. Amelogenin can adsorb onto
specific faces of crystals to affect their growth [82, 97]. Another study using solid-state NMR showed
that the hydrophilic C-terminal is oriented next to the HAP surface to exert control over crystal growth
[30]. In the absence of MMP-20, full-length amelogenin will persist in the enamel matrix for extended
period of time, which may inhibit the normal process of enamel crystal formation. Margolis et al.
showed that the mineralization events triggered by the proteolysis of amelogenin play an important role
in enamel mineral formation and that degradation products like P148 prevent uncontrolled
mineralization [98]. Proteolytic digestion of amelogenin by MMP-20 increases the capacity of
amelogenin to promote nucleation and crystal growth [99]. Thus, timely processing of amelogenin by
MMP-20 is an important event in normal enamel formation.

43

2.4 Methods and materials:
Animals
Mmp-20 heterozygous mouse (Mmp-20
+/-
) with a C57BL/6J background were obtained from the Mutant
Mouse Regional Resource Center (MMRC) and housed in the University of Southern California vivarium.
This study was approved by the University of Southern California Institutional Animal Care and Use
Committee.
Enamel crystal isolation
Porcine: un-erupted third and fourth molars were dissected from 6-month-old pig mandibles. The teeth
were washed in phosphate-buffered saline (PBS) with protease inhibitors: 1mM
phenylmethanesulfonylflouride (PMSF), 1mM sodium fluoride (NaF) and 1mM benzamidine. The teeth
were then air-dried and the enamel was scraped using a razor blade onto a glass slide.
Figure 12. Scheme showing the methods and steps involved in the isolation of enamel
crystals from WT and Mmp-20 null mice enamel.
44

Murine: The Mmp-20 heterozygous males and females were mated to obtain Mmp-20
+/+
and Mmp-20
-/-
colonies. The Mmp-20
+/+
males and females were used to obtain WT colonies which were used as
controls in this study. The Mmp-20
-/-
males and females were mated to obtain Mmp-20 null mouse,
which were used for all the experiments. The females were checked for vaginal plugs every day. The
pups were weaned, tagged and genotyped at the age of three weeks. Genotyping was performed by
Transnetyx using the following primers: Mmp-20 null 5’GCCGAGGATTTGGAAAAAGTGTTTA3’ and
3’TTCATGACATCTCGAGCAAGTCTTT5’ and WT 3’ATACCCCAAAAAGCATGAAGAGACT3’ and
3’CAAGTTTTAAAGGTTGGTGGGTTGT5’. Mandibular and maxillary incisors from adult Mmp-20 null and
WT mouse were dissected. The incisors were de-pulped and cleaned in simulated enamel fluid (SEF) by
sonicating them 3 times in a water bath for 30 seconds each, with a one-minute interval in between
each sonication. The SEF was changed each time. The incisors were air-dried and the secretory-stage
enamel was isolated using a scalpel and collected in pre-weighed tubes. The WT incisors were freeze-
dried for 12 hours before the isolation of the maturation-stage enamel. Maturation-stage enamel from
the Mmp-20 null mouse was easily removed by scraping it with a scalpel and was therefore not freeze-
dried prior to isolation.
Amelogenin preparation
Recombinant mouse amelogenin rM179 was expressed and purified as previously described [100]. The
amelogenin obtained is an analogue to M180. It lacks the first methionine and is non-phosphorylated.
Extraction of enamel matrix proteins
The isolated enamel from both porcine and murine teeth samples were weighed and washed with a
series of protein extraction buffers (phosphate buffer 0.1M pH 7.4; 50mM tris+4M urea pH 7.4 and
phosphate buffer 0.1 M pH 7.4) as shown in Figure 12 to wash all the adsorbed proteins from the
enamel surface. This was followed by washings with distilled water to remove the remaining buffer. The
45

supernatant from each wash was collected and stored for further evaluation. The enamel crystals were
then dissolved in 1M HCl. Supernatants collected from the second wash of 4M urea + 0.5 M tris (W1UT,
W2UT and W3UT) and dissolved crystals (W1HCl) were desalted using Microcon Centrifugal Filter
Devices MW: 3000Da at 10000 rpm for 40 minutes at 4C. All the supernatants were freeze-dried and
reconstituted in 200 µl of distilled water for further analysis. Protease inhibitors (1mM PMSF, 1mM NaF
and 1mM Benzamidine) were added to all the buffers to prevent the extracted proteins from
degradation.
UV-adsorption and Western blot
Supernatants collected from each wash (Figure 12) were used for protein quantification using a
Nanodrop ND-1000 spectrophotometer (NanoDrop Products, Wilmington, DE) at λ=280nm. The
supernatants were dissolved in 1X loading buffer, loaded in 12% acrylamide gels and run at 120 V. One
gel was used for silver staining and a second gel was used for Western blotting. The gel was transferred
to a PVDF membrane using a wet transfer at 120 V for 1 hour, incubated with 5% non-fat milk in PBST
(0.01% Tween-20), immunostained with primary antibody and then visualized using an enhanced
chemiluminescence plus Western blotting detection system (GE Healthcare). The antibody used was a
polyclonal antibody raised against recombinant mouse amelogenin (rM 179) in rabbit [101].
Mass spectrometry
The W1HCl freeze-dried samples obtained from porcine enamel were also used for mass spectrometry
analysis. Mass spectrometry was performed on a QSTAR Pulsar instrument (Applied Biosystems/MDS
Sciex, Toronto, Ontario, Ca) in liquid chromatography tandem mass spectrometry (LCMSMS) mode at
the University of California, San Francisco. Samples were eluted with solvent A (0.1% formic acid) and
solvent B (50% acetonitrile).
46

Raman spectroscopy
The Raman spectra were collected from 100 to 4000 cm
-1
under backscattering geometry by a LabRAM
ARAMIS confocal Raman Microscope at the Pacific Northwest National Laboratories (HORIBA scientific,
Japan) operated at a resolution of 2 cm
-1
with an excitation wavelength of 532 nm and laser power of
2.5 mW. A ×60 objective with numerical aperture of 0.75 was used to focus the sample and to collect
the spectra for 20 seconds. The experiment was repeated 5 times for each sample and the results were
averaged.
Atomic force microscopy (AFM)
A muscovite mica disc (diameter 9.9 mm, Ted Pella, Inc.) was freshly cleaved and used as a supporting
surface. Fifty µl poly-L-lysine solution (0.1% w/v, Ted Pella) was used to wash the mica surface for 5
minutes, thoroughly rinsed with water and dried by a stream of nitrogen gas to remove any impurities
stuck to the disc. The isolated WT and Mmp-20 null enamel crystals from the maturation and secretory
stages (as described Figure 12) were dispersed in water by sonication for 10 minutes to prevent
aggregation of the crystals. Three µl of this suspension was placed on the poly-L-lysine functionalized
mica. All samples were imaged in air by a NanoScope 8 Atomic Force Microscope equipped with a J
scanner to measure the thickness of the enamel crystals. Thereafter, both the enamel crystals and AFM
cantilever were treated with plasma to remove residual organic material adsorbed on the surface. Time-
lapse AFM images were continuously collected in an AFM equipped with a fluid cell after thermal
relaxation for 10 minutes.
All in situ AFM images were captured in ScanAsyst mode at room temperature (23°C) with a NanoScope
8 Atomic Force Microscope located at the Pacific Northwest National Laboratories in collaboration with
Dr. James DeYoreo and Jinhui Tao (E scanner, Bruker) using silicon nitride tips (TR400PSA triangular
lever; k=0.08 N/m; tip radius ~20 nm; resonance frequency 34 kHz in air; Asylum Research, Inc.). The
47

signal-to-noise ratio was maintained above 10. The scanning speed was 1-2 Hz. The peak force set point
was carefully tuned to minimize the average loading force (~50 pN) during in situ imaging. Force curves
between the silicon nitride tips and enamel crystals were measured in order to determine the organic
material trapped inside the crystals. Force curves were collected after etching the outer surface of WT
and MMP-20 null enamel crystals with 200 µl HCl at pH 4.6 followed by washing with 400 µl of water.
The measurements before and after etching were performed in water. A constant approach and
retraction velocity of 500 nm/s and dwell time of 1 s were used and more than 50 individual force curves
were collected for each surface condition. The average adhesion force was used to determine the
organic content.

