Cite this article Research Article Keywords: biomimetic materials/

Hanafy RA, Mostafa D, Abd El-Fattah A and Kandil S Paper 1900008 biosystems/chitosan
Biomimetic chitosan against bioinspired nanohydroxyapatite for repairing enamel surfaces. Received 17/01/2019; Accepted 03/07/2019
Bioinspired, Biomimetic and Nanobiomaterials,
https://doi.org/10.1680/jbibn.19.00008 ICE Publishing: All rights reserved

Bioinspired, Biomimetic and Nanobiomaterials

Biomimetic chitosan against bioinspired

nanohydroxyapatite for repairing enamel
surfaces
Rania Ahmed Hanafy PhD Ahmed Abd El-Fattah PhD
Department of Materials Science, Institute of Graduate Studies and Department of Materials Science, Institute of Graduate Studies and
Research, Alexandria University, Alexandria, Egypt; Department of Dental Research, Alexandria University, Alexandria, Egypt; Department of
Biomaterials, Faculty of Dentistry, Alexandria University, Alexandria, Egypt Chemistry, College of Science, University of Bahrain, Sakhir, Kingdom of
Dawlat Mostafa PhD Bahrain
Department of Dental Biomaterials, Faculty of Dentistry, Alexandria Sherif Kandil PhD
University, Alexandria, Egypt (corresponding author: Department of Materials Science, Institute of Graduate Studies and
dwlat.mostafa@yahoo.com) Research, Alexandria University, Alexandria, Egypt

In this study, chitosan was employed as a novel biomimetic mineralization model to repair damaged enamel to
compare its performance with that of bioinspired zinc-doped nanohydroxyapatite. Fifty human premolar tooth slices
were prepared, and artificial caries lesions were induced to produce demineralized enamel surfaces. The etched slices
were randomly divided into two groups: a chitosan-hydrogel-treated group and a zinc-doped nanohydroxyapatite-
treated group. In vitro assessment using energy-dispersive X-ray analysis, X-ray diffraction analysis and scanning
electron microscopy was conducted at the baseline, demineralization and remineralization stages. Baseline results
were matched with those for normal enamel; a marked reduction in the calcium (Ca)/phosphorus (P) ratio to 1·12
and the lack of the characteristic hydroxyapatite diffraction peaks were detected for demineralized enamel. The
remineralization stage revealed evident recovery of the mineral contents (the calcium/phosphorus ratio was 1·61 for
the chitosan-treated group and 1·58 for the bioinspired-nanohydroxyapatite-treated one), with apparent distinctive
X-ray diffraction patterns of hydroxyapatite in both groups. Scanning electron microscopic analysis showed the
absence of etched enamel porosity, with the formation of a newly formed rod-like apatite layer, similar to natural
enamel, which extended over the treated enamel surfaces of both groups. Chitosan hydrogel is recommended as a
biomimetic mineralization smart system for repairing demineralized carious enamel.

Notation common clinical approach to repairing the acid-etching of teeth is

D average crystallite size
K constant related to the crystallite shape, normally taken
as 0·9
q Bragg angle
l wavelength of the copper (Cu) Ka radiation
1. Introduction
Enamel forms the exterior layer of the mammalian tooth and is
composed of numerous nanorod hydroxyapatite (HA) crystals
arranged into well-organized microarchitectural units called
enamel rods. Tooth enamel exhibits unique physicochemical
properties due to its hierarchical structure from the nanoscale up
to the microscale.1,2 Previous studies have shown that amelogenin
(a 90% organic material) is of great significance for the formation
of tooth enamel in the process of biomineralization. However, this
protein is almost completely decomposed following maturation of
the enamel,3 and, therefore, etched enamel is incapable of
regenerating. Dental caries, a common disease caused by bacteria,
is the main cause of toothache with tooth loss, and people may be
susceptible to this disease all of their life.4 The term ‘caries’
means both the process of acid-etching and the result of this
etching process, which is known as caries lesion. Thus, in respect
of these two processes, the main clinical treatment is to control
the bacterial growth and repair the hard tissue.5 The most
to use dental restorative materials, which involve metals,
composite resins and ceramics. These filling materials can partly
relieve symptoms and increase the aesthetic appearance after
filling the tooth cavity.6
Over time, modern dentistry has evolved to a minimally invasive
methodology, in which caries is managed as an infectious disease,
deferring operational intervention as long as possible. The focus is
on maximum conservation of demineralized, non-cavitated
enamel and dentin. Moreover, the replacement of lost tooth
substance with biomimetic materials is considered as one of the
fundamentals of minimal intervention dentistry.7 With the goal of
remineralizing incipient lesions, several methods have been
introduced to remineralize the early tooth structure.8,9 Over the
past 25 years, fluorides have been instrumental in bringing about
a decline in the incidence of dental caries in most industrialized
countries. However, remineralization by topical fluorides attempts
to only reduce apatite dissolution rather than aiming to promote
remineralization of apatite crystals or replacement of lost
minerals.10 Nevertheless, concerns have been raised that, with the
wide array of both prescription and over-the-counter fluoride
products now being marketed worldwide, the total fluoride intake
has increased to perhaps harmful levels.11 The reservations that
exist about fluorides have led to the introduction of several agents

