Ceramics International 43 (2017) 11390–11402

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Ceramics International
journal homepage: www.elsevier.com/locate/ceramint

Biomimetic chitosan-hydroxyapatite hybrid biocoatings for enamel MARK

remineralization
⁎
Agripina Zahariaa, Viorica Muşatb, , Elena Maria Anghelc, Irina Atkinsonc, Oana-
Cătălina Mocioiuc, Mariana Buşilăb, Viorica Ghisman Pleşcanb
a
Faculty of Dentistry, Ovidius University of Constanta, 124 Mamaia Blvd., 8700 Constanta, Romania
b
Centre of Nanostructures and Functional Materials-CNMF, Department of Materials Science and Engineering, “Dunărea de Jos” University of Galati, 111
Domnească, 800201 Galaţi, Romania
c
Institute of Physical Chemistry “Ilie Murgulescu” of Romanian Academy, Spl. Independentei 202, 060021 Bucharest, Romania

A R T I C L E I N F O A BS T RAC T

Keywords: Development of biomimetic ceramic-based materials is currently a challenge in dental tissue engineering.
B. Surfaces Synthetic hybrid chitosan (CS)-hydroxyapatite (HAP) layers are regarded as candidates for teeth remineraliza-
B. Microstructure tion, protection against further demineralization ensuring also antibacterial activity. Thus, the aim of this work
C. Hardness was to obtain new biomimetic CS-HAP layers for restoration of damaged mature enamels and to pursue
D. Apatite, Biomimetic enamel-like layer
morphological, compositional, structural and hardness modifications of the grown layers by immersion for 4, 7
and 10 days into artificial saliva (AS) under CS-Emdogain (EMD) hydrogel action. SEM-EDX, HRTEM-SAED,
FTIR and micro-Raman findings indicated formation of carbonate-substituted HAP, B-type, with c-axis
orientation in the newly formed CS-HAP coatings. Prolonged immersion span of 10 days caused increasing
CS content in the superficial grown layer while carbonate content diminished. Optimum Ca/P ratio (1.85 at%)
and hardness of 2.48 GPa were recorded for seven days growth using CS-EMD hydrogel. Subtle changes in HAP
lattice parameters were recorded for 10-day grown layer while c-axis orientation of HAP crystals at mesoscale
was preserved. Mechanism of CS interaction during in situ biomimetic synthesis and self-assembly of HAP
crystals under CS-EMD hydrogel presence is also discussed.

1. Introduction makes it hard and abrasion-resistant but also crack-tolerant and

Current challenges in biomaterials science consist in obtaining
complex multifunctional materials by tissue engineering. During
recent years [1,2], biomimetic strategy has been considered an ideal
solution for growing adherent remineralized layers onto mature
enamel affected by sub micrometer erosion and initial carious
lesions, whereas problems of secondary effects as marginal leakage,
hypersensitivity, weak adhesion over time and secondary caries at
the interface between the original enamel and the filling materials
were eliminated. Thus, growth of synthetic enamel-like materials
with good adhesion and dense interface to the original enamel of
teeth has been of high interest [3].
Natural dental enamel, a hybrid biomaterial containing about 95%
hydroxyapatite (HAP) with general formula Ca10(PO4)6(CO)z(OH)2-uFu,
about 4% water and only 1% organic compounds (enamel proteins) [4],
is characterized by anisotropic structure and mechanical behavior, which
chemically resistant [5]. In enamel tissue, the HAP crystals are organized
into a complex hierarchical structure, from nanometer scale to macro-
scopic level [1]. At nanoscale, enamel consists of nanorod-like HAP
crystals of few tenths of nanometers in diameter [6] with c-axis
preferential growth orientation, perpendicular to the dentinal–enamel
junction [5,7]. At the mesoscale level, the HAP nanocrystals assemble
themselves into three types of architectural units named rods (prismatic
enamel), interrods (interprismatic enamel) and aprismatic enamel,
which interconnect in well-organized woven microstructures with com-
pact arrangement from meso to macro scale [8]. This structure extends
through the thickness of the enamel, from dentin-enamel interface to the
tooth surface [9], providing the unique mechanical properties of the
dental enamel [10]. However, biomimetic regeneration of the complex
and highly organized enamel structure and morphology is not naturally
possible after the substantial mineral loss due to acellular structure and
protein-free composition of the enamel. In fact biomimetic regeneration,

⁎
Corresponding author.
E-mail address: viorica.musat@ugal.ro (V. Muşat).

http://dx.doi.org/10.1016/j.ceramint.2017.05.346
Received 4 April 2017; Received in revised form 14 May 2017; Accepted 29 May 2017
Available online 31 May 2017
0272-8842/ © 2017 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
A. Zaharia et al. Ceramics International 43 (2017) 11390–11402

Table 1
Structural, compositional and mechanical parameters of the new biomimetic layers and the natural (R1) and acid-etched (R2) enamels.

