# Surface Enamel Remineralization: Biomimetic Apatite Nanocrystals and Fluoride Ions Different Effects

Authors: Norberto Roveri; Elisa Battistella; Claudia Letizia Bianchi; Ismaela Foltran; Elisabetta Foresti; Michele Iafisco; Marco Lelli; Alberto Naldoni; Barbara Palazzo; Lia Rimondini
Journal: Journal of Nanomaterials
 (2009)

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## Abstract

A  new method for  altered enamel surface remineralization has been proposed . To this aim carbonate-hydroxyapatite  nanocrystals  which mimic for composition, structure, nanodimensions, and morphology dentine apatite crystals and resemble closely natural apatite chemical-physical properties have been used. The results underline the differences induced by the use of fluoride ions and hydroxyapatite nanocrystals in contrasting the mechanical abrasions and acid attacks to which tooth enamel is exposed. Fluoride ions generate a surface modification of the natural enamel apatite crystals increasing their crystallinity degree and relative mechanical and acid resistance. On the other hand, the remineralization produced by carbonate-hydroxyapatite consists in a deposition of a new apatitic mineral into the eroded enamel surface scratches. A new  biomimetic mineral coating, which progressively fills and shadows surface scratches, covers and safeguards the enamel structure by contrasting the acid and bacteria attacks.


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## Body

## 1. Introduction

Dental erosion is the
chemical wear of the dental hard tissue without the involvement of bacteria
[1]. Its clinical relevance is becoming wider and wider [2–6], and it is
considered one of the main tooth pathologies able to cause patient discomfort,
after periodontal diseases and caries.Its
aetiology is related to the enormous increase in consumption of soft drinks,
fruit juices and sport drinks consumption [7]. However, other acid sources such
as drugs containing syrups, analgesics and vitamin C intake and environmental
acid exposure in working conditions are claimed to be related to enamel erosion
[8–12].The mechanisms involved
in the damage of dental hard tissue are the acid attacks on the outer few
micrometers of the enamel, which brings to demineralization
and dissolution of the mineral phase [13–16].Hydroxyapatite is the main
constituent of the dental tissues representing in enamel and dentine the 95 wt% and 75 wt%, respectively.The primary determinant of dissolution rate is the solubility of hydroxyapatite (HA) which is related to pH, and the presence of salivary pellicle also appears to be important [17–19].Frequent application of a high concentration of topical fluoride may be of some benefit in preventing further demineralization and increasing the abrasion resistance of erosion lesions [20].In vitro studies have shown that synthetic carbonated-hydroxyapatite (CHA) dissolution inhibition is a logarithmic function of the fluoride concentration in solution [21].Systemic intake of fluoride during tooth formation has been claimed to be effective in caries prevention just by means of the apatite demineralization inhibition. According to recommended daily allowances (RDAs) panel of the European Food Safety Authority, an intake of 0.1 mg fluoride/Kg body weight/day in children up to the age of eight years can be considered as the dose below which there will be no significant occurrence of moderate forms of fluorosis in permanent teeth [22].However, it is important to consider that fluoridated water, fluoride supplements in diet, fluoride toothpaste, and topical fluoride applications have been identified as sources of enamel fluorosis [23]. Moreover, the “probably toxic dose” of fluoride—the dose which should trigger therapeutic intervention and hospitalization—is 5 mg/Kg of body weight, but as currently packaged, many dental products contain sufficient fluoride to exceed the “probably toxic dose” for young children [24].Most
of the products and devices used to contrast enamel
and dentine erosion, such as fluoride [25–28], behave by reducing apatite
dissolution rather than aiming to promote mineralization through apatite
crystallization or replacement of the lost
mineral. Hydroxyapatite, as well as in bone, is responsible for the mechanical
behavior of the dental tissues. Unlike bone, in enamel and dentine, when HA is
dissolved or abraded, it cannot spontaneously remineralize because enamel
contains no cells and dentine apposition occurs only toward the pulp tissues.
Therefore, both enamel and dentine can be reconstructed only by the application
of alloplastic materials providing a sort of prosthetic restoration. In view of
this situation, the demineralized area and micrometric sized scratches, which
normally occur on enamel surface as a consequence of microwear and acid attack
[29], cannot be repaired biologically nor prosthetically.Hydroxyapatite has been widely subjected to experiment as bone filler
and prosthetic coating due to its biocompatibility and osteoconductivity,
representing an elective material covering a wide range of applications for bone
substitution and interface [30]. Poorly crystalline HA nanocrystals, in
addition to the excellent biological properties of HA, such as nontoxicity and
lack of inflammatory and immunitary responses, have bioresorption properties
under physiological conditions. This property can be modulated by modifying its
degree of crystallinity, which is achieved by implementation of innovative
synthesis with a nanosize crystals control. In the last decade, advanced
technology has been utilized to synthesize a new generation of biomimetic
apatitic alloplastic materials which can optimize the interaction with
biological entities thanks to their strong surface bioactivity [31]. The aim of
the present study is to highlight the effect of the synthetic biomimetic hydroxyapatite
crystals respect the fluoride ions into the remineralization in vitro of human
enamel surface.


