An amelogenin–chitosan matrix promotes assembly of an enamel-like
layer with a dense interface
Qichao Ruana, Yuzheng Zhangb, Xiudong Yanga, Steven Nuttb, Janet Moradian-Oldaka,⇑
aCenter for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA 90033, USA
bMork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA 90089, USA
a r t i c l ei n f o
Received 18 January 2013
Received in revised form 4 March 2013
Accepted 1 April 2013
Available online 6 April 2013
a b s t r a c t
Biomimetic reconstruction of tooth enamel is a significant topic of study in materials science and den-
tistry as a novel approach to the prevention, restoration, and treatment of defective enamel. We have
developed a new amelogenin-containing chitosan hydrogel for enamel reconstruction that works
through amelogenin supramolecular assembly, stabilizing Ca-P clusters and guiding their arrangement
into linear chains. These amelogenin Ca-P composite chains further fuse with enamel crystals and even-
tually evolve into enamel-like co-aligned crystals, anchored to the natural enamel substrate through a
cluster growth process. A dense interface between the newly grown layer and natural enamel was formed
and the enamel-like layer improved the hardness and elastic modulus compared with etched enamel. We
anticipate that this chitosan hydrogel will provide effective protection against secondary caries because
of its pH-responsive and antimicrobial properties. Our studies introduce an amelogenin-containing chito-
san hydrogel as a promising biomaterial for enamel repair and demonstrate the potential of applying pro-
tein-directed assembly to biomimetic reconstruction of complex biomaterials.
? 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Enamel is the exterior layer of the mammalian tooth and a hard
biomaterial with significant resilience that protects the tooth from
external physical and chemical damage . The remarkable
mechanical properties of enamel are associated with its hierarchi-
cal levels of structure from the nanoscale to the macroscale . The
building blocks of enamel, the enamel rods, are densely packed
arrays of elongated apatite crystals organized into an intricate
interwoven structure . Cellular activity and the protein-con-
trolled process of mineralization are key to achieving such pre-
cisely organized structures . The proteins that mediate the
mineralization of apatite crystals are gradually degraded and even-
tually removed during enamel maturation [1,3,4]. Mature enamel
is non-living and cannot regenerate itself after substantial mineral
loss, which often occurs as dental caries or erosion. Currently the
conventional treatments for carious lesions include refilling with
amorphous materials like amalgam, ceramics, or composite resin
. However, even after those treatments secondary caries often
arises at the interface between the original enamel and the filling
materials due to weakening adhesion over time . There is there-
fore a need for alternative restorative materials with improved
adhesion to the tooth surface. One such alternative is a synthetic
enamel-like material that can be prepared by biomimetic regrowth
on the enamel surface.
Various biomimetic systems have been developed to repair en-
amel defects, including liquids and pastes that contain nano-apa-
tite or different organic additives, for the remineralization of
early, sub-micrometer sized enamel lesions. A glycerine-enriched
gelatin system has been used to form dense fluorapatite layers
on human enamel [7,8]. Growth in small cavities of enamel-like
hydroxyapatite has been achieved in vitro , and a compacted
fluorapatite film with a prism-like structure was synthesized on
metal plates using a hydrothermal technique . Formation of
enamel-like structures under ambient conditions was also per-
formed in vitro using a liquid and pastes with different organic
additives [11–15]. Recently an electrospun hydrogel mat of amor-
phous calcium phosphate (ACP)/poly(vinylpyrrolidone) nanofibers
was developed for the in vitro remineralization of dental enamel
. These investigations constitute significant progress in the
study of enamel-like structures. Overall, however, biomimetic
strategies still face an ongoing challenge in the fields of dentistry
and material science.
In natural enamel the formation of apatite crystals occurs in an
amelogenin-rich matrix that plays a critical role in controlling the
oriented and elongated growth of apatite crystals [4,17–20].
enamel-like materials that contain nano- and microstructures
using amelogenin to control the crystallization of biomimetic
1742-7061/$ - see front matter ? 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
⇑Corresponding author. Tel.: +1 323 442 1759; fax: +1 323 442 2981.