48

Chapter 3: Analysis of size and morphology of enamel crystals
3.1 Background and objectives
Enamel formation is a complex process that involves precise proteolytic degradation of the enamel
matrix proteins (EMP) to form a final hardened structure with less than 5% organic matter. It has been
shown that amelogenin, along with other EMPs, helps to organize crystallite orientation and direction of
growth [6]. An in vivo study on amelogenin knockout and transgenic mouse showed that in the absence
of amelogenin, the length of the crystallites decreased compared to the WT. Interestingly, the width-to-
thickness aspect ratio remained normal in the amelogenin knockout mouse, showing that amelogenin is
critical for ultimate crystal size but has no role in maintaining the width-to-thickness ratio [102].
Proteases along with EMPs are also crucial for normal enamel formation. Mmp-20 null mouse, which
have abnormal enamel formation, provide evidence that ECM protein processing is crucial for proper
enamel formation [54].
Nanosphere formation by amelogenin has been extensively studied. Using DLS, AFM and TEM it was
shown that amelogenin undergoes self-assembly to generate spherical structures of 15-20 nm in
diameter by hydrophobic interactions [103, 104]. The size of the nanospheres increases when the
hydrophilic C-terminal of amelogenin is removed, as has been shown for both porcine and murine
amelogenin [105]. HAP crystallites in the presence of assembled amelogenin grow in a fashion that is
more organized and parallel along the c-axis. Processing of amelogenin by MMP-20 leads to the
formation of nanorods and other morphological patterns. The truncated hydrophobic amelogenin
products play a vital role in nanorod formation. These nanorods guide the mineral morphology and
orientation of enamel during its formation [16, 99]. Crystal growth experiments done with HAP and
octacalcium phosphate (OCP) also give evidence for the importance of proteolysis. Growth of OCP
crystals in the presence of recombinant full-length amelogenin rM179 and C-terminally cleaved
49

degradation product rM166 resulted in rod-like crystals. However, the presence of rM166 alone resulted
in OCP crystals which were longer and had a larger length-to-width ratio than crystals grown with rM179
[106]. Moreover, it was shown that full-length amelogenin actually inhibits crystal growth, giving further
evidence that proteolytic processing during the secretory stage is vital to normal enamel formation [94].
All the above-mentioned studies show that amelogenin plays a crucial role in enamel crystallite
formation and that its proteolytic processing is equally important in normal enamel formation.

In Chapter 2, using Mmp-20 null mouse enamel crystals, I gave evidence for occlusion of proteins within
the crystals, which affects the overall growth of enamel. In this chapter I will show how the
morphological characteristics of isolated enamel crystals from Mmp-20 null mouse differ from those of
WT mouse. I will also give further evidence of the occlusion of proteins within the enamel crystals using
electron microscopy. This chapter will further elucidate and give in vivo evidence for importance of
proteolytic processing during the secretory stage of amelogenesis and its effect on enamel.
3.2 Results
The differences in size and morphology between WT and Mmp-20 null mouse enamel crystals were
studied using various microscopic techniques.
3.2.1 Morphology of enamel crystals of WT and Mmp-20 null mouse
It was much harder to isolate and separate the Mmp-20 null mouse enamel crystals than it was for the
WT, due to the increased amount of protein associated with them. When observed under SEM (Figure
13 A-D), the Mmp-20 null crystals appeared more heterogeneous in length. Most crystals were shorter
than the WT crystals, but there was more variability. The Mmp-20 enamel crystals also appeared wider
and thinner. TEM (Figure 13 E-H) showed the WT crystals to be long with dark bands present at various
50

intervals. These dark bands are normal in enamel crystals and represent slight bends in the long crystal
which cause a difference in density, leading to oblique views [107]. Mmp-20 null crystals appeared

much wider and shorter compared to the WT. The Mmp-20 null enamel crystals also exhibited ragged
edges with kinks and pits (shown by white arrows in Fig 13H) whereas WT crystals were smooth with
continuous edges. The Mmp-20 null crystals give an appearance of a more plate-like morphology rather
than the long hexagonal crystals of the WT.
3.2.2 Width and thickness of enamel crystals of WT and Mmp-20 null mouse
The isolated enamel crystals of WT and Mmp-20 null mouse were observed under AFM for a systematic
analysis of their size distribution (Figure 14). A histogram was calculated using 137 isolated crystals from
WT mouse and 28 crystals from Mmp-20 null mouse. 52.5% of WT enamel crystals ranged from 50-90
nm in width, whereas in the Mmp-20 null mouse, 54.5% crystals ranged from 60-120 nm in width. This
Wild Type MMP-20 null
E
H F
G
Wild Type MMP-20 null
A
D B
C
Figure 13. Comparison of WT and Mmp-20 mouse enamel morphologies using SEM and TEM. A-B Wild Type and C-D
Mmp-20 null mouse. The Mmp-20 null mouse enamel crystals are more heterogeneous with a plate like morphology as
compared to WT which are long thin and more homogenous.
51

confirms the SEM and TEM findings that the Mmp-20 enamel crystals appeared wider than those of the
WT. When comparing the thickness of the enamel crystals, 66% of WT enamel crystals ranged from 20-
40 nm whereas the Mmp-20 null enamel crystals were slightly thinner, with 60% crystals ranging from
15-30 nm.

3.2.3 Analysis of areas of imperfections in the Mmp-20 null enamel crystals
HRTEM was used to observe crystal lattice patterns in the isolated WT and Mmp-20 null enamel crystals
(Figure 15). The Mmp-20 null enamel crystals appeared shorter and wider, as observed earlier (Figures
13 and 14). Panels B and E in Figure 15 are higher magnifications of the area represented by the red box
in panels A and D. The insets in panels B and E are diffraction patterns of the highlighted regions (red
boxes in panels A and D) created by Digital Micrograph 2.30.542.0 software. The 002 and 004 diffraction
Figure 14. Width and thickness of enamel crystals of (A-B) Wild Type and
(C-D) Mmp-20 null mouse. The Mmp-20 null mouse enamel crystals were
wider, 60-120nm, and thinner, 15-30nm, as compared to the WT which
varied from 50-90nm in width and 20-40nm in thickness.
Wild Type Mmp-20 null
A
B
C
D
52

patterns show that these crystals are HAP. Panels C and F show higher magnifications of the highlighted
regions (red boxes) in panels B and E. The WT enamel crystal shows a very characteristic pattern of HAP
crystal lattice. The Mmp-20 null enamel crystal shows a similar pattern overall but has a notable area in
which the lattice pattern is absent (highlighted by black dotted lines).

Such irregularities were seen in all the observed Mmp-20 null enamel crystals. These areas represent
imperfections that result from occluded proteins within the crystal. These imperfections also showed
the absence of a diffraction pattern when a Fourier transformation was performed using the digital
micrograph software. These imperfections, observed in enamel crystals from both maturation and
Figure 15.HRTEM of WT (A-C) and Mmp-20 null mouse (D-F). A and D show images of isolated
enamel crystals of WT and Mmp-20 null mouse respectively. B and E are higher magnification
images of each enamel crystal. The inserts in panels B and E show the diffraction pattern of
marked regions. C and F are higher magnification images of each individual crystal showing the
crystal lattice pattern. The dotted area in F shows an area of imperfection in Mmp-20 null mouse
enamel crystal.
A B
C
D E
F
53

secretory stages, included a sudden loss of crystal lattice pattern (Figure 16 A and B ), splitting of the
crystal lattice in two (also known as screw dislocation [108]; Figure 16 C) and narrow-angle boundary
where the crystal lattices are out of alignment (Figure 16 D). These imperfections were also analyzed
quantitatively by comparing the areas of imperfections in the WT and Mmp-20 null mouse. 31% of the
surface of the null mouse crystals were covered by imperfections, as compared to only 10% for the WT,
which was a statistically significant difference (p<0.005), as shown in Figure 16 E.

3.2.4 Crystallinity of Mmp-20 null enamel crystals
The crystallinity of a solid refers to the degree of structural order in that solid. Due to the presence of
occluded proteins inside the Mmp-20 null mouse crystals, crystallinity of the samples was measured and
0
5
10
15
20
25
30
35
*
n=6
WT
Mmp-20
null
Mmp-20 null
A B
Figure 16. Analysis of areas of imperfections in enamel crystals of Mmp-20 null mouse. A-D show examples of
imperfections observed in the Mmp-20 null mouse. A-B show sudden loss of crystal lattice pattern (indicated by
red arrows). C shows an example of screw dislocation. D shows the misalignment in the crystal lattice pattern
also known as small angle boundary. E) Analysis of areas of imperfections in the WT and Mmp-20 null mouse.
C D
E
54

compared to the WT (Figure 17). The full width of half maximum (FWHM) of the Raman peaks in totally
symmetric stretching mode (ν1) of the tetrahedral PO 4 at 960 cm
-1
was used as a measure of crystallinity
in this study. The WT maturation-stage crystals showed the lowest FWHM, representing the maximum
crystallinity of all the samples. Both the secretory- and maturation-stage enamel crystals of Mmp-20 null

mouse showed a higher FWHM than the WT maturation stage, indicating that their crystallinity is lower
than the WT enamel crystals. Interestingly, during the secretory stage, the crystallinity of WT and Mmp-
20 null mouse is almost the same. The difference emerges at the maturation stage, where it increased
only half as much in Mmp-20 null mouse as it is in the WT.