## 2. Materials and Methods

### 2.1. Chemicals

All the chemicals
reagents used were of high chemical grade from Sigma-Aldrich, Mo, USA.


### 2.2. Synthesis of Carbonate-Hydroxyapatite Nanocrystals

Plate-acicular shaped carbonate-hydroxyapatite nanocrystals about 100 nm in size were synthesized according to a modification of the method
previously reported [32] and patented [33]. CHA nanocrystals
about 100 nm in size were precipitated from an aqueous suspension of Ca(OH)2 (0.17 M)
by slow addition of H3PO4 (0.15 M). The reaction
mixture was stirred at 37°C for 12 hours, and then stirring was suspended allowing the deposition of CHA
nanocrystals. Synthesized CHA 100 nm sized nanocrystals were
isolated by filtration of the mother liquor, repeatedly washed with water and
freeze-dried. The fraction of plate-acicular shaped
crystals about 100 nm in size with a granular dimensions ranging from 100 to
150 μm was selected for the study. Aliquot of plate-acicular shaped crystals about 100 nm in size has been
allowed, after synthesis to intergrowth in the reaction mixture up to the
formation of clusters having dimension ranging from about 0.5 to 3.0 μm. The
cluster aggregation process has been stopped by adding a surfactant (Protelan
MST35 8 wt%) in the mother solution.Plate-shaped carbonate-hydroxyapatite nanocrystals about 20 nm sized were synthesized according to the method of Liou et al. [34]
with some modifications. The nanocrystals were precipitated from an aqueous
solution of (NH4)3PO4 (5.1 mM) by slow addition of
an aqueous solution of Ca(CH3COO)2 (8.5 mM) keeping the pH at a
constant value of 10 by addition of a NH4OH solution. The reaction
mixture was kept under stirring at room temperature for 24 hours, and then
stirring was suspended allowing the deposition CHA nanocrystals. Synthesized CHA nanocrystals were isolated by filtration of the solution,
repeatedly washed with water, and freeze-dried. The fraction of plate shaped
crystals about 20 nm in size having granular dimensions ranging from 100 to 150 μm was selected for the study.


### 2.3. Morphological Characterization

Transmission electron microscopy (TEM)
investigations were carried out using a Philips CM 100 instrument. The powdered
samples were ultrasonically dispersed in ultrapure water, and then a few
droplets of the slurry deposited on holey-carbon foils supported on
conventional copper microgrids.Scanning electron microscopy (SEM) observation was carried out by an SEM (Zeiss
EVO, 40 XVP) using secondary electrons at 25 kV and various magnifications.


### 2.4. Structural Characterization

X-ray diffraction
powder and enamel surface patterns were collected using an Analytical X'Pert
Pro equipped with X'Celerator detector powder diffractometer using Cu Kα radiation generated at 40 kV and 40 mA. The instrument was configured with a 1° divergence and 0.2 mm receiving slits. The
samples were prepared using the front loading of standard aluminium sample
holders which are 1 mm deep, 20 mm high ,and 15 mm wide. The degree of HA crystallinity was evaluated according to the formula
[35] (1)crystallinity=(XY)100, where X= net area of diffracted peaks, and Y=
net area of diffracted peaks  + background area.Crystal domain size along the c direction was calculated
applying Scherrer's formula(2)L(002)=0.94λ[cosθ(Δr2−Δ02)], where θ is the diffraction angle
for plane (002), Δr and Δ0 are the widths in radians of reflection (002) at
half height for the synthesized and the reference HA materials, respectively,
and λ=1.5405 Å.


### 2.5. Infrared Microscopy Spectral Analysis

ATR-IR spectra were recorded on a Thermo Nicolet 380 FT-IR spectrometer
equipped with a commercial ATR accessory.The infrared spectra were registered from 4000 to 400 cm−1 at 2 cm−1 resolution using a Bruker IFS 66v/S spectrometer using KBr pellets.ATR spectra were recorded with the cell empty to be used as a blank for
subsequent experiments. Samples were made by placing the powder sample onto the
Ge ATR crystal. Spectra were collected by averaging 32 scans at 4 cm−1 resolution.