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 Bioinspired, Biomimetic and Nanobiomaterials
and compounds, such as bioactive formulations of tricalcium
phosphate, bioactive glass (calcium sodium phosphosilicate),12
casein-based products13 and nanohydroxyapatite (nHA).14
Synthetic nHA might be termed ‘bioinspired’ rather than
‘biomimetic’, as it seeks to retain the features of natural HA and
translate them into medical technology with improved
performance.15 Bioinspired nHA has received considerable
attention for the remineralization of enamel due to its excellent
bioactivity, biocompatibility and ability to remineralize the initial
enamel lesion by forming a new layer of synthetic enamel around
the tooth, instead of hardening the existing layer with fluoride.16
Several studies have reported the considerable potential of nHA
to remineralize incipient caries lesions.17–22 Using nHA alone
as a first line of defense, however, is not enough because
HA does not have antibacterial properties. Consequently, the
addition of antibacterial agents to nHA could be an effective
strategy for providing a long-term cariostatic effect.23 Biomimetic
mineralization is a smart approach in dentistry to the treatment of
defective enamel.24 The key to restoring damaged enamel is
finding appropriate organic templates to induce enamel-like tissue
regeneration.25
Various biomimetic mineralization systems have been developed to
repair enamel defects – for example, amelogenin,26 peptide
amphiphiles,27 glumatic acid combined with nHA particles28 and
carboxyl-terminated poly(amidoamine) dendrimers29 have been
used to obtain a biomineralized layer containing organized needle-
like HA crystals on the surface of etched enamel. An attractive
biomimetic organic template is chitosan, the only pseudo-natural
cationic polymer, obtained from the deacetylation of chitin. It has
exceptional properties, including low cost, biodegradability,
biocompatibility, non-antigenicity and antibacterial effects, as well
as flexibility in hydrated environments.30,31 In addition, the positive
charge of chitosan allows it to adhere to negatively charged
surfaces, including demineralized enamel. Accordingly, the
chelating property of the amino group, as well as the tight
adsorption to enamel, supports chitosan in acting as a perfect
template for calcium (Ca2+) and phosphate (PO43−) ions to
regenerate new enamel-like mineralized tissue.32,33 Tian et al.34
investigated an N-methylene phosphoric chitosan model in order to
synthesize tooth-like HA. In another study, a small amelogenin-
derived peptide was incorporated into chitosan hydrogel for enamel
reconstruction.35 Most of the data reported in this study, however,
have limitations, such as difficulties in preparing proteins/peptides
and the complicated multiple steps in clinical applications.
Accordingly, the need to develop a simple strategy for mimicking
the organic matrix function in order to induce biomineralization
on the surface of the tooth enamel with antimicrobial activity is
still a crucial demand. From this perspective, the innovative
approach adopted in the current study was the preparation and
evaluation of chitosan hydrogel loaded with inorganic ions as a
simple low-cost biomimetic mineralization smart system to repair
initially carious enamel lesions in comparison with bioinspired
2
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Biomimetic chitosan against bioinspired
nanohydroxyapatite for repairing enamel
surfaces
Hanafy, Mostafa, Abd El-Fattah and Kandil
nHA doped with an antibacterial agent. The remineralization of
etched demineralized enamel using a chitosan biomimetic
mineralization system and bioinspired nHA is therefore proposed.
2. Materials and methods
2.1 Materials
Chitosan powder (93% deacetylation with high molecular weight)
was obtained from Sigma-Aldrich Chemical Co. Ltd, Darmstadt,
Germany. Calcium hydroxide (Ca(OH)2) and orthophosphoric
acid (H3PO4) were received from Oxford Co., India, and were
used as the starting material for calcium (Ca) and phosphorus (P)
precursors, respectively. Ammonia, ethanol, glacial acetic acid,
sodium hydrogen phosphate (Na2HPO4), calcium chloride
(CaCl2), poly(ethylene glycol) (PEG), sodium hydroxide (NaOH)