Sample Soaking 2θ (°) FWHM (°) Ca/P ratio (EDX A1070/A960 Crystallite Size χc (002) Lattice parameters Hardness
period days) (002) data) (at%/at%) (Raman data) (002) (nm)
a=b (Å) c (Å) (GPa)

Biomimetic CS-HAP hybrid layers grown under CS-EMD hydrogel

S2-1 4 25.93 0.3551 2.01 ± 0.05 0.1167 24.0 (1.0) 0.31 9.366(19) 6.885(9) 2.41
S2-2 7 25.92 0.3304 1.85 ± 0.05 0.1190 25.7(1.6) 0.35 9.416(15) 6.884(13) 2.48
S2-3 10 25.98 0.3371 1.33 ± 0.06 0.1057 25.3 (0.2) 0.36 9.399(5) 6.883(3) 2.15

Biomimetic HAP layers grown under EMD hydrogel

S1-1 4 25.95 0.3447 2.52 ± 0.06 0.1312 24.7 (0.5) 0.34 9.332(13) 6.883(7) 2.61
S1-2 7 25.97 0.3335 1.85 ± 0.10 0.1256 25.5(0.4) 0.37 9.355(15) 6.884(7) 2.76
S1-3 10 25.95 0.3360 1.95 ± 0.10 0.0708 25.3(0.4) 0.36 9.409(9) 6.897(11) 2.92

Natural enamel substrate, reference samples (R1-as selected, R2-acid etched)

R1 – 26.03 0.3128 1.71 ± 0.05 0.0472 27.2(0.5) 0.46 9.414(2) 6.902(19) 3.18
R2 – 25.96 0.3755 1.28 ± 0.03 0.0587 22.7(1.0) 0.28 9.347(8) 6.884(8) 1.82
Hydroxylapatite –crsytal system hexagonal; space group P63/m(176)-00-09-0432 9.418 6.884

mimicking natural process of enamel formation in vivo, is an organic
matrix mediated process of oriented nucleation and growth of HAP
crystallites [11]. Biomineralization occurs at the interface with the
organic matrix being controlled by molecular interactions between
organic components and inorganic mineral [12]. Various organic matrix
systems (liquids and hydrogels), named enamel matrix derivatives
(EMDs), have been investigated as biomimetic systems for enamel
remineralization [13]. Special attention was paid to proteins as the most
promising enamel matrix derivatives for regeneration of the complex
enamel-like structured hydroxyapatite layers. Ruan et al. [3] proposed
porcine amelogenin rP172 for organized growing of the enamel-like
apatite crystals. Lately Cao et al. [13] has used for the first time a
commercially available Emdogain product used in regeneration of soft
tissues, product based on specialized proteins containing mostly amelo-
genin [14], as enamel matrix derivative for the enamel-like tissue
regeneration. However, the use of the Emdogain® in biomimetic
synthesis of apatite-based coatings for restoration of mature enamel
lesions is not fully explored.
An important strategy in prosthetics is to combine biocompatibility
and antimicrobial properties of new restorative materials. Thus,
chitosan, an osteoconductive, biodegradable and nontoxic material
for human immune system [15] has already been used in dental
medicine for dental caries prevention, growing of bioactive coatings
required by osseo integration of dental and orthopedic implants [16],
bactericidal and/or bacteriostatic activity against various pathogenic
bacteria in stimulation of wound healing process [17]. At pH in the
range of 5.5-5.0, positive charged layer of protonated amino group of
chitosan interacts with amelogenin and hence prevents diffusion of
hydrogen ions towards mineral surface and enamel demineralization
[1,18,19]. Biomimetic systems containing nano-apatites and EMD-
chitosan have been proposed in preventive dentistry for enamel
remineralization [1,12,17]. There are some challenges in using Cs-
Amel hydrogel in obtaining new potential restorative bioceramic
materials for prosthodontics, among which the preservation of highly
organized rod-interrod enamel-like structure responsible for unique
combination of the enamel properties by controlling its synthesis [3].
This paper focuses on systematic study of compositional, structural
and nano- to mesoscale morphological changes occurring between 4
and 10 days of biomimetic growth of synthetic HAP and CS-HAP layers
grown directly onto demineralized natural enamel by using commercial
Emdogain as source of proteins in preparation of extracellular hydro-
gels through a modified method. Variation of the surface microhard-
ness of the newly biomimetic layers grown without or under chitosan
presence is also discussed.
2. Materials and methods
2.1. Reagents and acid-etched tooth slices preparation
Commercially available Straumann® Emdogain (0,15 mL syringe,
30 mg/mL EMD) from Basel consisting of amelogenin-based matrix
premixed with propylene glycol alginate was used as source of enamel
matrix derivative (EMD) proteins. 75–85% deacetylated medium
(190–310 kDa) molecular weight chitosan powders, purchased from
Sigma-Aldrich, was used for CS-EMD hydrogel preparation. The
commercial Blue Etch 10 mL, 36% o-phosphoric acid gel fabricated
by PPH Cerkamed was used for etching the human molars slices. The
other chemicals, from Sigma-Aldrich and Merck, were “pro analysi”
(p.a.) or “chemically pure” (CP) grade.
Selected human molars without caries or restored caries, collected
from local dental clinics (‘Ovidius’ University of Constanta, Faculty of
Dentistry) according to the ethic protocol with information and
consented of patients, were treated with sodium hypochlorite solution
(3 wt%) and phosphate buffer saline to remove bacteria. The clean
molars were cut longitudinally (approximately 2 mm thick slices) using
low speed diamond device under water cooling. The slices were
sonically cleaned with demineralized water and then demineralized
for 60 s using Blue Etch acid. After deminetalization, the slices were
washed and rinsed 3 times for 2 min with deionized water in
ultrasound bath, air dried and stored at 4 °C before remineralization.
2.2. EMD and CS-EMD hydrogels matrices preparation
The preparation method of EMD and CS-EMD hydrogels mimick-
ing extracellular matrix was inspired by the protocol developed by
Ruan and Moradian-Oldak [3]. Unlike this study, we used as source of
amelogenin the commercial Straumann® Emdogain gel product cur-
rently used in regeneration of soft tissues. There are also other
differences between our method and that used by Ruan et al., i.e. the
use of 1% acetic acid solution during the preparation of EMD hydrogel
matrix in order to achieve similar conditions as in the case of CS-EMD
hydrogel matrix preparation, and a higher protein concentration in the
final hydrogel matrices. These issues also differentiate our protocol
from that reported in the few studies using Emdogan as source of EMD
proteins [13].
EMD hydrogel used for biomimetic growth of HAP samples from
S1-series (Table 1) was obtained by adding 0.15 mL of EmdogainR over
960 μL of 1% (v/v) acetic acid solution and mixing until the solution
was clear. To this solution, 25 μL of 0.1 M CaCl2 and 15 μL of 0.1 M
Na2HPO4 solutions were added and the pH of the EMD solution was