Full Width of Half Maximum (FWHM)
WT Maturation
Stage
Mmp-20 null Secretory
Stage
Mmp-20 null
Maturation Stage
WT Secretory
Stage
Figure 17. Full width of half maximum (FWHM) of Raman peaks of WT and Mmp-20 null
secretory and maturation stage enamel crystals. The crystallinity is in the following order:
Mmp-20 secretory stage < WT secretory stage < Mmp-20 maturation stage < WT maturation
stage.
FWHM =2 2𝑙𝑛 2𝜎
55

3.3 Discussion
The unique mechanical properties of enamel are attributed to its composition and hierarchical structure.
In humans, 96-97% of enamel is composed of fluoridated HAP crystals while the remainder is organic
material. Human dental enamel has a keyhole structure which is 5µm in diameter with HAP crystals
approximately 68 nm in length, 26 nm in diameter and 2 nm in thickness [109]. In rodents, the HAP
crystals are arranged in a decussating pattern where the rods and interrods are interwoven with each
other.
The length and shape of the HAP crystals in enamel affect its mechanical properties. In this study, it was
found that the length, width and thickness of Mmp-20 null mouse enamel crystals differed from those of
WT mouse. When HAP crystals were grown in the presence of full-length porcine amelogenin (P172) or
its partially degraded ‘20K’ product (P148), it was found that the full-length amelogenin had an
inhibitory effect on the growth of the HAP crystals while no such effect was observed with P148 [110]. In
another in vitro study, growth of OCP crystals in the presence of purified bovine amelogenin extract
resulted in more elongated crystals as compared to other proteins like albumin and gelatin. Amelogenin
suppressed the OCP growth in the following order: b-axis > c-axis > a-axis, resulting in prismatic and
elongated rod-like OCP crystals [111]. The length-to-width ratio of OCP crystals increased in the
presence of rM166 (which lacks the C-terminal), the major degradation product of Mmp-20, when
compared to those grown in the presence of full-length amelogenin [106]. In an in vivo report with
amelogenin knockout mouse, it was shown that the enamel crystal thickness and width were markedly
reduced compared to WT mouse.
In this chapter, it was shown that Mmp-20 null mouse enamel crystals had more areas of imperfection
than WT crystals. Such imperfections have been reported previously in enamel HAP crystals [108].
Studies with enamel crystals of amelogenesis imperfecta patients have shown that the crystals in
affected individuals have a wide range of morphology. Some amorphous aggregates which gave no
56

electron diffraction pattern were also observed among the crystals in these studies. Fragile areas within
the crystals which were prone to electron beam damage and appeared as electrolucent spots were also
observed [112]. In the present study, Mmp-20 null enamel crystals were also observed to have
imperfections which gave no diffraction pattern. Our results indicate that such areas are where enamel
matrix proteins have become occluded within the crystals. Although another serine protease is secreted
during the transition and maturation stages in the Mmp-20 null mouse, it is not able to compensate fully
for the loss of Mmp-20.
Although the results in our study showed the presence of full-length amelogenin within enamel crystals
of Mmp-20 null mouse, the presence of amorphous calcium phosphate (ACP) in the areas of
imperfections cannot be completely ruled out. ACP is a transient calcium phosphate phase which is
formed during the secretory stage of amelogenesis. Full-length recombinant amelogenin rP172 has been
shown to transiently stabilize ACP nanoparticles. Such a phenomenon was not observed with rP148, the
C-terminally cleaved product of amelogenin [113, 114]. Beniash et al. also showed that full-length
amelogenin and ACP particles coexist in the youngest layers of enamel near the secretory faces of
ameloblasts, whereas in the older, more mature layers of enamel, along with degradation of
amelogenin, ACP phase transformation into HAP occurs [114]. Another in vitro study showed that P148
inhibits crystal nucleation and stabilizes ACP. Thus, P148 prevents unwanted crystal nucleation during
the secretory stage of amelogenesis [113, 115]. During the formation of apatite crystals in the presence
of glycine and glutamine, an intermediate phase of moldable ACP which could be rearranged to needle-
like crystals by the addition of biological additives was observed [95]. All the above observations suggest
that ACP is a transient stage during enamel formation and that the presence of full-length amelogenin
and the absence of Mmp-20 could stabilize this phase. This phenomenon may result in an enamel crystal
which has areas of occluded proteins and possibly ACP, as observed in our findings.
57

Another important observation in the present study was the reduction in crystallinity of the Mmp-20
null maturation stage crystals as compared to that of the WT. Such a reduction in crystallinity could be
due to the presence of impurities in the Mmp-20 null crystals. The crystallinities of bone and dentin are
lower than that of enamel. This is due to the fact that both dentin and bone have more organic content,
30% in dentin and 35% in bone, as compared to enamel, which has only 2-5% organics. The other reason
for decreased crystallinity in bone and dentin may be the presence of ACP, which is speculated to be
about 30% in bone [116]. Although the mechanical properties of Mmp-20 null mouse enamel were not
tested in this study, the general handling of the tissue and the fact that enamel can easily be scraped off
from the underlying dentin shows that the presence of organic macromolecules within the crystals also
affects its mechanical properties.
3.4 Materials and methods
Isolation of enamel crystals from WT and Mmp-20 null mouse
The enamel crystals from WT and Mmp-20 null mouse were isolated using the same method described
in Chapter 2 and Figure 12. The isolated crystals were then washed with the extraction buffers to
remove the adsorbed proteins around them, followed by washing with water. They were air-dried and
used for microscopy and Raman spectroscopy experiments.
Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)
Enamel crystals isolated from adult incisors of Mmp-20 null and WT mouse as described earlier (Figure
12, step 4) were analyzed using SEM and HR-TEM. The enamel crystal samples for SEM were suspended
in 100% ethanol and placed on SEM mounds. They were gold-plated for 30 seconds and imaging was
performed in a field emission scanning electron microscope (JEOL JSM-7001F) operating at an
accelerating voltage of 10keV.
58

The samples for HR-TEM were suspended in 100% ethanol, placed on TEM grids (Carbon Type-B, Ted
Pella Inc.) and dried for at least 14 hours before imaging. HR-TEM images were obtained on a JEOL JEM-
2100 microscope using an accelerating voltage of 200keV.
Atomic force microscopy (AFM)
The enamel crystals were prepared as described in Chapter 2.4.The images were captured in tapping
mode with silicon tips (PPP-FMR rectangular cantilever, nominal spring constant k=2 N/m, tip radius <10
nm, resonance frequency 75 kHz in air, Nanosensors
TM
). Cantilevers were resonated at approximately 75
kHz with a free amplitude of 50 nm, and images were collected at a tapping amplitude of approximately
80% of the free amplitude. The images were used to calculate the width and thickness of the enamel
crystals manually using NanoScope Analysis 1.4 software and the histogram was plotted using Origin 8.5
(OriginLab Corporation).
Statistical analysis of the areas of imperfections in the HAP crystals
The imperfections in the WT and Mmp-20 null mouse enamel crystals were calculated by taking a
Fourier transformation of the areas where no discernable lattice pattern was observed in HRTEM using
Digital Micrograph software. Each area of interest measured 8.4x8.4 nm
2
. All the areas showing an
absence of crystal lattice and subsequent diffraction pattern were calculated as areas of imperfections
(AOI). All such areas from 6 different enamel crystals (1 from secretory stage, 2 from maturation stage
and 3 mixed populations) from both WT and Mmp-20 null mouse were calculated and averaged. An
unpaired T-test assuming unequal variances was used to calculate the significance of the difference
between the Mmp-20 null and WT mouse. A value of p < 0.05 was considered statistically significant. All
statistical analyses were performed in Microsoft Excel 2010.

59

Raman spectroscopy
Raman spectra were collected as described in Chapter 2.4. To evaluate the relative crystallinity of
enamel crystals of WT and Mmp-20 null animals in both secretory and maturation stages, the Raman
peak of totally symmetric stretching mode (ν 1) of the PO 4 group located at ~960 cm
-1
was used for
FWHM analysis [117]. Gaussian functions of the form:
where y 0 is the
base of the peak, µ is the center of the peak, A is the area of the peak, and σ is the standard deviation or
Gaussian RMS width were used to fit the Raman peak at 960 cm
-1
with convergence of the R
2
=0.99 by
Origin 8.5 (Origin Lab Corporation). In this case, the FWHM was equal to
2 2ln 2 
.