and zinc oxide (ZnO) nanoparticles (nZnO) were purchased from
Sigma-Aldrich. Artificial saliva was obtained from Khalil
Pharmacy, Alexandria, Egypt.
2.2 Preparation of tooth slices
A total of 18 sound human premolars in the age range 18–25
years (12 males and six females), which were frequently extracted
for orthodontic purposes, were selected for this study. This is
because orthodontic patients suffer from teeth crowding and other
irregularities and have a treatment plan that requires extraction to
relieve crowding and align their teeth. The teeth were inspected
with the naked eye, where teeth with any visible caries,
restorations, cracks, stains and white spot lesions were excluded.
The teeth were thoroughly cleaned of debris and stored in a
solution containing 0·12% thymol to prevent bacterial growth
until the start of the study (used within 1 month following
extraction). After removal of debris, calculus and soft tissue from
the tooth surface, the teeth were stored in 10% formalin solution
until further use. The teeth were then sectioned 1 mm below the
cementoenamel junction with a slow speed diamond disk.
The roots were discarded, and the crowns were used for the study.
The specimens were stored in antifungal solution containing 0·1%
thymol solution until the experimental procedure was initiated.
Then, the teeth were collected, cleaned and embedded in methyl
methacrylate resin blocks. Each tooth block was mounted on a
microtome equipped with a water-cooled diamond saw to be
sectioned longitudinally from the proximal side to produce
approximately three tooth slices of 2 mm thickness each. After
cutting, all tooth slices were cleaned ultrasonically for 2 min,
rinsed and stored in deionized water.
2.3 Enamel demineralization protocol: induction of
caries lesion
To simulate carious lesions, all of the tooth slices were acid-
etched with 37% phosphoric acid for 1 min in a specific area on
the enamel surfaces.36 Following this procedure, a white chalky
lesion was developed on each enamel surface of the demineralized
slices, indicating the induction of early enamel caries. Following
the demineralization procedure, all demineralized tooth slices
were washed with copious quantities of deionized water.
 Bioinspired, Biomimetic and Nanobiomaterials
2.4 Preparation of the enamel remineralization regimens
2.4.1 Chitosan biomimetic mineralization hydrogel
loaded with inorganic ions
Chitosan powder (0·25 g) was dissolved in acetic acid solution (1%,
25 ml), then stirred at 80°C for 2 h and finally left to cool to room
temperature. Following this, the solution was loaded with inorganic
ions by mixing chitosan solution (960 ml), sodium hydrogen
phosphate solution (15 ml, 0·1 M) and calcium chloride solution
(25 ml, 0·1 M).24 The pH of the mixed chitosan solution was adjusted
by adding sodium hydroxide solution (1 M) drop by drop until pH
6·5 was reached, at which point the chitosan hydrogel was formed.
2.4.2 Bioinspired zinc-doped nHA paste
First, pure nHA powder was synthesized by the wet-chemistry
method according to the Bianco B protocol.37 Briefly, solution A of
calcium hydroxide (0·5 M) and solution B of orthophosphoric acid
(0·3 M) were prepared. Solution B was added dropwise at a constant
rate (3 ml/min) to solution A with vigorous stirring at 40°C, and then
aqueous ammonia (25%) was used to adjust the pH of the solution
to 9·4. The resulting precipitate was aged overnight, filtered, washed
with deionized water and finally calcined at 650°C for 2 h. Second,
zinc(Zn)-doped nHA paste was obtained in two steps: (a) initially,
the composite powder was prepared by mixing nZnO (0·1 g) with
the prepared nHA powder (4·9 g) and (b) then the paste was
obtained using composite powder (g), water (ml) and the
flocculating agent (ml) in the ratio 5:4·5:0·2. PEG was used as the
flocculating agent to obtain a paste of the appropriate consistency.
The prepared nHA was analyzed for its chemical composition using
energy-dispersive X-ray (EDX) analysis Inca Penta FETX3, Oxford
Instruments, model 6583, UK). Analysis was performed on uncoated
powder samples at 15 kV for 60 s. The calcium/phosphorus molar
ratio of the sample was determined as the average of five runs.
