Results in Engineering 15 (2022) 100583

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Results in Engineering
journal homepage: www.sciencedirect.com/journal/results-in-engineering

Engineering of biomimetic mineralized layer formed on the surface of

natural dental enamel
Pavel Seredin a, b, *, Dmitry Goloshchapov a, Anna Emelyanova a, Nikita Buylov a,
Vladimir Kashkarov a, Anatoly Lukin a, Yuri Ippolitov c, Tatiana Khmelevskaia c,
Iman A. Mahdy d, Manal A. Mahdy e
a
Solid State Physics and Nanostructures Department, Voronezh State University, University sq.1, 394018 Voronezh, Russia
b
Scientific and Educational Center “Nanomaterials and Nanotechnologies”, Ural Federal University, Mir av., 620002 Yekaterinburg, Russia
c
Department of Pediatric Dentistry with Orthodontia, Voronezh State Medical University, Studentcheskaya st. 11, 394006 Voronezh, Russia
d
Physics Department, Faculty of Science (Girls), Al-Azhar University, 11754 Nasr City, Cairo, Egypt
e
Solid State Physics Department, National Research Centre, 12622 Dokki, Giza, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: The problem of engineering a biomimetic mineralized layer on the surface of native dental tissue (bio-template)
Biomimetic was considered in our work.
Mineralization The formation of the mineralized layer on a biotemplate is achieved with the use of nanocrystalline carbonate-
Enamel tissue
substituted calcium hydroxyapatite (HAp), calcium alkali, and a complex of polyfunctional organic and polar
AFM
FESEM
amino acids.
microRaman spectromicroscopy By applying the set of structural and spectroscopic methods of analysis we have confirmed the formation of a
Nanoidentation mineralized biomimetic HAp layer on the surface of bio-template with properties resembling those of natural
hard tissue. The thickness of the biomimetic mineralized layer varies from 300 to 500 nm, while the direction of
some ncHAp nanocrystals coincides with that of the apatite crystals in the enamel.
We also demonstrated that the engineered mineralized HAp layer was characterized by homogeneous
micromorphology and enhanced nanohardness in the region of the enamel rods exceeding those of native
enamel.
The development of a strategy for biomimetic engineering and a technique for enamel surface pre-treatment to
enable tissue mineralization has huge potential in dental applications.

1. Introduction substituted hydroxyapatite HAp (Са10(PO4)6-x (CO3)x (OH)2-x, 0.1 < x

Recently, enormous efforts have been made to study the mechanisms
and approaches to effective engineering for the directed remineraliza­
tion of dental enamel [1–4] and human hard tissues [5,6]. Thus, it has
already been demonstrated that the assembly of an apatite-like inor­
ganic oriented structure on the surface of natural enamel can be orga­
nized using calcium phosphate nanoparticles [7–9]. However, inducing
the qualitative growth of hydroxyapatite (HAp) crystals commensurate
with natural enamel apatite crystals using nanoclusters on the bio­
template proved to be only partially possible.
The reproduction and successful restoration of dental tissue can be
effectively achieved with the use of nanocrystalline carbonate-
< 0.3) due to its maximum correspondence to the apatite of the dental
matrix [10–15]. As it has already been shown in several works [13,
16–19] related to the problem of dental tissue restoration, in order to
achieve the necessary crystal density, chemical resistance, and me­
chanical strength and hardness of the mineralized layers requires the
joint usage of HAp and polar amino acids for the formation of a bio­
mimetic mineralized layer, as well as the pre-determined conditions for
the interaction and conjugation of ncHAp crystals with an organic
enamel matrix [20–23].
It is likely that the key to solving this problem is choosing the optimal
approach to engineer a biointerface between the natural and biomimetic
material [2,24], and, in fact, a method of pretreatment of the natural

Abbreviations: HAp, hydroxyapatite; FESEM, field emission scanning electron microscope.