22
( ) /(2 )
0
()
2
x
A
f x y e




60

Chapter 4: In vitro growth of artificial enamel-like layer
4.1 Background and objectives
Unlike bone and dentin, which have around 20-30% organic matter, mature enamel contains very little
organic matrix (less than 5%). Enamel biomineralization involves secretion of a protein matrix by the
ameloblasts followed by mineral nucleation and eventual tissue maturation. Mature enamel does not
regenerate itself so development of synthetic enamel-like material is of significant interest for clinical
applications. Amelogenin protein has been used extensively in vitro to grow enamel-like structures for
the purpose of treatment of carious lesions and early enamel defects.
Several in vitro studies have been conducted to study the role of amelogenin on crystal morphology,
growth and organization [118]. Using AFM, it was shown that amelogenin has a tendency to adhere to
the ‘sides’ of the apatite crystals to control growth and morphology of the crystals [119]. Beniash et al.
showed that the hydrophobic portions of amelogenin affect the growth of crystals and that the C-
terminal domain is important for the parallel arrangement of the crystals with their c-axes parallel to the
long axes of the crystal bundles [120]. The HAP crystals formed early during the mineralization process
also have very intimate relationships with the stepwise degradation of amelogenin by MMP-20. A
previous study showed that the rate of amelogenin proteolysis by MMP-20 decreased in the presence of
apatite during crystal growth experiments [121]. Furthermore, it was demonstrated that amelogenin has
high affinity to the (010) side face of the crystals even after the cleavage of its C-terminus [121].
During the last decade, attempts have been made to synthesize biomaterials with enamel-like structure
using amelogenin protein. Remineralization of human enamel samples in the presence of amelogenin
and fluoride resulted in the formation of bundles of needle-like fluoridated HAP crystals [122, 123].
Small carious lesions were filled using a white crystalline plate of modified HAP which formed an
artificial enamel like layer 10µm thick with crystals parallel to the underlying tooth surface [124].
61

Another study used nanoscale apatite particles and glutamic acid to grow an enamel-like layer.
Glutamine selectively absorbed onto the apatite 001 faces, similar to amelogenin, and induced apatite
aggregation on natural enamel [3]. Different investigators used fluoridated HAP micro particles, EDTA
and fluoride ions to make an enamel-like structure via mesoscale assembly. EDTA was used as a
chelating agent to facilitate the growth of mesocrystals which then fused to form a single crystal of
fluoridated HAP [125]. Small peptides resembling the structure of amelogenin have also been used to
synthesize enamel-like layers. A β-sheet-forming peptide which generated 3D biomimetic scaffolds was
used for nucleating HAP crystals. This material was used in human enamel lesions to facilitate
remineralization [126]. Another group used anionic oligopeptide amphiphilic (OPA) with the hydrophilic
domain of amelogenin, which fostered biomimetic remineralization of demineralized human enamel.
We used LRAP, an alternatively spliced isoform of amelogenin, to induce faster growth of organized
needle-like HAP crystals with a robust interface between the new growth and the natural enamel
surface [5].
In our group, we have used amelogenin-containing chitosan (CS-Amel) hydrogel for the synthesis of an
organized, enamel-like layer on the natural enamel surface. We used the potential of amelogenin to
control the growth of apatite, imitating the natural phenomenon of amelogenesis. The Ca-P clusters in
the matrix were stabilized in the CS-Amel hydrogel and formed linear chains. These chains fused with
enamel crystals to form a co-aligned structure anchored to the natural enamel substrate. This treatment
increased the hardness and modulus of elasticity of the enamel by 9 and 4 times, respectively [127]. The
new enamel-like layer does not restore the complete mechanical properties of natural enamel,
however, due to the presence of organic material and the lack of hierarchical prismatic-interprismatic
structure [128].
62

In this chapter, I adopted the strategy of harnessing MMP-20 action in amelogenesis to mimic the
natural growth process and “re-grow” a biomimetic layer with improved mechanical properties on a
tooth surface. I used a chitosan-amelogenin hydrogel system to synthesize an enamel-like layer on
human enamel sections. MMP-20 is introduced in the system to mimic the early secretory stage of
amelogenesis and also prevent occlusion of proteins within the newly formed HAP crystals, as shown in
Chapters 2 and 3. This chapter is composed of two specific objectives:
 Part I: To identify optimum conditions for the action of MMP-20 in the chitosan hydrogel system
using HPLC and SDS-PAGE.
 Part II: To develop and characterize artificial enamel-like layer on human tooth sections.
4.2 Results
4.2.1 Proteolysis of amelogenin rP172 by MMP-20 in the presence of 2% chitosan solution
Because our biomimetic model contains 2% chitosan, proteolysis experiments were performed to test
the action of MMP-20 on recombinant porcine amelogenin (rP172) in 2% chitosan solution. The HPLC
results show (Figure 18A) that amelogenin was completely degraded within 24 hours. At the end of 14
hours, a major peak of full-length amelogenin was seen along with another peak representing a major
proteolytic product lacking the hydrophilic C-terminal 25 amino acids, namely rP148 (Figures 18A and 2).
The SDS-PAGE (Figure 18B) results show the presence of a full-length amelogenin band along with 2
more bands at 20KDa and 13KDa after 14 hours of proteolysis. Even after 24 hours, a full-length
amelogenin band along with another band at 13KDa still persisted. The discrepancy between HPLC and
gel electrophoresis results could be due to the sensitivity of the two techniques. The silver staining used
for SDS-PAGE is more sensitive than HPLC. The discrepancy could also be due to the difference in the
amounts of proteins loaded on the HPLC and SDS-PAGE. The HPLC samples were diluted with 0.2% TFA
due to the presence of chitosan in them.
63

4.2.2 Enamel “ r e- gro w t h ”
Figure 19 shows the enamel microstructure of human molar tooth sections. The enamel is composed of
highly organized HAP crystals growing preferentially along the c-axis from the DEJ to the enamel surface.
Enamel windows of 4 mm
2
prepared on sagittal tooth sections were used for this study (also refer to
materials and methods in Chapter 4.4). After mineralization in artificial saliva for 5 days, newly formed
needle-like apatite crystals were observed on the etched enamel surface. In the samples maintained in
solution containing only chitosan hydrogel (Figure 19D-F), the apatite crystals appeared more
heterogeneous, with varying lengths and direction, although the growth appeared dense and covered
the entire tooth surface window. When amelogenin was added to the remineralization chitosan
hydrogel, the apatite crystals became more homogenous and appeared to grow perpendicular to the
underlying enamel surface (Figure 19G-I). The crystals were well-organized, closely stacked and needle-
rP172
2-148
0 Hr
3 Hr
6 Hr
24 Hr
14 Hr
25

20

15

10
Figure 18. A) Reverse phase chromatography showing the amelogenin proteolysis products at various time
intervals. B) SDS-PAGE after silver staining showing peptides obtained after proteolysis of amelogenin by rhMMP-
20 at 3, 14 and 24 hours. Red arrow represents the ‘20k’ peptide rP148 and blue arrow shows full-length
amelogenin (rp172). Also refer to figure 2.
A
B
64

like, similar to what was observed in our previous study [4]. Recombinant MMP-20 was added to the
remineralization gel prior to application to simulate natural conditions during the secretory stage of
amelogenesis [6]. MMP-20 cleaves full length amelogenin to yield the amelogenin polypeptide rP148
that has been shown previously to promote crystal growth [99]. After 5 days, the newly grown layer
appeared very dense with well-organized needle-like apatite crystals organized parallel to each other
A B C
F E D
G
H I
J K
L
Etched
Enamel
Chitosan
Gel
Amelogenin-
chitosan
hydrogel
MMP-20
containing
amelogenin
-chitosan
hydrogel
Figure 19. SEM images showing etched enamel (A-C), and newly grown HAP crystals in chitosan
hydrogel (D-F), amelogenin-chitosan hydrogel (G-I) and MMP-20 containing amelogenin-chitosan
hydrogel (J-L).
65

along their c-axes and either perpendicular to the underlying enamel or at various angles from the
enamel surface (Figure 19J-L).
Another unique feature that was observed under SEM was the direction of the bundles of apatite
crystals at low magnification. As shown in Figure 20, the samples re-mineralized only with chitosan gel
(Figure 20A, D&G) did not show any defined direction in which the crystal growth occurred. When
A B C
F
E
D
G H I
Chitosan
hydrogel
Amelogenin-chitosan
hydrogel
MMP-20 containing
amelogenin-chitosan
hydrogel
Figure 20. SEM images showing direction of growth in the newly grown HAP crystals in chitosan
hydrogel (A, D, G), amelogenin-chitosan hydrogel (B, E, H) and MMP-20 containing amelogenin-
chitosan hydrogel (C, F, I). The direction of the growth is marked by red and white arrows. The
panels (D-I) enclosed in yellow box are transverse sections. Rest is sagittal sections (A-C).
66

amelogenin was added to the system, some degree of organization was observed. The bundles
appeared to follow a certain direction (marked by green arrows) along the underlying enamel prisms
(Figure 20B, E&H). Regrowth with MMP-20 in the chitosan gel resulted in more crystal organization
compared to the two control samples. The apatite crystals grew along the underlying enamel rods,
giving them a ribbon-like appearance (Figure 20C, F&I and marked by blue arrows). To confirm this
finding, re-mineralization experiments were also repeated on transverse tooth sections (Figure 20 D-I,
panels enclosed by yellow box). Similar findings were observed as described earlier with sagittal
sections. The apatite crystals followed an organized pattern of growth along the underlying enamel rods.
When I looked at these crystals at higher magnification (Figure 21), they also appeared needle-like and
were arranged in parallel along their c-axes. In the samples containing MMP-20, the apatite crystals
appeared to be arranged at different angles from the underlying enamel surface, but the overall
direction of these crystals was along the enamel rods (Figure 21 E-F).