### 2.6. Specific Surface Area Determination

Measurements
were undertaken using a Carlo Erba Sorpty 1750 instrument by measuringN2 absorption at 77 K and adopting the well-known BET procedure [36].


### 2.7. Spectrophotometric Analysis

Phosphorus contents were determined
spectrophotometrically as molybdovanadophosphoric acid using 1 cm quartz cell [37].Calcium contents were
measured using inductively coupled plasma (ICP) optical emission spectrometry
(OES) using a Perkin Elmer Optima 4200 DV.


### 2.8. X-Ray Photoemission Spectroscopy (XPS)

Analyses
were performed in an M-Probe Instrument (SSI) equipped with a monochromatic Al
Kα source (1486.6 eV) with a spot size
of 200×750μm
and pass energy of 25 eV, providing a resolution for 0.74 eV. With a
monochromatic source, an electron flood gun was used to compensate the build up
of positive charge on the insulator samples during the analyses. 10 eV
electrons were selected to perform measurements on these samples. The accuracy
of the reported binding energies (BEs) was estimated to be ±0.2 eV. The
quantitative data were also accurately checked and reproduced several times (at
least ten times for each sample), and the percentage error was estimated to be ±1%.


### 2.9. Statistics Analysis

Determination of HA crystallite
domain size along thec direction,
bulk and surface Ca/P ratio, and specific surface area were carried out 5 times
on the same synthesis product. Data are presented as mean value ±SD.
Differences were considered statistically significant at a significance level
of 90%.


### 2.10. In Vitro Enamel Remineralization Procedures

Slabs of enamel (3×3 mm) were
obtained from interproximal surfaces of premolars extracted for orthodontic
reasons. After the extraction, the teeth were cut with diamond disks, and the obtained
slabs were sonicated for 10 minutes in 50% ethanol in order to remove any debris.
Then, the specimens were etched with 37% phosphoric acid for 1 minute. Etched slabs
were washed in distilled water for 10 minutes under stirring and then air
dried. Two different in vitro remineralization procedures have been performed.The first in vitro remineralization procedure utilizes a 10 wt% CHA in the range from 100
to 150μm granules slurries constituted of both 20 nm or 100 nm sized
nanocrystals. CHA nanocrystals aqueous slurries were applied for 10 minutes on the
surfaces of the enamel slabs at room temperature with 100% relative humidity and
then removed by water washing and air dried.The second in vitro remineralization process utilizes toothpaste containing
both fluoride ions and CHA 0.5–3.0μm sized clusters constituted by
intergrowth of 100 nm sized
nanocrystals.Comparable etched enamel slabs were
divided into 3-groups of treatment using, respectively, fluoride or CHA-based
toothpaste and only water (control). Each slab was brushed three times a day
for a period of 15 days. The intervals between brushing sessions were at least
5 hours. Every brushing session has been performed for 30 secons by an electric
toothbrush using constant pressure and a bean sized toothpaste aliquot wetted
with tap water, closely resembling the in vivo usual tooth brushing procedure.
After every treatment, the single enamel slab was washed with tap water using a
cleaned toothbrush in order to remove residual toothpaste.


## 2.1. Chemicals

All the chemicals
reagents used were of high chemical grade from Sigma-Aldrich, Mo, USA.


## 2.2. Synthesis of Carbonate-Hydroxyapatite Nanocrystals

Plate-acicular shaped carbonate-hydroxyapatite nanocrystals about 100 nm in size were synthesized according to a modification of the method
previously reported [32] and patented [33]. CHA nanocrystals
about 100 nm in size were precipitated from an aqueous suspension of Ca(OH)2 (0.17 M)
by slow addition of H3PO4 (0.15 M). The reaction
mixture was stirred at 37°C for 12 hours, and then stirring was suspended allowing the deposition of CHA
nanocrystals. Synthesized CHA 100 nm sized nanocrystals were
isolated by filtration of the mother liquor, repeatedly washed with water and
freeze-dried. The fraction of plate-acicular shaped
crystals about 100 nm in size with a granular dimensions ranging from 100 to
150 μm was selected for the study. Aliquot of plate-acicular shaped crystals about 100 nm in size has been
allowed, after synthesis to intergrowth in the reaction mixture up to the
formation of clusters having dimension ranging from about 0.5 to 3.0 μm. The
cluster aggregation process has been stopped by adding a surfactant (Protelan
MST35 8 wt%) in the mother solution.Plate-shaped carbonate-hydroxyapatite nanocrystals about 20 nm sized were synthesized according to the method of Liou et al. [34]
with some modifications. The nanocrystals were precipitated from an aqueous
solution of (NH4)3PO4 (5.1 mM) by slow addition of
an aqueous solution of Ca(CH3COO)2 (8.5 mM) keeping the pH at a
constant value of 10 by addition of a NH4OH solution. The reaction
mixture was kept under stirring at room temperature for 24 hours, and then
stirring was suspended allowing the deposition CHA nanocrystals. Synthesized CHA nanocrystals were isolated by filtration of the solution,
repeatedly washed with water, and freeze-dried. The fraction of plate shaped
crystals about 20 nm in size having granular dimensions ranging from 100 to 150 μm was selected for the study.