* Corresponding author. Solid State Physics and Nanostructures Department, Voronezh State University, University sq.1, 394018 Voronezh, Russia.
E-mail address: paul@phys.vsu.ru (P. Seredin).

https://doi.org/10.1016/j.rineng.2022.100583
Received 28 June 2022; Received in revised form 8 August 2022; Accepted 9 August 2022
Available online 13 August 2022
2590-1230/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
P. Seredin et al.
tissue surface (biotemplate) for its subsequent effective mineralization
[25,26].
It is quite clear that in the ideal case the mineralized layer should
imitate not only the chemical, structural, and mechanical properties of
the native tissue [5], but at the same time it must have the corresponding
characteristics at the microscopic level, and even at the nanolevel.
Therefore, while applying biomineralization to the dental tissue it seems
preferable to use the biomimetic approach. It provides a number of
advantages when compared to more widespread processes. This is due to
the use of biologically active materials and reactive environments as due
to the natural conditions of the proceeding [27]. However, the bio­
mimetic strategy of mineralization requires placing special focus on the
previous pretreatment stage (modification) of the biotemplate surface
[28].
All of these questions, in our opinion, have been given rather
insufficient attention, and so they remain open, topical, and require non-
trivial resolution. Therefore, our work aimed to develop a concept for
the engineering of a biomimetic mineralized layer on the surface of
natural dental tissue as well as the study of its structural-spectroscopic
features.
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Results in Engineering 15 (2022) 100583
2. Methods of production and study of the samples
2.1. Obtaining enamel samples
In our study, we used dental tissue samples taken from patients aged
20–25 to create biotemplates. Healthy human teeth were collected at the
Burdenko Voronezh State Medical University in compliance with the
rules and regulations of the Helsinki Convention and ethical protocols.
The patients were physically healthy based on outpatient records and
had no unhealthy habits, including smoking. The teeth were extracted
from the patients for orthodontic reasons. The collected teeth were
immediately mechanically cleaned of plaque, rinsed repeatedly with
distilled water using a compressor, and placed in distilled water, where
they were stored at +4 ◦ C.
2.2. Sample preparation
Dental segments were prepared for our studies. Using a low-speed
(120 rpm) water-cooled diamond blade [29,30], the tooth samples
were divided into segments. The thickness of each segment was ~2 mm.
An ultrasonic bath (transmitter power ~25 W) was used to remove re­
sidual contamination from the surface of the enamel segments. The
treatment was performed in distilled water once for 60 s.
A total of n = 20 segments were selected, which were further divided
into 2 groups.
Fig. 1. Engineering of a mineralized layer on the
surface of bio-templates. Sample S1: natural enamel
without mineralization; Sample S2: after surface
pretreatment with Са(ОН)2 and a complex of poly­
functional organic and polar amino acids, and the
subsequent mineralization. Engineering stages: I –
stage of preliminary treatment of enamel surface with
the use of Са(ОН)2; II – stage of preliminary treatment
of the enamel surface with the use of a complex of
polyfunctional organic and polar amino acids; III –
stage of the enamel surface treatment with HAp so­
lution. Schematically in the right part of the figure the
expected state of the enamel surface is shown after
each stage of the treatment.
P. Seredin et al.
2.3. Sample groups
Sample type S1: Samples of this type were used as standards of
healthy enamel.
In accordance with the design of our experiment, engineering of
biomimetic mineralized layer on the surface of natural dental enamel
was subjected to the specified pretreatment procedure (see Fig. 1).
Sample type S2: Two pretreatment steps were performed for each of the
samples in this group. In the first step, samples from each group was
placed for 30 s in an alkaline solution of Са(ОН)2. Next, each sample was
washed with distilled water and placed for 30 s in a complex of poly­
functional organic and polar amino acids. Then samples were placed in a
freshly prepared solution that contained nanocrystalline carbonate-
substituted hydroxyapatite (HAp). The pH value of the solution was
8.5. The mineralization procedure was performed for 24 h at room
temperature. The enamel segments (samples) were then washed in
distilled water and stored at 4 ◦ C until the experiments were performed.
3. Materials
The process of obtaining calcium hydroxide involved high-
temperature annealing of bird shells (2 h at 950 ◦ C), during which the
Fig. 2. Microscopic images of the surface areas of the samples. a,c,e - healthy tooth enamel without treatment (Sample S1); b,d,f-after surface pretreatment with Ca
(OH)2 and a complex of polyfunctional organic and polar amino acids, and subsequent mineralization (Sample S2); a, b - Optical images; c,d - AFM images; e,f – AFM
phase contrast images.
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Results in Engineering 15 (2022) 100583
organic component of the shell burns and highly active calcium oxide
remains. A solution of Са(ОН)2 was obtained using distilled water
titration of calcium oxide. The aqueous solution of highly dispersed
nanosized carbonate-substituted calcium hydroxyapatite (HAp) was
obtained by chemical precipitation. A complex of polyfunctional organic
and polar amino acids includes a complex of low-concentration (up to
12%) of saturated and unsaturated polyfunctional organic acids (maleic
acid, polyacrylic acid, citric acid, distilled water) and polar amino acids
(arginine - 0.2%–1.6%, lysine - 0.05%–0.4%, histidine - 0.01%–0.2%).
3.1. Experimental set-up
Optical images of the surfaces of S1 and S2 samples were obtained
using an Olympus CX40 optical microscope. The surface morphology
was studied using a Femtoscan-001 NT MDT scanning probe microscope
in atomic force microscopy mode.
The surface morphology and cross-sections of samples before and
after mineralization were investigated with a field emission scanning
electron microscope (FESEM) Quanta FEG 250, Czech Republic). Sec­