A C E
F
D
B
Chitosan hydrogel
Amelogenin-chitosan
hydrogel
MMP-20 containing
amelogenin-chitosan
hydrogel
Figure 21. SEM images showing higher magnification of the newly grown HAP crystals in chitosan hydrogel
(A-B), amelogenin-chitosan hydrogel (C-D) and MMP-20 containing amelogenin-chitosan hydrogel (E-F).
The HAP crystals in panels E and F appear to be at least 1µm in length.
67

4.2.3 Characterization of the newly grown layer

The mineral phase and orientation of the newly grown crystals were confirmed by X-ray diffraction
(XRD) (Figure 22). All of the diffraction peaks at 2Ɵ = 25.8 (002), 2Ɵ = 31.6 (211) and 2Ɵ = 32.6 (300) can
be readily indexed to hexagonal phase hydroxyapatite (JCPDS 09-0432). XRD of etched enamel was
measured for comparison. The presence of sharp 002 peaks in both the chitosan-amelogenin and MMP-
20-containing chitosan-amelogenin samples indicated that the apatite crystals were parallel to each
other along the c-axis. HAP crystal orientation was calculated by analyzing the ratios of I (002)/I (211), which
were 0.5, 0.95 and 1.6 for samples containing chitosan hydrogel, amelogenin-chitosan hydrogel and
MMP-20-containing amelogenin-chitosan hydrogel, respectively (Table 3). Full width of half maximum
(FWHM) was calculated for all the samples and showed that the HAP crystals formed after addition of
MMP-20 had the highest crystallinity (Figure 23). The length-to-thickness ratio was determined by
2Ө
angle
Etched Enamel
Chitosan Gel
Chitosan+Amelogenin
Chitosan+Amelogenin+MMP-20
Intensity (counts)
002
211
300
400
Figure 22. XRD spectra of newly grown layer grown in chitosan hydrogel, amelogenin-chitosan
hydrogel and MMP-20 containing amelogenin-chitosan hydrogel. Diffraction peaks at 2Ɵ = 25.8
(002), 2Ɵ = 31.6 (211) and 2Ɵ = 32.6 (300) can be readily indexed to hexagonal phase
hydroxyapatite (JCPDS 09-0432).
68

calculating the I (002)/I (300) peak ratio. HAP crystals grown in the presence of MMP-20 showed the highest
length-to-thickness ratio (Table 3). In summary, HAP crystals grown in the presence of full-length
amelogenin and MMP-20 are long, needle-like, arranged in parallel along their c-axes and have good
crystallinity.

Ratio I (002)/I (211) I (002)/I (300)
Chitosan hydrogel 0.5 0.57
Amelogenin-chitosan
hydrogel 0.95 1.3
MMP-20 containing
amelogenin-chitosan
hydrogel 1.6 2.1
Table 3. Ratios of intensities at I
(002)
/I
(211)
and I
(002)
/I
(300)

to determine the crystallinity and length to thickness
ratio respectively. The HAP crystals obtained in the
presence of MMP-20 and amelogenin show more
orientation in the c-direction and are more in length as
compared to that grown in amelogenin only.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Full Width of Half Maximum
Figure 23. Peak obtained at 2Ɵ = 25.8 (002) was used for the calculation of
FWHM. The crystals grown in the presence of MMP-20 and amelogenin
show the highest crystallinity among all the samples.
Chitosan
hydrogel
MMP-20
containing
amelogenin-
chitosan hydrogel
FWHM =2 2𝑙𝑛 2𝜎
Amelogenin-
chitosan
hydrogel
69

4.2.4 Mechanical testing
Figures 24 and 25 show the hardness and elastic modulus of the HAP crystal layer in the following
samples: chitosan hydrogel, amelogenin-chitosan hydrogel and MMP-20-containing amelogenin-
chitosan hydrogel. The hardness and modulus of natural healthy enamel are 4.0 and 70 GPa,
respectively, as shown in the literature [129]. Acid etching by 30% phosphoric acid for 30 s was used as a
model for de-mineralization and resulted in a significant reduction in the hardness and modulus values
[4]. After mineralization in chitosan hydrogel, not much difference could be seen in the mechanical
properties of the newly formed layer (pink bar, Figure 24 and 25) A 2-fold increase in the elasticity
(Figure 24) and a 1.3 fold increase in hardness (Figure 25) were observed in the HAP crystals grown in
the presence of amelogenin. When MMP-20 was added to the system, there was a significant
improvement in the mechanical properties of the newly grown layer: a 5-fold increase in elasticity
(Figure 24) and a 3-fold increase in the hardness (Figure 25) as compared to the etched enamel.
0
10
20
30
40
50
60
70
80
GPa
Modulus of elasticity
Figure 24. Graph showing modulus of elasticity for the newly grown crystals and their
comparison with healthy and etched enamel.
***
***
***
Chitosan
hydrogel
Amelogenin-
chitosan
hydrogel
MMP-20
containing
amelogenin-
chitosan hydrogel
Etched
enamel
Healthy
enamel
70

To analyze the amount of organic content associated with the newly grown layer, FTIR was performed.
Figure 26 shows the FTIR spectra of the remineralization experiments with chitosan gel only, chitosan-
amelogenin gel and MMP-20-containing chitosan-amelogenin gel. The FTIR spectra of etched enamel
were included for comparison. The spectra showed characteristic bands of PO43- at 560-600 cm
-1
, which
correspond to Ѵ4 bending modes, and 1000-1120 cm
-1
, which correspond to Ѵ3 bending modes.
Absorption bands at 1453 and 1256 cm
-1
correspond to amide II and III bands of proteins, respectively.
The re-mineralization experiments with chitosan-amelogenin and MMP-20-containing chitosan-
amelogenin gel were compared directly to calculate the amount of protein associated with the newly
grown layer. The ratios of PO 4 (Ѵ4) to amide III and PO 4 (Ѵ3) to amide III bands were calculated to
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
GPa
Hardness
***
***
***
Figure 25. Graph showing hardness for the newly grown crystals and their
comparison with healthy and etched enamel.
Chitosan
hydrogel
Amelogenin-
chitosan
hydrogel
MMP-20
containing
amelogenin-
chitosan hydrogel
Etched
enamel
Healthy
enamel
71

determine the ratio of apatite to protein, as shown in Table 4. It was observed that the amount of
protein in the newly grown layer of MMP-20-containing chitosan-amelogenin samples was less than in
the chitosan-amelogenin re-mineralization sample.

Ratio (n=2) PO 4 ( ν4 at 600
cm
-1
)/Amide III
PO 4 ( ν3 at 1028 cm
-
1
)/ Amide III
Chitosan-amelogenin 0.49 1.82
MMP-20 containing
chitosan-amelogenin
0.68 1.78
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
400
520
640
760
880
1000
1120
1240
1360
1480
1600
1720
1840
1961
2081
2201
2321
2441
2561
2681
2801
2921
3041
3161
3281
3401
3521
3641
3761
3881
Absorption
Wavenumber (cm
-1
)
Chitosan+Amelogenin
Chitosan+Amelogenin
+MMP-20
Etched Enamel
Chitosan gel
Hydrogen-
phosphate v4
Hydrogen-
phosphate v3
Amide III
Figure 26. FTIR spectra of the newly grown HAP layer in the chitosan-hydrogel system.
Table 4. Ratios of absorbance of the PO4 peaks at 600 and 1028 cm-1 with
amide III peak. The HAP crystals obtained in the presence of MMP-20 and
amelogenin have a higher ratio showing that it has less amelogenin associated
with its crystals.
72