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adjusted to 6.5 by adding 1 M NaOH solution to obtain the hydrogel
matrix. Concentration of about 3.5 mg/mL EMD in the final hydrogel
matrices was achieved.
CS-EMD hydrogel used for biomimetic growth of CS-HAP of
samples from S2-series (Table 1) was obtained by adding 0.15 mL of
EMD over 960 μL of 1% chitosan solution, and then mixed for 5 min
using a vortex until the solution was clear. 25 μL of 0.1 M CaCl2
solution was added to the resulted solution followed by 15 μL of 0.1 M
Na2HPO4 solution and mixing for 5 min. The pH of the CS-EMD
solution was adjusted to 6.5 by carefully adding 1 M NaOH solution to
obtain the hydrogel matrix. The chitosan solution was prepared by
dissolving 1% (w/v) chitosan in a 1% (v/v) acetic acid solution followed
by stirring at 80 °C. After cooling to RT, the solution was filter using a
0.8 µm filter.
2.3. Artificial saliva (AS) solution preparation
The artificial saliva solution (AS) was prepared according to the
recipe proposed by Fletcher et al. [20] as follows: 0.2 mM MgCl2, 1 M
CaCl2, 4 mM Na2HPO4, 16 mM KCl and 4.5 mM NH4Cl were dissolved
in 20 mM HEPES (4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic
acid) buffer. The pH of the resulted solution was adjusted to 7.0 with
NaOH and solution was stored at 4 °C. Sodium fluoride (300 ppm) was
added at the solution before using.
2.4. Biomimetic growth of HAP and CS-HAP layers
For biomimetic growth of HAP and CS-HAP layers onto the acid-
etched enamel slide substrates, 20 μL hydrogel of EMD (Series S1) or
CS-EMD hydrogel (Series S2) were applied to cover the acid-etched
surface, dried at room temperature, transferred into a beaker contain-
ing AS solution and immersed at 37 °C for 4, 7 or 10 days, without
replacing the hydrogels or AS solution. At the end of the immersion
period, the tooth slices were removed, washed and air-dried. The
prepared biomimetic HAP and CS-HAP samples are presented in
Table 1.
2.5. Characterization of biomimetic HAP and CS-HAP layers
2.5.1. Morphology and composition characterization
The morphology and elemental composition of the investigated
samples were examined by scanning electron microscopy coupled with
energy dispersive X-ray (SEM/EDX) spectroscopy using a FEI Q 200
microscope in low vacuum. Before examination, the samples were
coated with 4 nm thick conducting layer of Au using a SPI-Module™
sputter coater system.
2.5.2. Crystalline structure characterization
Transmission electron microscopy (TEM) end selected area electron
diffraction (SAED) analysis was realized using a Philips CM120ST
microscope operated in transmission mode at 300 kV with TEM point
resolution of 2 Å and line resolution of 1 Å. Sample preparation
consisted in cutting cross-section slices with thicknesses between
1 ± 0.1 mm, followed by mechanical thinning and final ion etching up
to a thickness about 100 nm. For phase identification and calculation of
crystallographic parameters, the indexing presented in literature and
Cohen method with modified Riley-Nielson function has been used
[21].
The XRD patterns were collected by means of a Rigaku diffract-
ometer type Ultima IV equipped with thin film attachment for grazing
incidence X-ray measurements, at an incidence angle ω = 0.3°. The
source of the X-rays was a Cu tube (λ = 1.54056 Å) operating at 40 kV
and 30 mA. XRD data were recorded with a step size of 0.02 and a
speed of 5° (2θ)/min over a range of 10–80°. Rigaku's PDXL software
package connected to the ICDD database was used for the phase
identification and crystallite size calculation. XRD pattern was fitted
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using Whole Pattern Powder Fitting (WPPF) method [22]. The
diffraction peak profiles were modeled by a split-pseudo-Voigt function
and B-spline background model. Crystallite size, D, has been calculated
using Scherrer's formula along (002) direction, according to the
formula:
k ∙λ
D=
(FWHM )·cos (θ ) (1)
where: k is the shape factor taken as 0.94 for a spherical crystal with
cubic symmetry and width of the diffraction peak measured at FWHM
[23], FWHM is full width at half maximum of the intensity vs. 2θ
profile, λ is wave length of the Cu Kα radiation (1.54056 Å) and θ is
Bragg's diffraction angle.
The crystallinity degree, χc, corresponding to the fraction of
crystalline phase present in the sample has been calculated with the
relation [24]:
⎛ ⎞3
K ⎟
χc = ⎜⎜ ⎟
⎝ β(002) ⎠ (2)
where K is a constant (for a great number of hydroxyapatites, K is 0.24)
and β(002) is the peak (002) full width at half minimum (in degrees).
2.5.3. Spectroscopic structural characterization
Fourier transform infrared (FTIR) spectra of the investigated
samples were recorded in transmission mode, without other sample
preparations, within 400–1300 cm−1 domain with a sensitivity of
4 cm−1 using a Thermo Nicolet 6700 spectrometer.
Unpolarized Raman spectra of the enamel samples under discus-
sion were recorded by means of a LabRam HR spectrometer (Jobin-
Yvon–Horiba) within 50–4000 cm−1 range. The 514 nm line of an Ar+
laser was used as exciting radiation through a 100× objective of an
Olympus microscope in a backscattering geometry and at a confocal
hole of 200 µm and the grating of 1800 lines/mm. With a laser spot at
the surface pf the sample smaller than 2 µm spectral resolution was
better than 2 cm−1. Power of the laser was decreased to 8 mW to avoid
sample heating and degradation. The resulting spectra were back-
ground corrected and curve fitted by Gaussian-Lorenzian profile using
Igor 6.20 software.
2.5.4. Mechanical characterization
Microhardness measurements of the investigated films were per-
formed using a standard tester with a flat-field microscope type PMT-3,
LOMO, Russia, at X500, upgraded with digital camera and images
acquisition software. The indentation measurements were performed
with constant loads of 100 g for a period of 15 s. Five indentations were
taken for each microscopic site with specific aspect. Microhardness