E-mail address: firstname.lastname@example.org (J. Moradian-Oldak).
Acta Biomaterialia 9 (2013) 7289–7297
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calcium and phosphate [20–23]. The results have opened up the
promising possibility of remodeling complex enamel minerals in
an amelogenin-containing system.
Here we report development of a new amelogenin-containing
chitosan (CS-AMEL) hydrogel to synthesize an organized, enamel-
like mineralized layer on an acid-etched enamel surface used as
an early caries model. Compared with a previously developed ame-
logenin-containing system, CS-AMEL is easier to handle under clin-
ical conditions. It is biocompatible, biodegradable, and has unique
antimicrobial and adhesion properties that are practical for dental
applications [24–26]. Chitosan has been observed to have antimi-
crobial activity against fungi, viruses, and some bacteria, including
streptococci and lactobacilli, which are known as the principal eti-
ological factors of dental caries [27–29]. Therefore, we expect that
the ‘‘synthetic enamel’’ formed in the CS-AMEL hydrogel will have
antimicrobial properties that can prevent bacterial infection and
subsequent demineralization. In addition, chitosan is mucoadhe-
sive to both hard and soft surfaces . Importantly, the newly
formed crystals in the CS-AMEL hydrogel grow directly on the ori-
ginal enamel, achieving complete adhesion of the repaired layer to
the natural enamel with a dense interface. The robust attachment
of the newly grown layer demonstrated in the present work can
potentially improve the durability of restorations and avoid the
formation of new caries at the margin of the restoration.
2. Materials and methods
2.1. Amelogenin preparation
Recombinant full-length porcine amelogenin rP172 was ex-
pressed in Escherichia coli and purified as previously described.
The rP172 protein has 172 amino acids and is an analog of full-
length native porcine P173, but lacking the N-terminal methionine
as well as a phosphate group on Ser16 [20–23].
2.2. Tooth slice preparation
Human third molars (extracted following the standard proce-
dures for extraction at the Ostrow School of Dentistry of the Uni-
versity of Southern California and handled with the approval of
the Institutional Review Board) without any restored caries were
selected. Slices 0.1–0.2 cm thick (Fig. 1a) were cut longitudinally
using a water-cooled low speed diamond saw. To simulate early
caries lesions tooth slices were acid etched with 30% phosphoric
acid for 30 s and rinsed with deionized water.
2.3. Etched enamel repaired by the amelogenin-containing chitosan
The amelogenin-containing chitosan hydrogel was prepared by
mixing chitosan (medium molecular weight, 75–85% deacetylated,
Sigma–Aldrich) solution (960 ll, 1% m/v), Na2HPO4(15 ll, 0.1 M),
CaCl2(25 ll, 0.1 M) and amelogenin rP172 (200 lg), followed by
stirring at room temperature overnight. The pH value was adjusted
to 6.5 with 1 M NaOH. 20 ll of chitosan-based hydrogel was care-
fully applied to the enamel surface and dried in air at room temper-
ature. The tooth slices were then immersed in 30 ml of artificial
saliva (AS) solution (0.2 mM MgCl2, 1 mM CaCl2?H2O, 20 mM HEPES
buffer, 4 mM KH2PO4, 16 mM KCl, 4.5 mM NH4Cl, 300 p.p.m. NaF,
pH 7.0, adjusted with 1 M NaOH)  at 37 ?C for 7 days. After
the allotted time the tooth slice was removed from the solution,
rinsed with running deionized water for 50 s and air dried.
Scanning electron microscopy (SEM) imaging was performed in
a field emission scanning electron microscope (JEOL JSM-7001F),
operating at an accelerating voltage of 10 keV. X-ray diffraction
(XRD) patterns were recorded in a Rigaku diffractometer with Cu
Karadiation (k = 1.542 Å) operating at 70 kV and 50 mA with a step
size of 0.02?, at a scanning rate of 0.1?s?1in the 2h range 10–60?.