4.3 Discussion

In humans, dental enamel damage is irreversible under natural conditions. Thus, strategies to develop
artificial enamel or enamel–like structures are of great importance. There are various amorphous
substitutes available, such as amalgam, metal alloys, calcium phosphate cements and ceramics, which
are used to fill enamel defects. However, they are not ideal restorative therapies because they lack the
natural mechanical properties of enamel and are susceptible to further degradation, leading to
secondary decay. Recently, many strategies have emerged for biomimetic synthesis of enamel-like
structures which resemble natural enamel in both their microstructure and mechanical properties [4, 5,
130]. Despite all these studies, enamel repair using these systems is not yet conceivable. Recently, an
amelogenin-chitosan hydrogel system was developed in our laboratory and used to synthesize enamel-
like hydroxyapatite crystals on human tooth sections. In this part of my study, by adding MMP-20 to the
amelogenin-chitosan system, I attempted to synthesize an enamel-like structure with improved
microstructure and mechanical properties.
MMP-20 was used in the amelogenin-chitosan hydrogel system following the findings from in vivo
investigation of Mmp-20 null mouse. In Chapters 2 and 3, it was shown that the absence of Mmp-20
during amelogenesis resulted in the occlusion of amelogenin inside the HAP crystals, which affected
their shape, morphology and mechanical properties. The introduction of MMP-20 in the hydrogel
system simulated natural conditions, in which amelogenin is cleaved by MMP-20 during the process of
HAP crystal formation [6].
Stepwise processing of amelogenin at the C-terminus by MMP-20 is crucial to normal enamel formation
[6]. As shown in Figure 18, the 25KDa amelogenin is cleaved at the C-terminus to produce a 20KDa
polypeptide that has decreased affinity to apatite crystals [121, 131]. In vitro processing of amelogenin
by MMP-20 has been shown to produce major cleavage products similar to the native amelogenins such
73

as 20 kDa (Met1-Ser148), 13 kDa (Leu46-Ser148) and TRAP (Met1-Trp45) [31]. There is also a correlation
between the presence of enamel proteases and apatite crystal growth [132]. Aoba et al. reported that
the full-length porcine amelogenin adsorbed onto the surfaces of HAP crystals, inhibiting their growth.
The degraded products P148 and the 13KDa midsection showed reduced adsorption capacities and
inhibition of crystal growth [133]. A study on X-linked AI with a truncated amelogenin (lacking the C-
terminal) showed hypomineralized enamel with discoloration in such patients [134]. It was further
suggested that full-length amelogenin plays an active role in amelogenesis and that partial degradation
of amelogenin to C-terminally cleaved P148 regulates the mineralization process prior to full
degradation and its removal from the enamel matrix [133].
The formation of nanospheres by amelogenin was first reported by Fincham et al., who suggested that
amelogenin aggregates to from spherical structures due to intermolecular hydrophobic interactions
which interact with apatite [103]. Another in vitro study showed the formation of oligomers, mediated
by the N-terminal of amelogenin. Nanospheres break up to form oligomers to increase the surface area
for apatite binding [135]. The nanospheres have the ability to assemble into chains in vitro [136]. These
chain-like structures showed orientation along the c-axis of apatite crystals, suggesting that they
mediate apatite crystal growth during mineralization [136]. In another in vitro study it was shown that
amelogenin nanospheres adsorb onto HAP crystals with a good correlation coefficient (R
2
=0.99)[25].
Several different types of in vivo evidence for nanosphere-like structures have also been reported in the
literature. A finely granular “stippled material” between the faces of the secretory ameloblasts and the
mineralization front was reported and found to be continuous with the organic matrix [137]. A helical
structure varying in width from 15 to 30nm was also reported by Smales. He suggested that these
helices are arranged parallel to each other and to the enamel prisms [138]. Using a freeze fracture
technique, globular or spherical structures 300-500Å in diameter were observed in the youngest
74

enamel. The authors concluded that these structures may be proteins and their linear organization may
preempt the formation of apatite crystals [139].
In the current study, we have shown that in the presence of amelogenin in the chitosan hydrogel
system, long needle-like HAP crystals are formed after 5 days of mineralization. Wen et al. showed that
elongated HAP crystals were formed on bioactive glass and titanium in the presence of amelogenin
[140]. Further TEM observations showed that the HAP crystals were oriented parallel to each other and
composed of bundles of 15-20 nm thick crystals, much like the long and thin crystals observed in the
very early stage of enamel formation [140]. Beniash et al. also showed similar results in which the HAP
crystals grown in the presence of assembled amelogenin were organized parallel to their c-axes [35].
Both in vivo and in vitro evidence shows that enamel matrix proteins organize in linear chains to guide
HAP crystal growth.
In the present study it is suggested that amelogenin assembly guides the formation of long needle-like
HAP crystals which are arranged in bundles parallel to each other along their long axes (Figures 20 and
21). The addition of MMP-20 to the amelogenin-chitosan hydrogel system further increased the
organization of these newly formed crystals. It can be seen in the SEM image that the HAP crystals
grown in the MMP-20-containing amelogenin-chitosan hydrogel system were at least 1µm in length
(Figure 21). Figure 20 shows the presence of bundles of crystals following the pattern of the underlying
enamel rods. This could be due to the result of MMP-20 processing amelogenin at its C-terminus. Figure
18 showed that amelogenin is processed in 6-14 hours to form rP148 (recombinant equivalent of P148,
also refer to figure 2). Even after 24 hours, some amount of full-length amelogenin and rP148 remained
in the mineralization system, which could be the source of a constant supply of amelogenin (full-length
and cleaved products) similar to what occurs in vivo during the process of HAP crystal formation. MMP-
20 showed more activity on rP172 in the presence of HAP crystals when it was compared with the
75

activity of KLK-4 on rP172 or rP148. This finding suggests that the HAP crystals may have a role in the
activity of MMP-20 on enamel matrix proteins during amelogenesis [141]. Adding MMP-20 earlier in the
mineralization system resulted in earlier precipitation of mineral and more intensive crystal growth.
More Ca and PO 4 ions precipitated in the mineralization system throughout the course of the
experiment in the presence of MMP-20 [99]. Similar events may be happening in my in vitro regrowth
experiments, resulting in more organized crystal growth in the presence of MMP-20.
X-ray diffraction of the newly grown layers on all the samples showed the presence of HAP crystals
without any ACP or other crystalline phases (Figure 22). The 002 peak in samples maintained in only
chitosan hydrogel showed some orientation of the apatite crystals along the c-axis. Addition of
amelogenin improved the crystal orientation along the c-axis. The ratio of I (002)/I (211) improved from 0.5
for chitosan alone to 0.95 when amelogenin was added to the hydrogel (Table 3). Previous reports have
shown a similar improvement in the orientation of the crystals after addition of amelogenin [4, 5]. The I
(002)/I (300) ratio was calculated for the measurement of the length-to-thickness ratio, which improved
with addition of amelogenin (Table 3). The crystals became longer after the addition of amelogenin
compared to the ones grown only in chitosan hydrogel.
Addition of MMP-20 to the hydrogel improved the crystal orientation as well as the length of the
crystals considerably (Figure 22). The I (002)/I (211) and I (002)/I (300) ratios improved to 1.6 and 2.1,
respectively (Table 3). This can be seen in the SEM images (Figure 21), where the crystals grown in the
presence of MMP-20 appear to be substantially larger and more organized than in the other two
samples. Similar to our observations, another in vitro study reported a significant improvement in the
preferential orientation of the newly grown crystals when the HAP crystals were grown in the presence
of amelogenin and fluoride [123].
76

The crystallinity of the HAP crystals improved with addition of MMP-20 to the system (Figure 23). In
Chapter 3, I showed that the crystallinity of mature enamel crystals isolated from MMP-20 null mouse
decreased due to occlusion of amelogenin within the crystals (Figure 17). Our present finding can be
interpreted in a similar way. The presence of MMP-20 prevents amelogenin occlusion by degrading the
full-length protein into smaller peptides.
A significant improvement in the hardness and modulus of elasticity of the newly grown crystals was
seen after the addition of MMP-20 to the re-mineralization system (Figures 24 and 25). Ruan et al.
showed an improvement in the hardness and modulus of the apatite layer after addition of amelogenin
[4]. Microhardness testing showed a significant improvement in the Knoop hardness number of re-
mineralized enamel in the presence of amelogenin and fluoride as compared to that of etched enamel
re-mineralized in the absence of amelogenin [122].
It is well known that mature enamel contains only 2-5% organic matter, most of which is deposited in
the interrod region [14]. FTIR was used in my re-mineralization experiments to qualitatively analyze the
amount of amelogenin associated with the newly formed crystals. The results indicated less organic
content associated with the crystals grown with amelogenin and MMP-20 compared to those formed
without MMP-20 (Figure 26). Although it has not yet been tested, the reduction in the amount of
amelogenin could be responsible for the organization in the crystals grown in the presence of MMP-20
(Figure 20). In a previous in vitro study, we showed a similar trend for the growth of calcite crystals.
Calcite crystals grown in the presence of amelogenin and MMP-20 contained less organic matter and
had fewer deformities associated with them as compared to calcite crystals grown in the presence of
amelogenin [81].
This chapter presents a direct application of the effects of MMP-20 on HAP crystal formation shown in
Chapters 2 and 3. The newly formed layer grown in the presence of both amelogenin and MMP-20 was
77

found to have improved mechanical properties and crystal morphology when compared to the ones
grown in amelogenin only. This shows that the addition of MMP-20, by the process of protein
degradation, helps to regulate crystal growth in vitro, possibly by preventing occlusion of amelogenin
within the newly formed crystals.
4.4 Materials and Methods