calculations were carried out according to the following equation [25]:
P
H = 1854x
C2 (3)
where H is the microhardness, P is the applied mechanical load, and C
is the diagonal magnitude [µM] of the indentation print. The length of
diagonal was determined using the application software Optika Vision
Lite 2.1 on the captured image of tested surface.
3. Results and discussion
3.1. Nano- to mesoscale morphology and composition
Fig. 1 shows SEM images of the natural enamel samples before and
after acid-etching. At the nanoscale, the natural enamel sample (R1)
consists of ordered arrays of HAP nanorods with diameter of about 30–
40 nm, grown perpendicularly to the tooth surface, which assemble
itself at mesoscale level into prismatic rods and interrods, woven into
complex hierarchical structure. When examined the surface of etched
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Fig. 1. SEM images of tooth enamel (R1) and acid-etched enamel (R2) surfaces.
enamel (R2) used as substrate for the biomimetic growth, nanorods
were seen to be discontinuous and broken, compared with those seen
on natural enamel (R1) (Fig. 1). SEM images of the newly HAP layers
formed within ten-day immersion into AS under EMD hydrogel matrix
(Fig. 2) show nanoscale and hierarchical mesoscale structures similar
with natural enamel (R1 in Fig. 1). Parallel bundles of nanocrystals of
about 25 nm in diameter were self-assembled into central rods with
diameters about 3–4 µm and marginal rod sheath with appearance of
“keyhole-like” mesostructures [6] of about 6–8 µm in diameters
(Fig. 2). The increase of the growth period from 4 to 7 days causes
an increase in the length of the nanorod crystals, while ten-day growth
lead to morphological transformation of the nanocrystals from needle-
like (S1-2) to belt-like and formation of outer spherical particles not
bonded to inner particles, that indicates that the concentration at the
surface of the newly HAP layer became saturated compared to the
concentration of the AS (S1-3). The belt-like crystals self-assembled
side-by-side [6,7,19] into“fish-scale” morphology of the marginal rod
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sheaths (Fig. 2). Unlike the layers grown in the absence of CS, the
newly grown hybrid layers under CS-EMD hydrogel action shows
gradual morphological changes (Fig. 3). Small aspect ratio (height/
diameter) nanorod crystals with diameter of about 40 nm perpendicu-
larly grown to the surface can be observed after four-day growth (S2-1).
The hybrid layer grown during seven-day (S2-2) shows a new rod
architecture consisting of nanocrystals arranged in parallel to each
other and embedded together into an enamel prism-like structure
elongated in the direction of the c-axis, instead of bundles of individual
nanorods. Despite these morphological changes at the nanoscale, the
mesoscale hierarchical structure is preserved. After 10 days of immer-
sion in AS under CS-EMD hydrogel, the SEM images show major
morphological changes. The elongated-shaped nanorods formed during
seven-day growth (S2-2) have been transformed into a compact
structure of spherical-shaped grains (150–300 nm in diameter) (S2-3
in Fig. 3).
The new enamel-like layers formed under EMD hydrogel show an
A. Zaharia et al.
Fig. 2. SEM images of the biomimetic HAP layers grown under EMD-hydrogel for 4 days (S1-1), 7 days (S1-2) and 10 days (S1-3).
increase of P and Ca content when increasing the period of immersion
into AS from 4 to 10 days (Fig. S1 from Electronic Supplementary
Information/ESI) and Ca/P ratio (Table 1) over the value for stoichio-
metric apatite (1.67) [26], suggesting presence of the carbonated HAP-
type B (Ca10(PO4)6−y(CO3)y(OH)2) [27] with degrees of substitution (y)
of about 2 after four-day (S1-1), or around 1 after seven-day (S1-2) and
ten-day (S1-3). When comparing with the layers grown without CS, a
continuous decrease of Ca/P ratio, from 2.01 ± 0.05 to 1.33 ± 0.06, was
observed with increasing the remineralization time under CS-EMD
hydrogel (Table 1), suggesting some relevant compositional changes
associated to the morphological modifications illustrated in Fig. 3.
Thus, a shift of CA/P ratio from values higher than that of stoichio-
metric HAP phase to 1.33 ± 0.06 indicates a shift from carbonated B-
type HAP structure formed during the first seven-day (S2-1 and S2-2)
to calcium-deficient hydroxyapatite phase [28] formed after 10-day
(S2-3) (Table 1). The new HAP layers formed without CS showed an
increase content of P and Ca with increasing the period of immersion
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into AS from 4 to 10 days (Fig. S1 from ESI). Increased amounts of
fluorine, sodium and potassium, or carbon, nitrogen and oxygen were
observed for samples immersed for 10 days in the presence of EMD
(S1-3) and CS-EMD hydrogel matrices (S2-3), respectively (EDX
spectra in Fig. S1 from ESI). From the SEM images and associated
EDX elemental maps (Fig. 4), one can observe a correlation between
the increased concentration of the above mentioned chemical elements
and morphological changes occurring after ten-day immersion in AS on
the top of the newly grown layers. For the layer formed without CS (S1-
3), high concentrations of F, K and Na were observed in the areas
(dotted line) of agglomerations of spherical particles with diameters in
the range of 400–600 nm (Fig. 4a). Increased F content suggests the
formation of spherical fluoroapatite phase during a longer immersion
in AS, by superposition of a secondary nucleation process from solution
[29]. The presence of Na+ and K+ associated to this sample (Fig. 4a)
also suggests a possible substitution of calcium ions [30]. For the
hybrid layer formed in the presence of CS (S2-3), increased concentra-
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Fig. 3. SEM images of the biomimetic CS-HAP hybrid layers grown under CS-EMD hydrogel for 4 days (S2-1), 7 days (S2-2) and 10 days (S2-3).