Thin sections (?100 nm) between enamel and the newly grown
layer for the TEM observations were prepared in a SEM/FIB (JEOL
JIB-4500) with an ion accelerating voltage of 30 kV. The device
was also equipped with an in situ lift-out system (Omniprobe
Autoprobe 200), which had a tungsten needle attached to a micro-
manipulator inside the FIB vacuum chamber. High resolution
transmission electron microscopy (HR-TEM) images were obtained
on a JEOL JEM-2100 microscope using an accelerating voltage of
200 keV. The hardness and elastic modulus were measured at 20
test points in each sample (n = 3) using a nano-indenter (Agilent-
MTS XP) with a Berkovich tip. Circular dichroism (CD) spectropo-
larimetry was performed using a J-815 spectropolarimeter (JASCO,
Easton, MD). The spectra were recorded between 190 and 260 nm
with a step size of 0.5 nm and a scan rate of 50 nm min?1. Fluores-
cence spectroscopy was performed using a PTI QuantaMaster QM-
4SE spectrofluorometer (PTI, Birmingham, NJ). The amelogenin
solutions were excited at 290 nm. The emission spectra were mon-
itored between 300 and 400 nm with a step size of 1 nm.
2.5. Antimicrobial evaluation
Human saliva was collected as described in the literature 
for the antimicrobial experimentation. Healthy adults were chosen
as the subjects for saliva collection. Subjects were asked to refrain
from eating, drinking, and oral hygiene procedures for at least 1 h
prior to collection. Subjects were given distilled drinking water and
asked to rinse their mouths out with it for 1 min. Five min after this
oral rinse the subjects were asked to spit into a 50 ml sterile tube,
which was placed on ice while more saliva was collected. The sub-
jects were instructed to tilt their head forward and let the saliva
run naturally to the front of the mouth; Upon collection of approx-
imately 5 ml of saliva from a subject the saliva sample was imme-
diately taken to the laboratory for processing. Twenty ll of saliva
were added to tubes with 1 ml of lysogeny broth (LB) medium
containing chitosan–amelogenin hydrogel or amelogenin, and then
incubated at 37 ?C overnight. The OD600of the overnight cultures
was measured using a Beckman DU-640 spectrophotometer.
2.6. Statistical analysis
Enamel remineralization experiments were repeated three
times. The mechanical tests and antimicrobial experiments were
conducted in triplicate and data were expressed as means ± stan-
dard deviations. For mechanical testing Student’s t-test was applied
to identify differences in the hardness and elastic modulus between
etched and repaired enamel (n = 3). For the antimicrobial experi-
ments (n = 3) the OD values were compared between control and
Fig. 1. (a) Optical micrograph of a tooth slice used in this work. (b) SEM image of an
acid etched enamel surface.
Q. Ruan et al./Acta Biomaterialia 9 (2013) 7289–7297
samples containing chitosan–amelogenin hydrogel or amelogenin
by the sametest. In all experimentsthe differences were considered
statistically significant at P 6 0.05 and highly significant at
P < 0.001. All the statistical analyses were carried out using Origin
8.0 (Origin Lab, Northampton, MA) and Microsoft Office Excel 2007.
3. Results and discussion
3.1. Enamel remineralization without CS-AMEL hydrogel
Dental caries is caused by an imbalance in the dynamic process
of demineralization–remineralization of enamel . Enamel
demineralization occurs at low pH caused by acids of bacterial ori-
gin. To produce artificial caries a tooth slice was etched with 30%
phosphoric acid. When examined the etched enamel crystals were
seen to be discontinuous and broken, resembling crystals from car-
in the saliva permit recovery of some lost enamel mineral, the
remarkably organized structure of enamel cannot be regained
without protein mediation. We prepared an AS solution to simulate
the oral environment for enamel remineralization. After soaking in
AS solution alone for 7 days a calcium phosphate coating with a
thickness of 1 lm had formed on the surface of enamel. As shown
in Fig. 2a and b, the remineralized crystals had a rod-like structure
and the coating was porous. A similar layer but with a thickness of
10 lm formed on the etched enamel soaked in chitosan hydrogel
withoutamelogenin. Thisremineralizedapatitelayeralso consisted
of loosely packed crystals with a porous structure (Fig. 2c and d).