Recombinant amelogenin rP172 expression
Recombinant amelogenin was expressed in E. coli strain BL21 and purified by ammonium sulphate
precipitation and reverse phase column chromatography as previously described [101, 142]. It is 172
amino acids in length, lacks the first methionine, is not phosphorylated and is an analogue of the full-
length porcine amelogenin P173.
Analysis of degradation products of rP172 by rhMMP-20
200µg recombinant porcine amelogenin rP172 was dissolved in 2% chitosan gel solution followed by
addition of 1.5mM Na 2PO 4 and 2.5mM CaCl 2. The pH of the solution was adjusted at 6.5. rhMMP-20
(Enzo Life Sciences, contains catalytic domain of human MMP-20) was added to the solution at a ratio of
1000:1 along with 20µM ZnCl 2 solution to activate the enzyme. For SDS-PAGE samples, chitosan was not
added to the solution. The solution was incubated at 37°C for 3, 6, 14 and 24 hours to check for
degradation of amelogenin.
i) High Performance Liquid Chromatography (HPLC)
A C4 Analytical column (Vydac Hesperia, CA USA) was used for detection of proteolytic products. The
chitosan-containing sample was diluted with 0.2% TFA to make it safe to perform HPLC. The peptides
were eluted with Buffer A (0.1% TFA) and Buffer B (buffer A+60% Acetonitrile) with a gradient of B over
75 mins. Liquid Chromatograph workstation version 6.41 was used for the detection of the peaks.
78

ii) Gel Electrophoresis
Proteolytic products of amelogenin by MMP-20 were detected by electrophoresis. They were dissolved
in 1X loading buffer, loaded in 12% acrylamide gels, run at 120 V in SDS buffer and stained with silver.
Tooth slice preparation
Extracted human third molars (following the standard protocols at the Herman Ostrow School of
Dentistry and approved by the Institutional Review Board at the University of Southern California)
without any restorations were selected for the experiment. They were cut into sagittal (2mm) and
transverse (4mm) sections using a water-cooled low-speed diamond saw. The tooth slices were polished
using a fine-grit paper and then sonicated in a water bath for 2 minutes. They were then immersed in
30% phosphoric acid for 30 seconds followed by 2-3 washes with deionized water. 2mm
2
windows were
defined on the enamel portions of the tooth slices using acid-resistant nail varnish followed by drying for
at least 4 hours.
Enamel regrowth by amelogenin-containing chitosan hydrogel
The hydrogel was prepared by mixing 2% chitosan solution (medium molecular weight, 75-85%
deacetylated, Sigma-Aldrich) to 200µg amelogenin (rP172) followed by addition of 1.5mM Na 2HPO 4 and
2.5mM CaCl 2 solution. The pH of the solution was adjusted to 6.5 with 1M NaOH. This solution was used
as a positive control. rHMMP-20 was added to this solution at a ratio of 1000:1 followed by 20µM of
ZnCL 2. 2% chitosan solution with 1.5mM Na 2HPO 4 and 2.5mM CaCl 2 was used as a negative control. 30µl
of each of these solutions were carefully applied to exposed enamel windows of the prepared tooth
slices and air dried for 15 minutes. The tooth slices were then immersed in 30 ml artificial saliva solution
(0.2mM Mgcl 2, 1mM CaCl 2.H 2O, 20mM HEPES buffer, 4mM KH 2PO 4, 16mM KCl, 4.5mM NH 4Cl, pH 7.0)
79

with a fluoride (F
-
) concentration of 1ppm at 37°C for 5 days. After the allotted time, the tooth slices
were removed from the solution, sonicated in a water bath for 10 mins and air dried.
Characterization
i) Scanning electron microscopy
The dried tooth slices were sputter-coated with gold for 30 seconds and imaging was performed in a
field emission scanning electron microscope (JEOL JSM-7001F) operating at an accelerating voltage of
10keV.
ii) Fourier Transform Infrared Spectroscopy
FTIR spectra were acquired from a Nicolet 4700 Spectrometer with a Gladi-ATR diamond crystal
accessory. The sample with newly grown crystals was pressed on the diamond crystal and scanned at 0.2
cm
−1
resolution, from 4000 cm
−1
to 500 cm
−1
, 36 times for each sample.
iii) X-ray Diffraction
The newly grown crystals were analyzed by X-ray diffraction (XRD; Rigaku diffractometer, Rigaku
Corporation, Tokyo, Japan) with Cu K λ radiation (λ=1.542 Å) operating at 70 kV and 50 mA with a
sampling step of 0.04 and 2Ɵ ranging from 5-65°. The peak at 2Ɵ=25.8° (002) was used for the
calculation of Full Width of Half Maximum (FWHM) using OriginPro 8.0. Jade Materials Data processing
6.5.26 (Materials Data Inc.) software was used for the calculation of I (002)/I (211) and I (002)/I (300) ratios.
iv) Nanoindentation
Hardness and elastic modulus were calculated using a nano-indenter (Agilent-MTS XP) with a Berkovich
tip at a depth of 2000 nm. Twenty-five test points in 2 different areas of enamel in each sample (n=4)
were measured.
80

Statistical Analysis
Enamel re-mineralization experiments were repeated 3 times and mechanical tests were conducted in
duplicates. The data was analyzed using Excel 2010. Single-factor ANOVA (analysis of variance) was used
to calculate the significance of the difference between the hardness and modulus of elasticity values
between the samples grown in chitosan hydrogel, amelogenin-chitosan hydrogel and MMP-20
containing amelogenin-chitosan gel. A value of p < 0.001 was considered statistically significant.

81

Chapter 5: Conclusion and future work

Earlier work on the enzyme MMP-20 in mice has shown that it is crucial for normal amelogenesis and its
absence results in malformed enamel [54]. The overall objective of my thesis was to understand the role
of MMP-20 in enamel biomineralization. This project was undertaken to elucidate the molecular
mechanism by which MMP-20 affects enamel crystal growth. In Chapters 2 and 3, in vivo experiments
were designed to explore the effects of MMP-20 on enamel crystal growth using Mmp-20 null mouse as
an animal model. In Chapter 4, in vitro experiments were conducted to re-grow an artificial enamel-like
layer on human 3
rd
molar tooth sections using the knowledge obtained from the in vivo part of my
study.
I first used porcine enamel crystals obtained from un-erupted 3
rd
molars to establish a reliable protocol
for isolation and extraction of adsorbed and occluded enamel matrix proteins from enamel crystals. I
then developed a protocol for the isolation of Mmp-20 null mouse enamel crystals from their
continuously growing incisors. Such a protocol has the potential to inform study of enamel crystals of
other genetically modified animals.
Since the main objective of this portion of my study was to observe occluded proteins inside the
isolated crystals, extraction buffers containing phosphate and urea were used to wash all the adsorbed
proteins from the surface of the enamel crystals. The crystals were then dissolved in 1M HCl. The
enamel crystals were separated according to the stage of amelogenesis from which they were obtained
in order to show that the serine protease Klk-4 does not fully compensate for the loss of Mmp-20.
Immunochemistry and UV-adsorption showed that with each extraction buffer wash, the amount of
adsorbed protein reduced. This is how we justified that protein obtained after crystal dissolution in 1M
HCL was actually occluded and not adsorbed on the surface. Immunochemistry confirmed the occluded
protein to be full-length amelogenin M180.
82