tions of C, N and O observed in the area (dotted line) of rod sheath tion patterns (Fig. 5c). The new layers grown during four and seven-
(Fig. 4b) suggest, in agreement with literature data [31], higher day (S2-1 and S2-2) show (002) diffraction line weaker and broader
chitosan content in superficial rod sheath areas. than (211) line, suggesting smaller scale ordered HAP crystals aligned
along the c-axis [32]. The (002) line of HAP phase became the most
3.2. Crystalline and molecular structure
intense in the pattern of the hybrid layer grown for ten-day (S2-3),
showing the presence of mesoscale extended ordered HAP crystals
The synthetic layers grown without CS display characteristic X-ray
aligned along the crystallographic c-axis. The values of the crystallinity
diffraction lines corresponding to hexagonal HAP phase (Fig. 5)
degree, lattice parameters and crystallite size of the new grown HAP-
according to the standard card of HAP (JCPDS files, PDF no. 01-
based layers, calculated from the broadening of the (002) diffraction
072-1243, 01-086-0740). HAP crystals exhibit high tendency to grow
line according to the Eq. (1) [22,24], are presented in Table 1. The
along c-axis demonstrated by a continuous increase of (002) diffraction
crystallite size, lattice parameters and crystallinity degree increased
line intensity with the growth duration (Fig. 5b). The presence of sharp
during the first seven days of immersion, but remained bellow the
and intense 002 and 004 diffraction lines indicates that the newly
values for the natural enamel (Table 1). A higher growth of lattice
grown crystals aligned along the crystallographic c-axis were extended
parameter "a" as compared with the parameter "c" can be highlighted
at mesoscale [26], in accordance with the microstructure observed by
when the duration of immersion into AS increases from four to seven
SEM (Fig. 2).
days (sample S2-2 in Table 1), that indicating a more pronounced
CS-HAP hybrid layers formed under CS-EMD hydrogel also consist
increase of the crystallite in [100] direction (diameter) than in the
of hexagonal phase HAP nanocrystals, as confirmed by X-ray diffrac-