These porous layers did not resemble natural enamel structure,
which has a high packing density of apatite crystals.
3.2. Enamel remineralization with CS-AMEL hydrogel
Fig. 3 shows the microstructures of human molar enamel and
the newly grown layer on an etched enamel surface soaked for
7 days in CS-AMEL hydrogel. At the nanoscale (Fig. 3a) natural en-
amel is made of highly organized arrays of apatite crystallites
growing preferentially along the c-axis, perpendicular to the sur-
face (black arrows in Fig. 3a). After mineralization for 7 days simi-
lar organized crystals were formed on the etched enamel surface
treated with CS-AMEL hydrogel. The crystals grown in CS-AMEL
hydrogel were composed of numerous nanorods oriented preferen-
tially along the c-axis with a diameter of ?50 nm, nearly parallel to
each other in the longitudinal direction (white arrows in Fig. 3b).
The newly grown layer, with a thickness of 15 lm, was tightly
bound to the surface of the natural enamel (Fig. 3b, inset). Exami-
nation at higher magnification revealed no obvious boundary at
the interface (Fig. 3c). The bulk of the newly grown layer contained
needle-like crystals that were bundled to form a fundamental orga-
nization unit analogous to that of natural enamel crystallites
(Fig. 3d and e). The HR-TEM image showed clear lattice fringes per-
pendicular to the nanorod axis with an interplanar spacing of
d = 0.3429 nm, in accordance with the distance between the
(002) crystal planes of hydroxyapatite (JCPDS 09-0432), which
suggests that the nanorods formed in CS-AMEL hydrogel grow in
the  direction (white arrow in Fig. 3e). Selected area electron
diffraction (SAED) of the newly grown layer resulted in an arc-
shaped pattern along the (002) diffraction plane, indicating a hier-
archical alignment of the c-axes of the newly formed crystals
The orientation and composition of the newly grown crystals
were further confirmed by X-ray diffraction (XRD) and energy dis-
persion spectroscopy (EDS) (Fig. 4). All of the diffraction peaks can
be readily indexed to hexagonal phase hydroxyapatite (JCPDS 09–
0432) crystals. The unsplit diffraction peak around 2h = 32? indi-
cates the poor crystallinity of newly formed apatite in the CS-AMEL
that the (001) faces are parallel to the surface (Fig. 4a), i.e. the crys-
tals align in an orderly fashion along the crystallographic c-axis, in
accordance with the microstructure seen by SEM and TEM observa-
tion (Fig. 3). EDS revealed the presence of calcium, phosphate and
fluorine ions in the newly grown layer (Fig 4b). The structural and
compositional analyses indicated that the newly formed layer con-
tain fluoridated hydroxyapatite with poor crystallinity .
Fig. 2. SEM images of the newly grown layer without amelogenin after remineralization in an artificial saliva solution for 7 days. (a, c) Top view; (b, d) side view. (a, b)
Without chitosan gel; (c, d) with chitosan gel.
Q. Ruan et al./Acta Biomaterialia 9 (2013) 7289–7297
3.3. Functions of chitosan and amelogenin in enamel remineralization
with CS-AMEL hydrogel
Comparing the morphologies of the remineralized layers
formed in the chitosan hydrogel with and without amelogenin,
we observed that disordered structures with a porous morphology
were formed without the protein (Fig. 2), but ordered enamel-like
structures were obtained in the presence of amelogenin (Fig. 3b–f).