ondary electron images are made at 10 kV, 0.1 nA.
The molecular properties of the biomimetically mineralized dental
enamel tissue were studied by microRaman spectroscopy. All spectra
P. Seredin et al.
were obtained in the range of 100–4000 cm− 1 using a Raman micro­
scope RamMix 532 with a spectral resolution of 1 cm− 1. Excitation was
performed using a 532 nm laser. The mechanical properties of the
mineralized hard tissue were investigated by surface nanoindentation
using the Nanoindentation Tester (CSM Instruments). To determine the
hardness value, the Oliver and Farr method was used. The results were
processed using the Indentation for CSM Instruments software.
4. Results and discussion
4.1. Microscopy
Fig. 2 shows the microscopic images of the characteristic surface
areas of the examined samples obtained by optical and atomic force
microscopy.
In Fig. 2,a one can observe the surface morphology typical of the
lateral surface area of healthy enamel (Sample S1). The main local el­
ements of this morphology are enamel rods extending orthogonally to
the enamel surface [22,31,32], between which there are protein-rich
areas (a protein-rich sheath) or the so-called interrod space. The diam­
eter of the observed enamel rods, based on the optical microscopy data
presented, is ~5 μm, whereas the size of a typical interstitial space is ~2
μm (Fig. 2). It should be noted that for the surface of a typical healthy
enamel specimen ( Sample S1), there is a specific distribution of the
heights of the exiting enamel rods, which is evident from the change in
contrast in the optical image (Fig. 2a).
After pretreatment of biotemplates using different approaches
(Fig. 1) and subsequent mineralization performed, the surface of Sample
S2 at the macro level shows typical for healthy enamel S1 morphology
areas (Fig. 2a and b). At the same time, images of the mineralized tissue
show different surface microrelief. In the case of two-step pretreatment
of biotemplate (Sample S2) first in alkaline medium and then in a
complex of polyfunctional organic and polar amino acids, the subse­
quent mineralization leads to a homogeneous distribution over the
surface formed on the basis of HAp mineralized layer without local in­
homogeneities (Fig. 2, b). The surface does not exhibit sharp differences
in height, which best corresponds to the morphological organization of
natural enamel (Fig. 2a).
Fig. 2,c shows an AFM image of a typical surface of intact enamel
sample (Sample S1) in a 20 × 20 μm area. The surface morphology has a
characteristic relief due to the complex hierarchical structure of enamel.
In general, the relief is determined by the existence of areas of the
enamel rods ~6 × 4 μm and the space between them (Fig. 2, c). The
nanoroughness of each of the enamel rods is determined by groups of
oriented hydroxyapatite nanoprisms bonded together by a protein ma­
trix [7,22,31,32]. The characteristic thickness of enamel prismatic
nanocrystals is less than 100 nm [33]. It should be noted that the surface
of healthy enamel samples (Sample S1) has a local roughness associated
with the exit rods on the enamel surface, as well as the array of apatite
nanocrystals forming them [34]. Large height differences across the
surface are associated with non-uniformity of natural mineralization of
the natural enamel surface, the hierarchical organization of apatite
nanocrystals within the enamel rod and in interrod regions [33].
The surface morphology of S2 samples (Fig. 2, d) deserves special
attention, which is characterized by a more homogeneous micromor­
phology of the mineralized layer. However, the formation of larger
particles ~500 nm as a result of the splicing of HAp nanocrystals into
agglomerates by the amino acid layer is observed on the surface. This
behavior may be a consequence of additional enamel treatment with a
complex of polyfunctional organic and polar amino acids solution,
which, as shown in several works [35,36], promotes directed aggrega­
tion of nanocrystalline hydroxyapatite. One should note that formation
of the mineralized layer on the surface of Sample S2 is confirmed by
AFM phase contrast images (Fig. 2, f).
The formation of mineralized layers is also confirmed by the results
of field emission electron microscopy. Typical images of the surface
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Results in Engineering 15 (2022) 100583
areas of the mineralized tissue and cross-section are presented in Fig. 3.
Based on the obtained results, FESEM data are in a good agreement with
the results of AFM imaging and, in addition, they allow us to visualize
the features of the morphology in the mineralized tissue accounting for
different techniques of biotemplate pre-treatment.
For example, Fig. 3,a demonstrates the surface of healthy enamel
(Sample S1) with a characteristic aprismatic surface layer while at the
macro-level one can see enamel rods and the inter-rod space. Note that
after the mineralization procedure it is possible to see morphology areas,
just as in AFM images (see Fig. 2) which are characteristic for the
healthy enamel. It is clearly observed that on the surface of the sample
after their mineralization there exists a layer formed by nanocrystals and
their size is comparable with that of hydroxyapatite (ncHAp) nano­
crystals of 50–70 nm. One can distinctly see that the surface morphology
of the mineralized layer for the Sample S2 is very close to the
morphology of native enamel (Fig. 3,b).
Cross-section studies of the native enamel samples (Sample S1,
Fig. 3c and d) and enamel with a mineralized layer (Sample S2, Fig. 3e
and f) including those with a greater magnification x100000 (Fig. 3e and
f) do both confirm and visualize the sizes of the formed mineralized
layer. It is clearly seen that for the sample of sound enamel (Sample S1)
SEM the cross-section image quite obviously demonstrates local hier­
archy of the enamel prisms. Fig. 3,d with a high-resolution micropho­
tograph for the region of the cross-section shows that apatite
nanocrystals of the intact enamel have a smaller length and order
compared to the internal layers, which is due to the processes of natural
mineralization.
In case of Sample S2 (Fig. 3,f) that corresponds to the formation of
the mineralized layer comprising of the particles and agglomerates of