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Fig. 4. SEM images and EDX elemental maps of biomimetic layers grown during 10 days under EMD hydrogel (a) and CS-EMD hydrogel (b).

Fig. 5. XRD patterns of natural enamel (R1), acid-etched enamel (R2) and biomimetic HAP layers (b) and CS-HAP hybrid layers (c).

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Fig. 6. HRTEM cross-section images of the acid-etched natural enamel R2 (a), S1-2 biomimetic HAP layer grown without CS (b) and S2-1 biomimetic CS-HAP hybrid layer grown with
CS (c).
[001] direction (height). This result is consistent with the SEM results
that indicated an increased diameter of the nanocrystals embedded in
the CS matrix, inside the hybrid composite CS-HAP nanorods in the
sample S2-2 (Fig. 3). A continuous growth trend for crystallinity degree
with the increase of immersion period in AS up to ten days was
observed for the synthetic CS-HAP layers (Table 1), suggesting a
positive effect of CS molecules concerning the ordered self-assembling
of HAP nanocrystals.
Fig. 6 illustrates HRTEM images taken from the acid-etched natural
enamel (Fig. 6a) and interface between the acid etched enamel and
newly grown remineralized layers formed under EMD (Fig. 6b) and CS-
EMD (Fig. 6c) hydrogels. Each HRTEM figure includes insets corre-
sponding fast Fourier transform (FFT) pattern (at the top) for crystal
orientation and selected area for electron diffraction spots rings
(SAED) pattern (at the bottom of the figure). The SAED pattern of
acid-etched enamel (R2) (inset of Fig. 6a) shows well defined spots
rings (SAED) pattern associated to single polycrystalline enamel phase
identified as hydroxyapatite (HAP) [21]. SAED patterns of the remi-
neralized layers in EMD and CS-EMD hydrogels show diffraction
figures characteristic to polynanocrystalline material identified as
hydroxyapatite (HAP), consisting of small spots on a ring, each spot
arising from Bragg reflection of an individual nanocrystallite together
with some amorphous scattering feature films (insets of Fig. 6b and c).
Higher contribution of amorphous phase was observed in the case of
the four-day grown hybrid layer under CS-EMD hydrogel (inset in
Fig. 6c). Fast Fourier transform (FFT) measurements have revealed
(100) and (010) HAP crystalline planes for the demineralized enamel
substrate (inset in Fig. 6a) and different orientation of the newly grown
HAP crystals in the remineralized enamel layers with respect to the
natural HAP nanocrystals of the enamel substrate (insets in Fig. 6b and
c). The measured angle between (220) plane of natural HAP mono-
crystal substrate and (110) plane of the new HAP-like crystallite grown
in EMD and CS-EMD hydrogels has values of 52° and 39°, respectively.
Fig. 7 illustrates the FTIR spectra of the investigated samples.
Natural enamel sample (R1) and acid-etched demineralized sample
(R2) (Fig. 7a) have similar bands in infrared spectra characteristic to
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hydroxyapatite but with different intensities. The characteristic bands
of PO43- group appear in FTIR spectra as absorption bands at
1135 cm−1 (ν3), 621-525 (ν4) cm−1 and at 443 cm−1(ν2). The bands
characteristic to CO32- group are present at 882 (ν2) and more intensive
at 1620 and 1640 cm−1(ν3). Similar bands for PO43- group and CO32-
group were recorded by others authors in bones and hydroxyapatite
spectra [33,34]. The bands for PO43- group of hydroxyapatite can be
shifted to higher or smaller wave number position depending on the
preparation route [34–38]. The bands from 3853 to 3500 cm−1 were
attributed to O-H vibrations in adsorbed water or as hydroxyl group.
The bands at 3399 cm−1 and 3053 cm−1 are characteristic to stretching
of N-H and C-H bonds in the amide from collagen. As mentioned
before, the intensities of hydroxyapatite bands in infrared spectra of R1
and R2 samples are different, the acid-etched demineralized
sample (R2) showing decrease of N-H and C = O bands as result of
demineralization. In case of new remineralized layers grown under
EMD hydrogel (Fig. 7b), one can observe the increasing of bands
intensities for hydroxyapatite (HA). In the spectrum of layer obtained
after seven-day immersion in AS (S1-2), the PO43- bands increased in
intensity confirming the formation of new grains of HA on the
remineralized surface enamel. The increase of growth duration to 10
days led to more intense and well defined bands corresponding to the
increasing of crystalline HA and decreasing of carbonate content, in the
spectrum of sample S1-3 the carbonate peaks were present only as
shoulders at 797 and 830 cm−1. In the case of hybrid CS-HAP layers
formed under CS-EMD hydrogel, positions of bands for PO43- group
are shifted (Fig. 7c), leading to the conclusion that interactions between
Cs and HAP phase are present. The FTIR show the increase of the
intensities of bands characteristic to carbonated hydroxyapatite after 7
days in saliva (samples S1-2 and S2-2, in Fig. 7b and c), followed by
increased HAP bands together with decreased CO32- band with further
increasing the period of immersion into AS up to 10 days. Decrease of
CO32- group band is higher in the case of hybrid CS-HAP layer (S2-3 in
Fig. 7c).
Raman spectroscopy is a powerful technique in investigation of
apatite-based materials as well as organic ones present in human
A. Zaharia et al. Ceramics International 43 (2017) 11390–11402

Fig. 7. FTIR spectra of natural enamel (R1), acid-etched enamel (R2) (a) and biomimetic HAP layers (b) and CS-HAP hybrid layers (c).