These results indicate that amelogenin mediation is an essential
factor for the formation of orderly enamel-like structure in the
chitosan hydrogel system. Although chitosan hydrogels has also
been reported as a mineralization matrix because of its charged
surface , chitosan molecules were not found to affect the func-
tion of amelogenin during synthesis of the repaired layer.
The chitosan–amelogenin interaction was studied using CD and
fluorescence spectroscopy at pH 3.5, 5.5 and 8.0 (Fig. 5). All the CD
spectra of pure amelogenin showed negative ellipticities around
203 nm, which are characteristic of unordered polyproline type II
structures . At pH 3.5 the intensity of the minima gradually
increased and the trough shifted to a higher wavelength with
increasing ratios of chitosan to amelogenin, indicating a possible
change in the conformation of amelogenin in the presence of chito-
san. In the corresponding fluorescence spectra red shifts of the
emission maxima were also observed with increasing amounts of
chitosan, indicating the exposure of tryptophan residues in amelo-
genin (Fig. 5a) [37,38]. These changes in the CD and fluorescence
spectra clearly illustrated that there was a direct interaction be-
tween amelogenin and chitosan at pH 3.5. At pH 5.5 both the inten-
sity of the negative dichroic signals and the positions of their
minima were changed on the addition of chitosan to amelogenin,
however, there was no shift in the fluorescence spectra (Fig. 5b).
When the pH reached 8.0 it was difficult to find the dichroic signal
or the emission band in the CD and fluorescence spectra of amelo-
genin in association with chitosan (Fig. 5c). The results from the CD
and fluorescence spectra revealed that the interaction between
chitosan and amelogenin is dependent on pH. At pH values below
the pKa of chitosan (6.5)  the amino groups were almost
completely ionized, and the charge density of chitosan increased;
Fig. 3. SEM and TEM images of natural enamel and the newly grown layer after remineralization in amelogenin–chitosan gel for 7 days. (a) Microstructure of native enamel.
Black arrows indicate the crystallographic orientations of the apatite crystallites in native enamel. (b) After 7 days remineralization with chitosan–amelogenin hydrogel an
enamel-like layer had formed on the surface of etched enamel. (Inset) Thickness of the newly grown layer. Rectangles 1 and 2 represent the selected areas corresponding to
(b) and (c). White arrows indicate the apatite orientations in the newly grown layer. (c) The newly grown layer was firmly bound to the surface of the enamel. (d) Bundles of
organized crystals were found inside the repaired layer. The arrows indicate a typical bundle of parallel crystals inside the newly grown layer. (Inset) The homogeneous
surface of the repaired layer. (e) HR-TEM image of a rod-like crystal taken from the area outlined by the red rectangle on the crystal bundle in the inset. The arrow indicates
the crystallographic direction of an apatite crystal along the c-axis. The HR-TEM image represents a typical bundle of parallel crystals. (f) SAED image of the newly grown
layer. (Inset) TEM image of the repaired layer prepared by focused ion beam (FIB) milling.
Q. Ruan et al./Acta Biomaterialia 9 (2013) 7289–7297
thus chitosan interacted with amelogenin through electrostatic
interaction. In contrast, when the pH was higher than 5.5 the inter-
action with chitosan was weak because of its low solubility and
Therefore, under our experimental conditions (pH > 6.5) amelo-
genin is the crucial factor in controlling the oriented growth of
fluoridated hydroxyapatite crystals. Even so, the role of chitosan
is likely more than just as an amelogenin carrier. Chitosan in the
CS-AMEL system could provide effective protection from enamel
erosion because of its pH responsiveness. The development of car-
ies is associated with a continuous pH change in the plaque biofilm
due to the accumulation of acid by-products from metabolism of
fermentable carbohydrates [32,40,41]. As the pH decreases (in
the general range 5.5–5.0) positive hydrogen ions from the acids
bind to the negative phosphate and hydroxyl ions from enamel
mineral, leading to mineral loss. The potential advantage of having
chitosan present on the enamel surface is that the amino groups of
chitosan could capture the acid hydrogen ions, forming a positive
protective layer preventing the diffusion of hydrogen ions to the
mineral surface, as well as interacting with amelogenin to avoiding
amelogenin loss into the saliva. When the normal pH is restored (to
the range 6.3–7.0) by saliva  the weakly interacting amelo-
genin would be released from the chitosan to regulate the remin-
eralization of enamel.