~50 nm in size is observed on the surface that corresponds to the sizes of
ncHAp nanocrystals [13] used for the layer formation. The thickness of
this layer varies from 300 to 500 nm (indicated in Fig. 3,f by arrows).
This result correlates quite well with AFM studies of the mineralized
layers.
4.2. Raman microspectroscopy
We studied the differences in the chemical composition between a
natural and mineralized enamel tissue using Raman microspectroscopy,
which is sensitive to distortions in the crystal structure of apatite [37].
Fig. 4,a shows typical Raman spectra of mineralized tissues (Samples
S1 & S2), as well as the spectrum of nanosized carbonate-substituted
calcium hydroxyapatite we used. The analysis of results shows that
the basic and most intensive modes in spectra of samples of artificially
mineralized and natural tissues, and also synthesized bioinspired
apatite, can be attributed to characteristic vibrations of carbonate-
substituted calcium hydroxyapatite, which is a basis of a mineral
component of investigated samples [38–43].
Thus, phosphate ions are associated with four groups of bands active
in the Raman spectrum of both mineralized tissue and nanocrystalline
hydroxyapatite HAp. The most intensive maximum here υ1 is symmetric
valence vibration PO3− 4 , localized in the region of 930–990 cm
− 1
[38,39,
41–44] (Fig. 4,a). It is necessary to pay attention to the different position
of the main maximum in the spectra of the studied samples.
The next group of bands is attributed to phosphate ions are the less
intense υ3 stretching oscillation, consisting of three overlapping maxima
around 1025 - 1045 cm− 1, and the υ2 and υ4 PO3− 4 bending modes
(Fig. 4,a). The υ2 PO3-
4 - bending mode appears as two well resolved peaks
at ~430 cm− 1 and 445 cm− 1, while the υ4 PO3- 4 - mode appears as a group
of four overlapping maxima at ~579 cm− 1, 590 cm− 1, 609 cm− 1, and
616 cm− 1 [38,39,41–44].
As for the carbonate groups present in the structure of apatite, in the
Raman spectra they are associated with the active vibrational band υ1,
which occurs due to the inclusion of carbonate anion СO2− 3 in the crystal
lattice of calcium hydroxyapatite. In this case when the carbonate anion
of the PO3−4 group is substituted (B-type substitution, typical for biogenic
P. Seredin et al.
Fig. 3. FESEM morphology and cross-sections typical images of the samples. a,c,e - healthy tooth enamel without treatment (Sample S1); b,d,f-after surface pre­
treatment with Ca(OH)2 and a complex of polyfunctional organic and polar amino acids, and subsequent mineralization (Sample S2); a, b – surface morphology; c,d,
e,f - cross-sections images.
materials) a maximum of about 1068–1070 cm− 1 is observed in the
spectrum (Fig. 4,a). At the same time, the inclusion of carbonate anion
СO2−3 in the lattice instead of the OH-group of apatite (A-type substi­
tution, characteristic for natural enamel [38,40,41]) leads to the
appearance of a peculiarity in the range 1001–1104 cm− 1 (Fig. 4,a).
It can be noted that the band profiles of natural enamel sample (S1)
and sample with mineralized layer S2, obtained using alkaline solution
of Ca(OH)2 and a complex of polyfunctional organic and polar amino
acids the most similar are.
To establish the influence of the biotemplate pretreatment method
on the chemical differentiation and spatial distribution of the mineral­
izing layer, we performed Raman mapping of 7 μ × 7 μ characteristic
surface areas of S1 and S2 samples, taking into account the morphology
of the enamel surface. For this purpose, an automated motorized 2-axis
stage was used, providing a minimum incremental displacement of 250
μm. Selected sample sections were scanned with a spatial resolution of
0.5 μm, and signal acquisition was performed with a 100× objective
lens. The spectrometer was carefully calibrated beforehand using the
silicon Raman mode. To increase the signal-to-noise ratio, the spectrum
at each map point was averaged based on 40 scans.
Chemical imaging (functional group mapping) of the surface areas
was then performed using the CytoSpec software. As a result, chemical
images were created, which are distributions in the color scale of the
value of the ratio of intensities in the selected frequency range. For
chemical imaging, we chose the R-ratio of the integral intensities of the
low-frequency and high-frequency components of the υ2 PO3− 4 doublet.
Typical chemical maps were obtained, as well as optical images of the
microsurfaces of Samples S1 and S2 for which mapping was performed,
5
Results in Engineering 15 (2022) 100583
and they are shown in Fig. 4b and c. An analysis of chemical imaging
maps showed that the value of the R-ratio chosen for chemical imaging
depends not only on the location on the surface of the mineralized tissue,
and thus on the chemical composition at a particular point, but also on
the type of biotemplate pretreatment. Thus, for healthy enamel (Sample
S1) this ratio varied from 0.49 to 0.64. At the same time, in the area of
the center of the enamel rods R took the minimum possible value, and in
the interrods area, where the deviation from the orientation of local
crystallites is great [45], this ratio was maximum.
The opposite picture is demonstrated by Sample S2. The R-ratio on
chemical images was maximal in the area of the center of enamel rods,
and it was minimal in the interstitial area (Fig. 4b and c). At the same
time, in typical areas of Sample S2, the R-ratio was higher and lay in the
range 0.55–0.75. The reason for this, in our opinion, is not only the
active growth of ncHAp crystals, for which the integral intensity of the
low-frequency satellite υ2 PO3− 4 doublet was significantly higher than
that of the high-frequency satellite (see Fig. 4,a) and was R~1.32, but
also their preferential orientation (texture) appearing in case of a pre-
treated biotemplate surface.
4.3. Nanoindentation
Since the hierarchical enamel substructure is formed by oriented
enamel rods consisting of tightly packed hydroxyapatite nanoprisms
bound together by protein and peptides, nanoindentation of natural and
mineralized tissue surfaces was performed both in the area of enamel
rods and in the adjacent interrods area (see Fig. 2). As a result of
nanoindentation, Vickers hardness was determined from the recorded
P. Seredin et al. Results in Engineering 15 (2022) 100583