Structural analyses of the nHA particles were performed by
powder X-ray diffraction (XRD) measurements38 using a
diffractometer (XRD-7000, Shimadzu, Japan). The X-ray beam
was copper (Cu) Ka radiation (l = 0·1540 nm), operated at 40 kV
and 38 mA. The XRD pattern was recorded in the 2q range from
5 to 100° at a scanning rate of 5°/min.
The size and morphology of nHA particles were studied by
transmission electron microscopy (TEM) using a Jeol JEM-100CX
(Japan) microscope. A few drops of the nHA particles (dispersed
in ethanol) were mounted on carbon-coated copper grids and
double-stained with freshly prepared uranyl acetate for 20 min and
then with lead citrate for 2–5 min prior to imaging by TEM.
2.5 Tooth enamel remineralization protocol
2.5.1 Grouping
Fifty demineralized tooth slices were randomly coded from 1 to
50 and then divided into two groups according to the
remineralization regimens as follows: a chitosan-hydrogel-treated
group (n = 30) and an zinc-doped nHA-treated group (n = 20).
No cells were employed in this study.
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Biomimetic chitosan against bioinspired
nanohydroxyapatite for repairing enamel
surfaces
Hanafy, Mostafa, Abd El-Fattah and Kandil
2.5.2 Enamel remineralization protocol of the chitosan
hydrogel biomimetic mineralization system
The chitosan hydrogel regimen was applied to each etched tooth
slice by injection through a plastic syringe, and then the slices
were left at room temperature until completely dry. Following
this, each treated tooth slice was transferred to a beaker
containing 30 ml of artificial saliva solution and incubated at
37°C. Finally, after the allotted incubation time, the remineralized
chitosan hydrogel tooth slices were removed from the artificial
saliva, rinsed with deionized water three times for 5 min each,
cleaned ultrasonically (25 Hz, 120 W) for 30 s and then dried at
room temperature. Only ten treated slices were kept for a short
incubation period of 7 d24 for morphological inspection with
scanning electron microscopy (SEM), while 20 treated slices that
represented group sample size were left in the incubator for 21 d
for laboratory assessment.
2.5.3 Enamel remineralization protocol of bioinspired
zinc-doped nHA paste
zinc-doped nHA regimen paste was applied to each demineralized
tooth slice by brushing using a soft toothbrush (Meta P & Bond
Microbrush) for 2 min, followed by washing with copious
quantities of deionized water to remove any remaining paste
adhering and then storing in deionized water for subsequent
application. This remineralization treatment protocol was repeated
two times per day (morning and evening) for 21 d.39 Finally, at
the end of the study period, all zinc-doped nHA-treated tooth
slices were cleaned ultrasonically (25 Hz, 120 W) for 30 s and air-
dried for laboratory assessment.
2.6 In vitro assessments
In vitro assessments were performed during this study at three
sequential stages: (a) baseline assessment for the prepared tooth
slices, (b) a second assessment to analyze the demineralized
damaged enamel surfaces and (c) analytical characterization of the
remineralized treated tooth slices prepared with either chitosan
hydrogel or zinc-doped nHA. Elemental analysis was completed in
the laboratory using EDX analysis. Additionally, XRD analysis
was used to analyze the crystallographic patterns. Moreover,
morphological imaging was performed with SEM (Jeol JSM-5300-
JSM, Tokyo, Japan). The samples were sputter-coated with gold in
a sputter-coating unit (JFC 1100 E) and observed by SEM at 20 kV
with a spot size of 5 nm.
2.7 Statistical analysis
The data obtained were subjected to statistical analysis using the
SPSS software. The results were analyzed by analysis of variance.
Multiple comparisons between groups were achieved by a paired
t-test. The level of significance was set at the value of p < 0·05.
3. Results
3.1 Characterization of the prepared nHA particles
The characterization results of the synthesized nHA particles are
presented in Figure 1. As shown in Figure 1(a), the TEM image
3
 Bioinspired, Biomimetic and Nanobiomaterials Biomimetic chitosan against bioinspired
nanohydroxyapatite for repairing enamel
surfaces
Hanafy, Mostafa, Abd El-Fattah and Kandil