Our HR-TEM and SEM analyses provide further insight into the
function of amelogenin in the remineralization of newly grown
mineral. In the original CS-AMEL hydrogel we observed linear
chains of ?10 nm nanoclusters, as shown in Fig 6a. The calcium
phosphate (Ca-P) clusters formed under Ca-P supersaturatedcondi-
tions are thought to be the building blocks of both amorphous Ca-P
as well as the apatitic mineral phase [43,44]. Generally, without a
stabilizing agent these Ca-P clusters aggregate randomly to form
plate-like mineral particles [45,46]. Indeed, we could not find any
orientedaggregation of Ca-P clusters in the original chitosan hydro-
gel in the absence of amelogenin. We suggest that the presence of
amelogenin provides an opportunity for stabilization of the precrit-
ical clusters at a minimum free energy, since the co-assembly of
amelogenin–Ca-P clusters imparts kinetic and thermodynamic sta-
bility to the system . As a result, and as in previous studies
[45,46], we suggest that amelogenin assemblies stabilized the Ca-
P clusters in the CS-AMEL hydrogel and guided arrangement of
the clusters into linear chains that eventually evolved into enam-
3.4. Continuous growth of newly formed crystals on the enamel
SEM images of the side view of a layer grown in CS-AMEL hydro-
gel for 3 days (Fig. 6b) indicate that the newly grown crystals are
mostly oriented perpendicular to the surface of the substrate and
in non-prismatic orientations (white arrows in Fig. 6b). The inter-
face betweenthe repaired layerand the enamel substrate,indicated
by the dotted line, reveals no apparent gap. To further explore the
interface microstructure we used a focused ion beam (FIB) tech-
nique to prepare TEM samples for higher resolution analysis.
Fig. 6c depicts a HR-TEM image of the interface where the new crys-
tals nucleate, clearly exhibiting lattice fringes from the (301) and
(10?3) planes of the enamel
d = 0.236 nm), as well as the (002) plane of the synthetic crystal
(d = 0.339). The corresponding fast Fourier transform (FFT) images
(inset in Fig. 6c) show two different patterns, indicating that the
crystals in the enamel and in the fused repaired layer grew with dif-
ferent orientations, which is consistent with the SEM observations
in Fig. 6b. Remarkably, the enamel and the newly grown crystals
fused togetherto form a seamless interface (black arrows in Fig. 6c).
Although the exact growth mechanism remains unresolved, it is
clear that amelogenin-stabilized clusters with orientated aggrega-
tion are crucial to the continuous growth of new crystals on the en-
amel. Recent research has shown that calcium-based biominerals
can be formed at a templating surface via stable pre-nucleation
clusters, with aggregation into an amorphous precursor phase
and transformation of this phase into a crystal [47,48]. Similarly
to these cluster growth models [48,49], the newly grown crystals
formed in CS-AMEL hydrogel in our experiments started with the
aggregation of pre-nucleation clusters leading to the nucleation
of amelogenin–Ca-P and then the development of oriented apatite
crystals. The possible repair processes are schematically presented
in Fig. 6d. Initially the pre-nucleation Ca-P clusters, stabilized by
amelogenin, aggregate to form linear chains in the CS-AMEL hydro-
gel. Subsequently parts of the cluster aggregates in contact with
the enamel surface become dense by adopting a closer packing of
the clusters. The continuation of this process leads to the formation
of an amorphous precursor phase that further fuses with enamel
crystals and ultimately transforms into crystalline apatite, which
is oriented along the c-axis as directed by amelogenin. As a result
the newly formed crystals continuously grow on the enamel crys-
tals and are oriented by amelogenin so that their long axes run per-
pendicular to the enamel surface, like natural enamel prisms .