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Fig. 8. Raman spectra of natural enamel (R1), acid-etched enamel (R2) (a) and biomimetic HAP layers (b) and CS-HAP hybrid layers (c).
mineralized tissues [3,39,40]. Fig. 8 illustrates the Raman spectra of
natural enamel sample (R1), acid-etched enamel (R2) and newly grown
remineralized layers without or with the presence of CS (Table 1).
Raman modes of phosphate at 960 cm−1(υ1), 420–450 cm−1 (υ2)
1000–1100 cm−1 (υ3) and 580–610 cm−1 (υ4) of carbonate at
1070 cm−1 (υ1) and faint stretching υ(OH) mode at about 3570 cm−1
indicate carbonated apatite [3,39–41], namely B-type apatite, in the
enamel samples in Fig. 8. Carbonate content of the apatite materials is
derived by area ratio of 1070 and 960 cm−1 bands while hydroxylation
degree from area ratio of 3570 and 960 cm−1 bands, respectively.
Intensity of the 427 cm−1 band gives information on apatite growing
towards c-axis direction [42]. More intense 427 cm−1 band is notice-
able for ten-day immersed S1-3 sample in comparison to S1-2 one.
Presence of fluorescent background [43] and 1370 and 1560 cm−1
bands of graphite are consequences of organic content of the enamel
samples investigated. Thus, band located at 2953 cm−1 (inset Fig. 8c) is
very likely to originate from protein [11,44] since the corresponding
band of chitosan was found at 2885 cm−1 [43]. As expected for inner
layers of enamels [44], etched surface of the R2 enamel gives a slightly
wider 959 cm−1 band and more CO32- content (Table S1 from ESI).
Slight increase of the FWHM for the 959 cm−1 band in comparison to
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references R1 (9.45 cm−1) and R2 (9.9 cm−1) points out to lowering of
the crystallinity degree of apatite for remineralized enamels of S1-1,
S1-2 and S1-3, respectively, confirming the XRD data (Table 1).
Although more carbonate content (except for S1-3) than R1 was
recorded for remineralized samples, it is smaller when remineralization
takes place into CS-EMD hydrogel matrix and diminishes when span of
immersed increases from 4 to 10 days.
3.3. Microhardness
The estimated microhardness values of the natural, acid-etched and
air-dried remineralized samples are shown in Table 1. One can observe
that hardness of the enamel surface declined till reaching about 57% of
that of natural tooth after 2 min acid-etching in 36% o-phosphoric acid
gel. When compared with the hardness of the demineralized sample
(R2), the hardness of the synthetic HAP layers grown under EMD
hydrogel for four to ten days (S1-1 to S1-3) reached 82–92% of natural
tooth. The behavior of synthetic hybrid CS-HAP layers is different when
compared with that of synthetic HAP layers without CS. Thus,
microhardness reached 76% and 78% of that of natural tooth for layers
obtained during four to seven-day immersion into AS under CS-EMD
A. Zaharia et al.
hydrogel, respectively (S2-1 and S2-2) and decreased at 67% for ten-
day immersion (S2-3) (Table 1). These values are comparable with
those reported in literature [13,19]. The slight decrease of hardness for
the CS-HAP layers obtained under chitosan for four and seven days
when compared with layers without CS can be explained by the
increased content of organics. The microhardness decrease for longer
durations of ten-day immersion in AS (sample S2-3) may confirm the
increased CS content in superficial layer (Fig. 4b) and formation of
calcium-deficient HAP phase (Table 1).
3.4. Mechanisms of biomimetic HAP and CS-HAP layers formation
Synthetic HAP layers grown in the presence of EMD hydrogel by
immersion into AS for four and seven days (Fig. 2) show nano- and
mesoscale morphologies similar to natural enamel (Fig. 1), confirming
the successfully biomimetic reconstruction of enamel-like structure
under amelogenin proteins present into EMD hydrogel [45]. Following
the biomimetic process of apatites formation from solution of Ca2+ and
PO43- ions, the initial co-precipitated amorphous calcium phosphate
(ACP) consisting of low calcium content hydroxyapatite (Ca-P) clusters
transforms into hydroxyapatite (HAP) according to the following
reaction [45]:
(ACP) Ca10−xH2x(PO4)6(OH)2 → (HAP) Ca10(PO4)6(OH)2
According to literature data, formation of ACP is initiated by
amelogenin phosphorylation through interaction of amine groups with
PO43- ions [2]. The resulted phosphorylated groups interact with Ca2+
ions from AS solution, producing stabilized Amel-Ca-P clusters [13,46],
which is then transformed into nanocrystals [19]. We illustrate in Fig. 9
the presumed mechanism of biomimetic growth of c-axis oriented
synthetic HA crystals in AS solution under amelogenin hydrogel onto
the etched natural enamel crystals used as template [1]. Recent
computational and experimental studies demonstrated that the com-
plex amelogenin macromolecules are characterized by two types
terminal domains, a hydrophobic amine N-terminal domain and a
hydrophilic charged carboxy C-terminus domain [47,48]. During the
amelogenin-assisted HAP formation, specific atomic-level interactions
Fig. 9. Schematic representation of biomimetic formation of synthetic enamel-like HAP layer (a) and hybrid CS-HAP layer (b) onto natural enamel tamplate during imersion into AS
solution.
Ceramics International 43 (2017) 11390–11402
occur between amelogenin molecules and HAP crystal faces.
Amelogenin molecules selectively adsorbed onto the (100) face of
HAP by strong molecular interactions between carboxylic groups of
their C-terminus and calcium ions of the (100) face of HAP [47,48].
This specific adsorption interfacial interaction between the structured
amelogenin macromolecules and the ACP phase play the key role in
lowering the activation energy of nucleation of (001) mineral faces
[46]. Thus, the selective adsorption of amelogenin has induced c-axis
elongation and formation of prismatic nanorod-like crystals by selec-
tively inhibiting HAP growth along (100) face and thereby promoting
growth along the basal (001) face which is free of adsorbed amelogenin
molecules and therefore in direct contact with AS solution (Fig. 9a).
Thus, the amelogenin macromolecules control the nanorod-like mor-
phology of the primary formed mineral phase by paralleling the c-axis
of HAP to their long axis [49] and formation of rod-like HAP
nanocrystals [13], as observed for samples S1-1 and S1-2 (Fig. 2).
The obtained nanorod-shaped amelogenin-HAP crystals acted as
hybrid building blocks for the new synthetic apatite phase [19],
becoming ordered by hydrophobic interactions between the amelogen-
in molecules selectively adsorbed onto the (100) face of neighboring
crystals [46]. Therefore, the structured amelogenin proteins have
controlled the morphogenesis of the primary HAP nanocrystals by