reference data (JCPDS PDF number 74-0566); the two patterns

(a)
matched closely and exhibited a good coincidence without
additional phases. XRD was considered as a convenient technique
for determining the mean size of the crystalline materials, as it is
sensitive to the crystallite size inside particles. Two crystalline
peaks at 25·90 and 34·17° were used to calculate the nHA crystal
15 nm
sizes, according to the Debye–Scherrer formula
130 nm
Kl
D¼
1. FWHM  cos q

HV = 80·0 kV Direct mag: ×10 000 100 nm

where D is the average crystallite size; K is a constant related to
2000 the crystallite shape, normally taken as 0·9; l is the wavelength of
(b)
Ca the copper Ka radiation (0·1540 nm), FWHM is the full width at
half-maximum of the XRD peak; and q is the Bragg angle. The
1500
crystal sizes at 2q = 25·97° (002) and 34·17° (300) correspond to
P the length and width of HA, respectively.
Counts

1000
The average crystallite size of the nHA particles was found to be
20·40 ± 2·53 nm, which is in good agreement with the actual
500 value for the enamel of human teeth.41

3.2 Characterization of the demineralized and

0
0 5 10 15 20 remineralized enamels
300 Energy: keV It was found that the baseline calcium/phosphorus molar ratio
(c) determined by EDX analysis was 1·67 ± 0·01, reflecting the ratio
of normal enamel. For the demineralized enamel surfaces, the
calcium/phosphorus ratio fell considerably to 1·12 ± 0·04,
Intensity: counts

250 suggesting mineral depletion on etched enamel slices. Following

200
150
20 30 40
2θ: º
Figure 1. Characterization of the prepared nHA: (a) TEM image;
(b) EDX chart; (c) XRD pattern
of the prepared nHA particles exhibits a nearly regular hexagonal
rod-shaped morphology with uniform particle size. The diameters
of these rods were about 15 nm, and the lengths were nearly
130 nm. The EDX analysis (Figure 1(b)) estimated that the
synthesized nHA powder had a calcium/phosphorus ratio of
1·65 ± 0·01, which is close to the actual value for HA (1·67).
Figure 1(c) shows the XRD pattern of nHA, which indicates the
main characteristic diffraction peaks of nHA crystals positioned at
2q angles of 21·86, 22·96, 25·90, 31·25, 32·96, 34·17, 35·58,
43·98, 45·42, 46·79, 48·173, 49·58, 50·59, 51·32 and 52·16°.40
This pattern was compared with the XRD pattern of nHA
remineralization, there was an apparent recovery in the calcium/
phosphorus ratio of the remineralized treated teeth surfaces in
both groups, which indicated the privilege effect of the
remineralized regimes used to reverse mineral loss. The calcium/
phosphorus ratio was 1·61 ± 0·01 for chitosan-hydrogel-treated
surfaces and 1·58 ± 0·01 for the zinc-doped nHA-treated group.
50 60 Figure 2 displays the baseline, demineralized and remineralized
XRD patterns for chitosan-hydrogel- and zinc-doped nHA-treated
groups. The baseline XRD spectrum of the enamel slices revealed
the characteristic peaks of normal enamel HA minerals at 2q =
25·90, 31·25, 32·96 and 34·17°, which are consistent with the (002),
(211), (112) and (300) peaks. Moreover, the sharp and intense (002)
XRD diffraction peak indicated what is called the (002) ‘preferred
orientation’, which is the distinct crystallographic orientation within
the normal enamel where the crystals prefer to align vertically along
the crystallographic c-axis.42 Lack of the characteristic XRD
diffraction pattern of HA, particularly around 2q = 31·8, 32·2 and
32·9°, was detected in the case of demineralized enamel surfaces,
with reduced sharpness and intensity of the (002) peak.
The XRD patterns of remineralized enamel treated with either
chitosan hydrogel or zinc-doped nHA demonstrated the
characteristic peaks of HA, as well as the (002) preferred
orientation, as previously discussed for normal enamel. It was

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 Bioinspired, Biomimetic and Nanobiomaterials Biomimetic chitosan against bioinspired
nanohydroxyapatite for repairing enamel
surfaces
Hanafy, Mostafa, Abd El-Fattah and Kandil

Remineralized
chitosan-treated
enamel

Remineralized
Intensity

zinc-doped nHA-treated
enamel

20 KV ×1000 10 µm
Demineralized
enamel
Figure 3. SEM image (×1000) of the baseline enamel surface
(211)

(002) (112)
(300)
Baseline (a)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85
2θ: º

Figure 2. Baseline, demineralized and remineralized XRD patterns

for chitosan-hydrogel- and zinc-doped nHA-treated groups

noted that all nHA XRD diffraction peaks were sharp, split and
clear, which implied good crystallinity following remineralization
and reflected the clear differences between the XRD spectra of
demineralized and remineralized enamel. Furthermore, the relative
intensities of the (002) XRD peaks of the regrown crystallites 20 KV ×5000 5 µm
were perceptibly enhanced over that of natural enamel (Figure 2).

An SEM image of the baseline enamel surface is shown in (b)

Figure 3. SEM analysis showed the smooth and homogeneous
morphological pattern of the normal enamel surface with well-
organized enamel rods. On the other hand, SEM observations of
demineralized etched enamel surfaces showed loss of the compact
enamel structure, with the presence of numerous interspaces,
demonstrating distinct roughness with micropores, and cavities all
over the demineralized enamel surfaces (Figure 4).