During the study of the mechanical characteristics, the values of

Young’s modulus were also determined for the characteristic areas of the
surface of sound enamel (Sample S1) and Sample S2: after surface pre­
treatment with Са(ОН)2 and a complex of polyfunctional organic and
polar amino acids, and the subsequent mineralization. Thereby, the
averaged modulus of elasticity for the intact enamel is of ~101 GPa, that
coincides as well with the known data from the literature [46]. At the
same time, measurements of Young’s modulus of the surface layer after
surface pretreatment with Са(ОН)2 and a complex of polyfunctional
organic and polar amino acids, and the subsequent mineralization with
use of nanosized carbonate-substituted calcium hydroxyapatite
demonstrated a reduction of the Young’s modulus value down to ~89
GPa. A decrease in the elasticity of the surface layer for the samples of
the S2 group at the same time with the increased value of hardness
relative to the native enamel (Sample S1) is a consequence of hy­
droxyapatite nanocrystals formation in the process of their crystalliza­
tion on the biotemplate of a dense mineralized layer with a small part of
organic component. Since the amino acid booster was utilized in our
experiments only for the modification of biotemplate surface, but it was
not incorporated into the composition of biomimetic layer, the surface
areas are mainly comprised of nanocrystalline hydroxyapatite. This is
supported by Raman microspectroscopy data (chemical images), and its
utilization confirmed the presence of only nano-cHAp on the surface of
biotemplates (Fig. 4). As it follows from the results of performed ex­
periments, in order to attain the values of elasticity modulus similar to
those ones in the natural enamel, it is necessary to develop a technology
for the simultaneous utilization of an amino acid booster, a Са(ОН)2
alkali, and a complex of polyfunctional organic and polar amino acids,
for the treatment of the surface as during subsequent mineralization.
Investigating this problem is the aim of our future studies.