Amelogenin has a tendency to aggregate at a neutral pH due to its hydrophobic and hydrophilic
interactions [14]. Immunochemistry alone could not conclude whether occluded proteins were present
inside the isolated enamel crystals. Therefore, we took advantage of other techniques such as Raman
and AFM force spectroscopy to confirm our hypothesis. The presence of multiple rupture curves in AFM
and a broad peak at 3000 cm
-1
in the Raman spectra associated with the Mmp-20 null mouse enamel
crystals confirmed the presence of occluded amelogenin inside these crystals. The use of isolated
enamel crystals and such techniques as AFM and Raman spectroscopy has the potential to detect
occluded proteins in spite of the problem of protein aggregation.
Using electron microscopy, I showed that the overall morphology of Mmp-20 null enamel crystals differs
from that of crystals from WT enamel. The Mmp-20 null mouse enamel crystals were smaller in length,
had a reduced thickness and were heterogeneous. HRTEM showed the presence of areas of
imperfections within the crystals which showed no discernable lattice or diffraction pattern. All such
areas of imperfection were measured and analyzed, and the total area of imperfections in the Mmp-20
null enamel was found to be significantly higher than in the WT enamel crystals. In the past, little work
has focused on the morphology of enamel crystals in knockout animal models. In vivo studies have
shown that the enamel structure in the Mmp-20 null mouse is altered. The characteristic decussating
pattern is lost and the enamel is thinner and weaker than in the WT [54]. Here, I present a systematic
analysis of isolated enamel crystals not only at the micro-level but also within the crystals to look at their
lattice pattern.
Besides occluded amelogenin, another explanation for the presence of imperfections in the Mmp-20
null mouse enamel crystals could be the presence of ACP along with full-length amelogenin. Although
we have not been able to confirm the presence of ACP inside the crystals at this time, in vitro studies
have shown that full-length amelogenin can stabilize ACP during the mineralization process. Further
83

enzymatic processing of full-length amelogenin is needed for the transformation of ACP to HAP [115].
Beniash et al. also showed that the initial mineral in enamel consists of a transient ACP phase, and that
its transformation into the crystal phase is triggered by proteolytic processing of amelogenin [114].
Thus, it is possible that due to the presence of full-length amelogenin, complete transformation of ACP
to HAP is not able to occur in Mmp-20 null mouse, which results in imperfections inside the enamel
crystals.
In the in vivo study presented here, I showed that absence of Mmp-20 causes occlusion of amelogenin
inside the enamel crystals. I used these findings and incorporated this idea into our in vitro chitosan-
hydrogel system to form an enamel-like layer for the purpose of restoration of enamel defects. Earlier,
our group showed that long, thin, needle-like HAP crystals grew in the presence of full-length
amelogenin in a chitosan hydrogel in the presence of calcium and phosphate ions. The newly grown
layer in the presence of MMP-20 and amelogenin showed long, thin HAP crystals which were arranged
in parallel along their c-axes. They grew along the rod pattern of the underlying enamel. Crystals grown
in the presence of amelogenin alone were thin, needle-like HAP but showed less organization than those
grown in the presence of MMP-20. FTIR confirmed that less amelogenin was associated with the layer
formed in the presence of both MMP-20 and amelogenin, which is similar to the findings obtained in my
in vivo study. From X-ray diffraction, I confirmed that the HAP crystals in the MMP-20-containing
amelogenin hydrogel showed more uniform orientation, higher crystallinity and a greater length-to-
thickness ratio than the remainder of the samples. Mechanical testing of the hardness and elastic
modulus of the samples showed significant improvement as the result of MMP-20 addition when
compared to the chitosan hydrogel and amelogenin-chitosan hydrogel samples.
84

Based on the above findings, I conclude that the action of MMP-20 on amelogenin has a major impact
on enamel biomineralization. This control over crystal growth is achieved by sequential processing of
amelogenin by MMP-20 during the secretory stage of amelogenesis to give intermediate products (such
as P148) that allow the enamel HAP crystals to grow normally. The absence of MMP-20 results in the
accumulation of excess of full-length amelogenin in the enamel matrix, which ultimately can become
occluded inside the forming HAP crystals and result in abnormal enamel formation. I propose that MMP-
20, along with other functions, plays a major role in the regulation of enamel formation by proteolytic
processing of enamel matrix proteins. Here I show a model (Figure 27) which depicts the function of
Secretory stage
Maturation stage
WT MMP-20 null
Me ch a n i c a l
Pr op e r t i e s
Figure 27. Proposed model showing function of MMP-20 during secretory stage of
amelogenesis. MMP-20 degrades full-length amelogenin to form C-terminally cleaved P148
which further gets degraded by KLK-4 to crate long HAP crystals. In the absence of MMP-20,
full-length amelogenin persists in the enamel matrix till the maturation stage and is
occluded in the forming HAP crystals affecting their structural and mechanical properties.
Full-length amelogenin
P148
MMP-20
KLK-4
85

MMP-20 during the secretory stage of amelogenesis in which we propose a novel function for this
enzyme in the prevention of occlusion of proteins inside crystals during their formation.
There are still gaps in the full understanding of enamel biomineralization. In the future, we can analyze
Klk-4 null mouse to study the effects how Mmp-20 regulates normal HAP formation by preventing
occlusion of amelogenin at the secretory stage. This animal could also be used as a model to study
enamel crystal formation at the maturation stage. Another interesting study would be to evaluate the
presence of other enamel matrix proteins like ameloblastin and enamelin inside the HAP crystals of
Mmp-20 null mouse. Further in vitro studies are needed to improve both the structural and mechanical
properties of the enamel-like material grown in the chitosan-hydrogel system. In the future we would
like to eliminate chitosan from the remineralization system to study whether MMP-20 has any effect on
chitosan or vice versa. Our ultimate goal is to synthesize an enamel-like material which can be used by
dentists for the restoration of enamel defects.

86

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Abstract (if available)

Abstract Introduction: Defects in enamel formation are an important topic of study in the world of dentistry and biomineralization. The formation of tooth enamel is a typical example of cell- and matrix-mediated biomineralization. Enamel is composed of a well-organized hierarchical structure of long apatite crystals which are arranged in parallel in prisms or rod-like structures. Amelogenesis is a dynamic process involving hydroxyapatite crystal nucleation and maturation concomitant with formation of an enamel matrix of proteins like amelogenin, ameloblastin, enamelin, etc. and proteases like matrix metalloprotease-20(MMP-20) and kallikrein-4 (KLK-4). These proteases degrade the enamel matrix (EMP) proteins to create space for the hydroxyapatite crystals to grow in width and length. I designed a series of in vivo experiments to study the effects of the absence of MMP-20 on HAP crystal formation using Mmp-20 null mouse as my animal model. Using the information that I extracted from in vivo experiments, I designed in vitro experiments to re-grow artificial enamel-like materials with improved structural and mechanical properties. ❧ Materials and methods: 6 month old pig third and fourth molars were used for the extraction of enamel crystals and establishment of a protocol. Enamel crystals were isolated from adult mandibular and maxillary incisors from wild type and Mmp-20 null mouse. The isolated enamel was washed with a series of extraction buffers to wash off adsorbed proteins followed by their dissolution in 1M HCl. Qualitative and quantitative analysis of the proteins associated with isolated enamel crystals of WT and Mmp-20 null mouse was done by using UV-adsorption, immunochemistry, Raman spectroscopy and in situ Atomic force microscopy (AFM). The morphology and structural changes in the Mmp-20 null mouse as compared to the WT mouse was studied by Scanning electron microscopy (SEM), high-resolution Transmission electron microscopy (HRTEM) and Atomic force microscopy. ❧ The in vitro experiments were performed on extracted human 3rd molar tooth slices. Recombinant porcine amelogenin rP172 was dissolved in 2% chitosan followed by the addition of Na₂HPO₄ and CaCl₂ solution. The pH of the solution was adjusted to 6.5 with 1M NaOH. Recombinant human MMP-20 (rhMMP-20) was added to this solution at a ratio of 1:1000. The chitosan hydrogel alone was used as a negative control and the solution containing rP172 only was used as positive control. 30µl of each of these solutions were carefully applied to exposed enamel windows of the prepared tooth slices and air dried for 15 minutes. The tooth slices were then immersed in 30 ml artificial saliva solution with a fluoride (F⁻) concentration of 1ppm at 37℃ for 5 days. After the allotted time, the tooth slices were removed from the solution, sonicated in a water bath for 10 mins and air dried. Characterization of the newly formed layer was done by SEM, X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and mechanical testing. ❧ Results: UV-adsorption and immunochemistry showed that there was a significant difference in the amount of proteins in the enamel of Mmp-20 null mouse as compared to that of the WT mouse. The dissolved crystals in the Mmp-20 null mouse showed the presence of full-length amelogenin which suggests that in the absence of MMP-20

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Creator Prajapati, Saumya (author)

Core Title Role of MMP-20 in preventing protein occlusion in enamel apatite crystals: relevance in enamel biomineralization and biomimetics

School School of Dentistry

Degree Doctor of Philosophy

Degree Program Craniofacial Biology

Publication Date 07/01/2016

Defense Date 05/03/2016

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

Tag biomineralization,hydroxyapatite crystals,MMP-20,OAI-PMH Harvest,protein occlusion

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Moradian-Oldak, Janet (committee chair), Frenkel, Baruch (committee member), Haworth, Ian (committee member), Nutt, Steven (committee member), Paine, Michael L. (committee member)

Creator Email saumya.praj@gmail.com,sprajapa@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-260417

Unique identifier UC11280391

Identifier etd-PrajapatiS-4497.pdf (filename),usctheses-c40-260417 (legacy record id)

Legacy Identifier etd-PrajapatiS-4497.pdf

Dmrecord 260417

Document Type Dissertation

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Rights Prajapati, Saumya

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

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

Repository Name University of Southern California Digital Library

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