Generally, inspection of the SEM images of remineralized

enamel repaired with either chitosan hydrogel or zinc-doped nHA
clearly revealed that both remineralization regimens induced the
20 KV ×5000 5 µm
remineralization of HA. SEM images of the enamel surfaces treated
with the chitosan hydrogel biomimetic mineralization system after 7
and 21 d are shown in Figures 5 and 6, respectively. Figure 5(a) Figure 4. SEM images of the demineralized etched enamel
surfaces: (a) reflected honeycomb-like morphological etched
shows partial coverage of the porous etched enamel with regenerated
enamel pattern; (b) loss of enamel compact structure with
mineralized tissue. A higher-magnification image (Figure 5(b)) observable loss of prism cores
indicates the newly regenerated layer similar to the microstructure of

5
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 Bioinspired, Biomimetic and Nanobiomaterials Biomimetic chitosan against bioinspired
nanohydroxyapatite for repairing enamel
surfaces
Hanafy, Mostafa, Abd El-Fattah and Kandil

(a) (a)

20 KV ×1000 10 µm

(b)
20 KV ×1000 10 µm

(b)

20 KV ×10 000 1 µm

Figure 6. SEM images of the enamel surfaces treated with the

chitosan hydrogel biomimetic mineralization system after 21 d at
different magnifications: (a) at ×1000; (b) at ×10 000
20 KV ×5000 5 µm

(c)
20 KV ×10 000 1 µm
Figure 5. SEM images of the enamel surfaces treated with the
chitosan hydrogel biomimetic mineralization system after 7 d at
different magnifications: (a) at ×1000; (b) at ×5000; (c) at ×10 000
natural enamel with rod-like structures that appear in a disorganized
pattern with relatively uniform size and shape. A still higher-
magnification image (Figure 5(c)) displays loosely packed rod-like
structures mineralized with HA crystals. Again, SEM images
confirm the newly regenerated enamel-like layer on treated enamel
surfaces with the chitosan hydrogel biomimetic mineralization
system after 21 d. Figure 6(a) shows complete coverage of the
etched enamel with a regenerated mineralized layer showing the
classical rod-like structure. The rods are arranged in parallel in a
compact, organized and smooth morphological pattern. Meanwhile,
the higher-magnification image (Figure 6(b)) shows the high packing
density of the rod-like structures compared with the 7 d observations.
Figure 7 shows the SEM images of the enamel surfaces treated
with the zinc-doped nHA bioinspired mineralization system after
21 d. Figure 7(a) presents the newly formed homogeneous and
smooth apatite layer coverage of the etched enamel. The higher-
magnification SEM image (Figure 7(b)) reveals aggregation of the
synthesized nHA clusters adsorbed on the repaired enamel surface.
It is worth mentioning that the morphological pattern of the newly
mineralized enamel-like layer regenerated on enamel surfaces
incubated according to the chitosan biomimetic mineralization model
for 7 d (Figure 5) or 21 d (Figure 6) differs distinctly from the
pattern of the layer formed on zinc-doped nHA-remineralized
enamel surfaces presented in Figure 7.