(I) electrostatic interaction with Ca2+ ions from (100) face through C-
terminus domain and contributed to their self-assembly at the mesos-
cale into enamel specific interwoven network (Fig. 2) by hydrophobic
interactions between their N-terminal domains (Fig. 9a).
The biomimetic CS-HAP hybrid layers grown under CS-EMD
hydrogel matrix show gradual morphological changes (Fig. 3) by
increases the duration of synthesis, when comparing with the natural
enamel (Fig. 1) or HAP layers grown without CS (Fig. 2), suggesting the
interference of chitosan macromolecules into one or more steps of the
mechanism of enamel-like CS-HAP layers formation. Thus, CS mole-
cules could interfere into the co-precipitation of ACP, formation of
hybrid nanorod-like crystals and/or in their mesoscale self-assembly,
involving interactions between CS molecules and ions from AS solu-
tion, interactions between CS and ACP or HAP nanocrystal, and also
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interactions between CS and amelogenin molecules. It is well known
that behavior of the deacetylated chitosan molecules is pH dependent
due to the presence of amine group [1]. At pH in the range of 5.0–5.5,
below the pKa value (6.5), the charge density of chitosan molecules
increases (overall positive charges) due to the protonation of amino
groups which favors formation of the electrostatic interactions. At pH
above 6.5 chitosan molecules are characterized by low protonation and
therefore their interactions are driven by CH and CH3 hydrophobic
groups [45–50] or by NH2 amine group chelating effect [48].
Kumar et al. reported that at pH above 6.5, chitosan molecules can
promote the co-precipitation of ACP by direct interactions with PO43-
ions from solution, in both acidic and basic solutions, with formation of
phosphorylated chitosan groups, according to the following reactions
[2]:
Cs-CH2OH + PO43- → Cs - CH2-O-PO33- (II)
Cs-NH2 + PO43- → Cs -NH-O-PO33- (III)
Like the protein phosphorylated groups (Amel-Ca-P) mentioned
before, the chitosan phosphorylated groups could chemically bind
calcium ions and therefore impose specific structural constraints for
the aggregation induced crystallization of ACP [2]. Our experimental
results showed some evidences of interactions between Cs molecules
and HAP phase, i.e. shifted positions for PO43- group bands in FTIR
(from 1042 cm−1 for HAP layers to 1092 cm−1 for CS-HAP layer,
Fig. 7), as well as increased diameter of the HAP nanocrystals
coprecipitated in the presence of CS molecules. However, taking into
consideration the biomimic activity of amelogenin molecules present
inside CS-EMD hydrogel, we consider that there is not enough evidence
of a major involvement of CS molecules into direct co-precipitation
from solution of Ca-P clusters. Thus, in the reaction environment of
6.5–7pH the CS effect suggested by the above mentioned experimental
results can be explained by the chelating effect of NH2 amine group
from CS molecules that can promote strong interactions with Ca2+ of
apatite phase [51]. As mentioned before, in the biomimetic
Amelogenin-Ca-P nanocrystals, Ca2+ ions from crystallographic Ca
sites existing onto the terminated basal (001) face are free of adsorbed
amelogenin molecules. These ions, noted Ca(2), have two unsatisfied
coordination valences, that can coordinate the amine group of CS,
being in the same time strongly bonded on the HAP structure by other
five coordination interactions, from a total coordination number of 7
[48], according to reaction [51]:
HAP-Ca2+(2) + 2CS-NH2 → (CS-NH2)2…Ca2+(2)-HAP (IV)
We consider that these coordination interactions are responsible for
orderly embedding HAP nanocrystals into c-axis elongated-shaped CS-
HAP hybrid nanorod (S2-2, Fig. 3). Following these interactions, Cs
molecules are aligned along the c-axis of HAP nanorod-like crystals
(Fig. 9b).
The presence of amelogenin molecules selectively adsorbed on the
(100) face of HAP nanocrystals not only controls the coordination
interactions of Ca(2) ions from the (001) face with NH2 group of CS,
but also can influence the effect of chitosan during the self-assembly of
HAP nanocrystals. Thermodynamic studies on chitosan-proteins inter-
actions showed that the binding process between chitosan and protein
molecules is spontaneous, resulting in complex formation. Non-cova-
lent interactions, as hydrogen bonding, hydrophobic and electrostatic
interactions may be driven between polysaccharide and protein mole-
cules in aqueous solution [41,50]. Following the coordination interac-
tion mentioned before, CS molecules get aligned along the c-axis of
HAP nanorod-like crystals and are disposed between the amelogenin
molecules absorbed on their (100) faces. Taking into consideration that
our experimental study was conducted at pH between 6.5 and 7, we
consider that the chitosan molecules aligned within HAP nanorods
interact by hydrophobic binding with the hydrophobic N-terminal
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groups of amelogenin molecules selectively adsorbed on (100) HAP
surface (Fig. 9b). Thus, HAP nanorod-like crystals are embedded in a
protein-polysaccharide hybrid matrix through strong non-covalent
interactions.
4. Conclusions
Biomimetic HAP and CS-HAP enamel-like layers were grown
directly onto acid-etched natural teeth in artificial saliva for four to
ten days under EMD and CS-EMD hydrogels extracellular matrices
prepared through a modified method.
SEM-EDX, HRTEM-SAED, FTIR and Raman data indicated for-
mation of B-type carbonated substituted hydroxyapatite (HAP) with c-
axis orientation, when carbonate content decreased as immersion time
increased from four to ten days. An optimum Ca/P ratio (1.85 at%) and
hardness of 2.48 GPa were recorded, as well as a well conserved
hierarchical rod-interrod structure was observed for seven-day remi-
neralization in CS-EMD hydrogel.
Chitosan caused major morphological modifications, subtle changes
in HAP lattice parameters and preserved c-axis orientation at mesos-
cale. SEM images highlighted interference of CS macromolecules in
HAP nanocrystals self-assembling at nano- and mesoscale. For the
formation of hybrid CS-HAP layers under amelogenin hydrogel, CS
macromolecules act as matrix for orderly embedding the structured
amelogenin-HAP nanocrystals by selectively binding Ca(2) from HAP
(001) faces via coordination interactions and amelogenin molecules
adsorbed onto (100) faces via hydrophobic contacts.
Synthesis of multifunctional biomimetic CS-HAP materials could
provide highly organized enamel-like structure for teeth remineraliza-
tion.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ceramint.2017.05.346.
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