5. Conclusions

In our work we have demonstrated that with the use of nano­

crystalline carbonate-substituted calcium hydroxyapatite (HAp), a cal­
cium alkali, and a complex of polyfunctional organic and polar amino
acids, a biomimetic mineralized layer can be formed on the surface of
the dental natural tissue (biotemplate).
The formation of a mineralized layer with properties resembling
those of natural hard tissue was confirmed by the results of field emis­
sion electron and atomic force microscopy, also chemical imaging of the
surface areas with help of Raman microspectroscopy. The thickness of
Fig. 4. Results of Raman microspectrosopy. (a) Typical Raman spectra of the
the biomimetic mineralized layer varies from 300 to 500 nm, while the
studied samples. Optical (left) and chemical (right) images of typical sample
direction of some ncHAp nanocrystals coincides with that of apatite
surface areas: (b) healthy tooth enamel without treatment (Sample S1); (c) after
surface pretreatment with Са(ОН)2 and an amino acid booster, and subsequent
crystals in the enamel. We also demonstrated that the engineered
mineralization (Sample S2). mineralized HAp layer was characterized by homogeneous micromor­
phology and enhanced nanohardness in the region of the enamel rods
exceeding that of native enamel.
load-displacement curve for each indentation test for Samples S1 and S2
Obtaining a mineralized layer with a similar hierarchy and cleavage
in the enamel core area and in the interrod area. It follows from the
characteristic of natural enamel, taking into account the peculiarities of
obtained results that the averaged value of the hardness of the surface
micromorphology of dental tissue, is an urgent problem for future
layer of a healthy enamel sample (S1) takes the value of HV~600, which
research.
corresponds to the data known from the literature [45]. Thus, hardness
The development of a strategy for biomimetic engineering and a
in the area of enamel rods (HV~810) practically twice exceeds that one
technique for enamel surface pre-treatment in order to provide tissue
for interrods (HV~393) of the value that is connected with orientation
mineralization has a huge potential for application in dental clinic
of apatite nanocrystals of natural enamel.
practices.
Hardness measurements for the S2 samples showed a significant
increase in the HV value (by more than 15%) in the enamel rod area
Ethical permission
(HV~930) as compared to intact enamel (specimens S1) and decrease in
the HV value in the interrod area (HV~370).
All experiments with human dental tissue and data collections were
The increased value of HV hardness in the area of enamel rods in
performed in accordance with relevant guidelines and regulations,
Sample S2 is apparently associated with changes occurring in the sub­
including that all human participants provided informed consent for
structure of the mineralized layer: with homogeneous crystallization of
data collection and handling followed by the Helsinki Declaration. The
hydroxyapatite nanocrystals on the biotemplate surface after treatment
study was approved by the Ethics Committee of Voronezh State Uni­
in alkaline solution, and the binding of individual nanocrystals and
versity (Permission no. Pr-002.003.2022, 3 March 2022).
agglomerates into a single complex by an amino acid booster (Fig. 2).

6
P. Seredin et al.
Funding
This work was funded by the Russian Science Foundation, grant
number 21-75-10005;
The access to scientific equipment and methodology was provided
under support of the Ministry of Science and Higher Education of Russia,
Agreement N 075-15-2021-1351.
Author contribution
P.S.: Conceived and designed the experiments, analyzed the data,
performed the experiments, contributed reagents/materials/analysis
tools and wrote the manuscript. D.G.: Contributed reagents/materials/
analysis tools, performed the experiments, analyzed the data, prepared
the figures and tables and wrote the manuscript. Y.I.: Contributed re­
agents/materials/analysis tools. A.E., N.B., T.K., V.K., A.L., I.M., and M.
M.: analyzed the data and wrote the manuscript. All authors have read
and agreed to the published version of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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