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 Bioinspired, Biomimetic and Nanobiomaterials
(a)
20 KV ×5000 5 µm
(b)
20 KV ×1000 1 µm
Figure 7. SEM images of the enamel surfaces treated with the
zinc-doped nHA bioinspired mineralization system after 21 d at
different magnifications: (a) at ×1000; (b) at ×10 000
4. Discussion
Enamel is unique in that the enamel-forming cells, the
ameloblasts, as well as the enamel organ stem cells, are lost at the
time of tooth eruption. Ameloblast cell culture models have
therefore proven to be challenging. Moreover, enamel crystal
nucleation and elongation require a complex array of
posttranslational protein modifications, while the organization of
enamel crystals into prisms and the formation of decussating
prism patterns depend on highly coordinated ameloblast
movements governed by mechanisms still to be elucidated.43
Regeneration of lost dental tissue can also be achieved by means
of stem cell biology or gene therapy technology. However, the
complexity and high cost of these approaches seriously limit their
wide application. As a result, to date there are no reports of
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Biomimetic chitosan against bioinspired
nanohydroxyapatite for repairing enamel
surfaces
Hanafy, Mostafa, Abd El-Fattah and Kandil
successful in vivo cell-based enamel tissue engineering. This
problem can be circumvented by cell-free biomimetic
remineralization using biomaterials. Thus, efforts have been made
to find organic templates with high efficiency and low cost to
regenerate enamel, such as gels, surfactants, proteins/peptides and
polymers.44,45
This study represents a successful in vitro cell-free approach to
the synthetic generation of parallel and elongated enamel-like
apatite crystals prepared using a chitosan hydrogel matrix
enriched with calcium and phosphate ions. This biomimetic smart
system is capable of regenerating mineralized enamel-like tissue
to accomplish enamel repair; subsequent assessment of its
remineralization behavior under simulated oral conditions was
carried out. Indeed, chitosan in the form of hydrogel was used, as
the physiochemical nature of the gel-like environment more
realistically mimics the unique environment of natural enamel.
Moreover, the hydrogel has advantages over a solution-based
system, as it is clinically easier to handle.46 On the other hand,
nHA was chemically synthesized following the Bianco B protocol,
which is considered to be the most suitable protocol for
mimicking HA found in dental tissue.37 Laboratory EDX and
XRD results revealed both the chemical and crystal phase purity
of the HA powder thus prepared. Also, the calculated crystal size
of the prepared HA particles was 20·4 nm, confirming the
production of nHA particles of almost the same size as human
tooth enamel. This outcome has a prospective application in
dentistry for the reconstruction of tooth enamel suffering from
mineral loss, as highlighted by Li et al.17 The rod-like
morphology of the prepared nHA was observed in the TEM
images. Based on these results, it was confirmed that the prepared
rod-like nHA was similar to its isotopes of normal enamel. The
subsequent procedure to dope nHA powder with nZnO was
performed to achieve effective enamel repair with bacteriostatic
properties. The bioinspired zinc-doped nHA regimen was used in
a paste-type formulation to be applied on the demineralized
enamel surfaces with disposable cotton-tip applicators. This was
done so as to be convenient for patients to replicate the procedure
using cotton-tip toothpaste applicators, and 2 min twice daily
application for 21 d was employed to make it clinically relevant.47
The EDX results displayed a calcium/phosphorus baseline ratio of
1·67, which was equivalent to the normal enamel ratio,36 while
the marked reduction in the calcium/phosphorus ratio to 1·12 ±
0·04 following demineralization revealed the successful induction
of carious lesions on the etched enamel surfaces. The enamel
surfaces repaired with either chitosan hydrogel or zinc-doped nHA
paste reflected a noticeable recovery in the calcium/phosphorus
ratio, hence confirming the successful remineralization of the
etched enamel surfaces.
It has been reported that HA crystals can be detected from their
distinctive peaks using XRD analysis. The peaks at 2q = 25·7,
31·8, 32·2 and 32·9°, which are consistent with the (002), (211),
(112) and (300) peaks, are characteristic of the XRD pattern of
HA.48 The XRD baseline patterns were analogous to those
7
 Bioinspired, Biomimetic and Nanobiomaterials
characteristic of HA. The demineralized etched enamel surfaces
exhibited a clear decrease in the intensities of the characteristic
XRD diffraction peaks of HA. Additionally, a reduction in the
sharpness and intensity of the (002) peak indicated that the surface
of the artificially demineralized enamel suffered the greatest
mineral loss and the remaining mineral phase lost its crystal lattice
orientation with its random distribution of crystallites. Similar
results have been reported by other biomimetic studies.24,29,34,36
The XRD patterns of the enamel surface following treatment with
chitosan hydrogel matched well with the expected peaks of HA,
hence confirming the newly regenerated HA layer. The relative
intensities of the (002) peak of the regrown crystallites were
distinctly enhanced over that of natural enamel, indicating
recovery of the crystal lattice of apatite and longitudinal growth
along the c-axis with the parallel orientation of the crystals. This
result is consistent with the results of the study by Rongquan
et al.,49 which used well-known analytical reagents such as
ethylenediaminetetraacetic acid (Edta) to chelate calcium ions and
to control the nucleation and growth of HA crystals. This in vitro
experiment indicated that Edta induced the assembly of hexagonal
prisms such as HA microcrystals along their c-axis direction
with enhanced intensity of the (002) peak over that of natural
enamel.49 XRD examination of the enamel surfaces treated with
bioinspired zinc-doped nHA revealed the same diffraction pattern
characteristics of the synthetic nHA powder, indicating its presence