crystal(d = 0.261 nmand
3.5. Bonding strength between the newly grown layer and enamel
The dense interface between the synthetic and natural enamel
crystals promoted strong bonding between the newly grown layer
Fig. 4. (a) XRD spectra of the newly grown layer after remineralization in a chitosan
gel with and without amelogenin for 7 days. (b) EDS spectrum of the repaired layer
after remineralization in a chitosan gel with amelogenin for 7 days.
Q. Ruan et al./Acta Biomaterialia 9 (2013) 7289–7297
and the tooth surface. Fig. 7 shows backscattered electron and sec-
ondary electron images of the newly grown layer after ultrasonic
treatment (42 kHz, 100 W) for 10 min. The results revealed that
the newly grown layer formed in the CS-AMEL hydrogel was
tightly bound to the enamel surface (Fig. 7a), and the organized
structure was unaffected by ultrasonic treatment (Fig. 7b). In con-
trast, following the same treatment we observed a large gap be-
tween the enamel and the repaired layer formed in a chitosan
hydrogel without amelogenin (Fig. 7c). In clinical dentistry bond-
ing strength is one of the most important attributes for enamel
restorative materials. Due to poor adhesion leading to gaps at the
enamel–restoration interface the currently available materials of-
ten have limitations in terms of their durability. These gaps in-
crease the possibility for bacterial leakage and secondary caries,
which are the main causes of restoration failure . In the present
study robust attachment of the newly grown layer formed in the
CS-AMEL hydrogel can potentially improve the durability of
restorations and avoid the formation of new caries at the margin
of the restoration.
3.6. Mechanical properties of the reconstructed enamel-like layer
repaired by CS-AMEL hydrogel
Fig. 8a shows the hardnesses and elastic modulus of healthy en-
amel, etched enamel, and the reconstructed enamel-like layer re-
paired by a chitosan hydrogel with and without amelogenin. The
hardness and modulus of a caries-free enamel slice were estimated
to be 4.0 and 70 Gpa, respectively , and both the hardness and
modulus were severely compromised by acid etching (nearly 88%
decrease in modulus and 98% decrease in hardness). After mineral-
ization in a chitosan hydrogel without amelogenin we observed
only slight increase in the hardness and modulus of the etched
enamel surface (Fig. 8a). Clearly the porous structure (Fig. 2c and
d) caused by conventional remineralization could not provide
Fig. 5. CD and fluorescence spectra measured at different mass ratios at pH (a) 3.5, (b) 5.5 and (c) 8.0, revealing that the interaction between chitosan and amelogenin is pH
Q. Ruan et al./Acta Biomaterialia 9 (2013) 7289–7297
satisfactory mechanical functions. However, after treatment with
amelogenin-chitosan hydrogel for 7 days the hardness and modu-
lus of the etched enamel surface increased significantly (P < 0.001).
The modulus increased by nearly four times and the hardness was
increased by nearly nine times (Fig. 8a). Although the mechanical
properties were not the same as those of native enamel, the re-
paired enamel treated with CS-AMEL hydrogel showed superior
properties compared with the control (without amelogenin) due
to the well-organized crystal orientation . The amelogenin
and chitosan residues in the repaired layer may limit its mechani-
cal performance, which could potentially be improved by removal
of the organic material using proteolytic enzymes [52–54]. More-
over, in clinical practice the mechanical properties of the repaired
layer could be further improved by repetitive application of CS-
AMEL hydrogel in order to achieve a thicker repaired layer. Further
work is needed in order to assess the stability of the CS-AMEL
hydrogel in the oral cavity.