on the enamel surface. On the basis of this finding, it could be
concluded that the brushing procedure failed to remove the new
HA formed, suggesting chemical bond formation between the
synthetic nHA and the natural HA enamel, with this bond allowing
the formation of a persistent HA coating whose morphology was
determined by SEM analysis. It is worth mentioning that the XRD
results for the baseline, the demineralized stage and the subsequent
remineralization were allied to the EDX results, thus confirming
the validity of these outcomes.
SEM is one of the most sensitive and least time-consuming
techniques for assessing and comparing changes before and after
application of the remineralizing agents in vitro, as reported in
earlier studies.50,51 The normal enamel morphological pattern was
observed and recorded as a baseline SEM result. The SEM
observations of demineralized enamel slices showed general loss
of normal enamel integrity, with the detection of numerous
interspaces and pores all over the etched enamel surfaces. This
etched pattern of enamel was comparable with those reported in
previous studies.19,20,26 Following repair of the enamel with
chitosan hydrogel loaded with inorganic ions, there was evidence
of an enamel-like layer firmly adhering to the damaged enamel
surface for both incubation periods (7 and 21 d). Similar results
were obtained by Tian et al.,34 who proposed the idea of using
chitosan gel linked with phosphate dental adhesive agent for
enamel mineralization using supersaturated calcifying solution
(SCS). Some authors have also reported deposition of long, thin
needle-shaped crystals of enamel-like calcium phosphate onto a
bioactive glass in an SCS containing recombinant porcine
amelogenins.52,53
8
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Biomimetic chitosan against bioinspired
nanohydroxyapatite for repairing enamel
surfaces
Hanafy, Mostafa, Abd El-Fattah and Kandil
A different mechanism, however, was adopted in this study
whereby artificial saliva was used instead of SCS to mimic the
physiological conditions. This was challenging because of the
limited number of ions available in saliva for remineralization.
Thus, chitosan hydrogel was loaded with inorganic calcium and
phosphate ions to act as a reservoir for replenishing the mineral
precursor. This concept was further advanced by exploring the
effect of biomimetic mineralization using the hydrogel for two
different incubation periods. The most striking feature of the
current study was the time-dependent regeneration effect shown
by chitosan. Following 7 d of incubation, a newly grown enamel-
like layer was formed on the repaired enamel surface. The
regrown layer showed a rod-like structure that was not highly
organized, and samples of it displayed an amorphous surface.
These observations are in accordance with those of Ruan et al.,24
who developed amelogenin-containing chitosan hydrogel for
enamel reconstruction, and also with those of Zhang et al. in
2018,54 who investigated the remineralization of white spot
lesions for a 7 d period with chitosan and a Bioglass + polyacrylic
acid slurry. On extending the incubation period to 21 d, the newly
formed enamel-like layers were densely packed and oriented
parallel to each other in a manner very similar to the normal
enamel pattern. Furthermore, the regenerated calcium phosphate
coating persisted, firmly adhered to the enamel surface even under
ultrasonic conditions (25 Hz, 120 W, 30 s), and an etched enamel
surface was not observed. Although the exact mechanism remains
unresolved, it is clear that extending the incubation period is
crucial to the continuous growth of a regenerated layer of apatite
crystals on repaired enamel surfaces.
Recent studies have reported that a newly generated HA layer can
be formed on a template surface through stable prenucleation
clusters, with aggregation into an amorphous precursor phase and
transformation of this phase into a crystal.55,56 Similarly, chitosan
hydrogel loaded with inorganic ions in the current study acted as a
nucleating template that successfully gathered a high concentration
of calcium and phosphate ions and guided their arrangement into
HA crystals, producing the newly regenerated enamel-like layer.
On the other hand, bioinspired zinc-doped nHA achieved a perfect
remineralization pattern through adsorption of the nHA clusters
that blocked pores on the damaged enamel surfaces, resulting in
the formation of a superior apatite surface texture of smooth
remineralized enamel with complete disappearance of the etched
enamel features. This could be attributed to the similarity of the
prepared HA rod-like nanoparticles to the apatite crystals of tooth
enamel in morphology, crystal structure, size and crystallinity.
Similar findings have been observed by other studies.17–19
Regarding the comparison between the enamel remineralization
patterns of chitosan and bioinspired zinc-doped nHA regimens,
although chitosan did not possess the same physicochemical and
crystallographic properties of natural tooth enamel as zinc-doped
nHA for enamel remineralization, chitosan in the form of a
biomimetic mineralization hydrogel could be considered as an
innovative treatment to repair demineralized enamel simply
through its biomimetic ability to provide a remineralization
 Bioinspired, Biomimetic and Nanobiomaterials
environment with regeneration of a new apatite layer reflecting the
architecture of normal enamel.
5. Conclusions
Compared with bioinspired nHA as the prevailing
remineralization regimen, the chitosan biomimetic mineralization
system is a simple and innovative low-cost approach to repairing
demineralized and damaged enamel surfaces by regenerating a
new HA crystal layer possessing properties similar to those of
natural enamel. Further studies are needed to evaluate the
practical performance in different dental fields of the enamel
remineralization regimes recommended by this study, particularly
following long-term orthodontic bracket therapy, which is
considered as one of the major causes of enamel demineralization.
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