3.7. Antimicrobial properties of the CS-AMEL hydrogel
Human saliva used as a source of bacteria was cultured in LB
medium to examine the antimicrobial properties of CS-AMEL by
observing the optical density (OD) values and turbidity (Fig. 8b).
After overnight culture the medium without chitosan gel was opa-
que due to presence of bacteria, while the medium with chitosan
gel was clear (inset in Fig. 8b). The OD value was significantly re-
duced when CS-AMEL hydrogel was added to the LB medium
(P < 0.001, Fig. 8b). These results demonstrate that the CS-AMEL
hydrogel can effectively inhibit bacterial growth. We believe that
the antimicrobial effect of the CS-AMEL hydrogel was attributable
to chitosan. Chitosan has been observed to have antimicrobial
activity against a wide variety of bacteria, including streptococci
and lactobacilli, which are known as the principal etiological fac-
tors in dental caries. Moreover, chitosan has several advantages
Fig. 6. (a) TEM image of the original CS-AMEL hydrogel showing the elongated nanochain-like structure (white arrows). (b) Cross-section SEM image of the repaired layer
after remineralization in amelogenin–chitosan gel for 3 days fused to the surface of the natural enamel. The white and black arrows indicate the crystallographic orientations
of the crystals in the newly grown layer and natural enamel, respectively. The dotted line shows the boundary of the natural enamel and the newly grown layer. (c) HR-TEM
image of the interface between the enamel and regrown crystal, showing seamless growth of the repaired crystal on the enamel. The black arrows indicate the interface
between regrown and enamel crystals. (Inset) FFT images corresponding to enamel and regrown crystals. (d) Schematic illustration of the enamel repair process.
Fig. 7. SEM images of reconstructed enamel-like layers after ultrasonic treatment.
(a) Backscattered electron image of a cross-section and (b) second electron image of
the surface of an ultrasonically treated newly grown layer obtained in chitosan–
amelogenin hydrogel. (Inset) The typical morphology of the surface at higher
magnification. (c) Backscattered electron image of a cross-section of an ultrason-
ically treated newly grown layer obtained in chitosan hydrogel without
Q. Ruan et al./Acta Biomaterialia 9 (2013) 7289–7297
over other types of antiseptic agents, including a higher antibacte-
rial activity, a broader spectrum of activity, a higher killing rate,
and lower toxicity to mammalian cells . Therefore, we expect
that clinical application of the CS-AMEL hydrogel can not only fulfil
superficial enamel reconstruction, but also effectively suppress
bacterial infection and subsequent demineralization.
In summary, taking advantage of the potential of amelogenin to
control the organized growth of apatite crystals and the potential
antimicrobial activity of chitosan we have developed a new amelo-
genin-containing chitosan hydrogel for superficial enamel recon-
struction. Amelogenin assemblies stabilized Ca-P clusters in a CS-
AMEL hydrogel and guided their arrangement into linear chains.
These amelogenin–Ca-P composite chains further fused with en-
amel crystal and eventually evolved into enamel-like co-aligned
crystals, anchored to the natural enamel substrate through a clus-
ter growth process [47,48]. The continuous growth of crystals
formed an excellent bond between the newly grown layer and
the enamel. Furthermore, the hardness and elastic modulus of
etched enamel were increased by nine and four times after treat-
ment with CS-AMEL hydrogel. We anticipate that this chitosan
hydrogel will provide effective protection against secondary caries
because of its pH-responsive and antimicrobial properties. Our
studies introduce a promising CS-AMEL hydrogel method for
superficial enamel repair and demonstrate the potential of apply-
ing protein-directed assembly to the biomimetic reconstruction
of complex biomaterials.
This research was supported by NIH-NIDCR grants DE-13414
and DE-020099 to J.M.O. The authors would like to thank Dr Sisi
Liu for assistance with the transmission electron microscopy, and
the Center for Electron Microscopy and Microanalysis (CEMMA)
at USC for electron microscopy.
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