## 2.3. Morphological Characterization

Transmission electron microscopy (TEM)
investigations were carried out using a Philips CM 100 instrument. The powdered
samples were ultrasonically dispersed in ultrapure water, and then a few
droplets of the slurry deposited on holey-carbon foils supported on
conventional copper microgrids.Scanning electron microscopy (SEM) observation was carried out by an SEM (Zeiss
EVO, 40 XVP) using secondary electrons at 25 kV and various magnifications.


## 2.4. Structural Characterization

X-ray diffraction
powder and enamel surface patterns were collected using an Analytical X'Pert
Pro equipped with X'Celerator detector powder diffractometer using Cu Kα radiation generated at 40 kV and 40 mA. The instrument was configured with a 1° divergence and 0.2 mm receiving slits. The
samples were prepared using the front loading of standard aluminium sample
holders which are 1 mm deep, 20 mm high ,and 15 mm wide. The degree of HA crystallinity was evaluated according to the formula
[35] (1)crystallinity=(XY)100, where X= net area of diffracted peaks, and Y=
net area of diffracted peaks  + background area.Crystal domain size along the c direction was calculated
applying Scherrer's formula(2)L(002)=0.94λ[cosθ(Δr2−Δ02)], where θ is the diffraction angle
for plane (002), Δr and Δ0 are the widths in radians of reflection (002) at
half height for the synthesized and the reference HA materials, respectively,
and λ=1.5405 Å.


## 2.5. Infrared Microscopy Spectral Analysis

ATR-IR spectra were recorded on a Thermo Nicolet 380 FT-IR spectrometer
equipped with a commercial ATR accessory.The infrared spectra were registered from 4000 to 400 cm−1 at 2 cm−1 resolution using a Bruker IFS 66v/S spectrometer using KBr pellets.ATR spectra were recorded with the cell empty to be used as a blank for
subsequent experiments. Samples were made by placing the powder sample onto the
Ge ATR crystal. Spectra were collected by averaging 32 scans at 4 cm−1 resolution.


## 2.6. Specific Surface Area Determination

Measurements
were undertaken using a Carlo Erba Sorpty 1750 instrument by measuringN2 absorption at 77 K and adopting the well-known BET procedure [36].


## 2.7. Spectrophotometric Analysis

Phosphorus contents were determined
spectrophotometrically as molybdovanadophosphoric acid using 1 cm quartz cell [37].Calcium contents were
measured using inductively coupled plasma (ICP) optical emission spectrometry
(OES) using a Perkin Elmer Optima 4200 DV.


## 2.8. X-Ray Photoemission Spectroscopy (XPS)

Analyses
were performed in an M-Probe Instrument (SSI) equipped with a monochromatic Al
Kα source (1486.6 eV) with a spot size
of 200×750μm
and pass energy of 25 eV, providing a resolution for 0.74 eV. With a
monochromatic source, an electron flood gun was used to compensate the build up
of positive charge on the insulator samples during the analyses. 10 eV
electrons were selected to perform measurements on these samples. The accuracy
of the reported binding energies (BEs) was estimated to be ±0.2 eV. The
quantitative data were also accurately checked and reproduced several times (at
least ten times for each sample), and the percentage error was estimated to be ±1%.


## 2.9. Statistics Analysis

Determination of HA crystallite
domain size along thec direction,
bulk and surface Ca/P ratio, and specific surface area were carried out 5 times
on the same synthesis product. Data are presented as mean value ±SD.
Differences were considered statistically significant at a significance level
of 90%.


## 2.10. In Vitro Enamel Remineralization Procedures

Slabs of enamel (3×3 mm) were
obtained from interproximal surfaces of premolars extracted for orthodontic
reasons. After the extraction, the teeth were cut with diamond disks, and the obtained
slabs were sonicated for 10 minutes in 50% ethanol in order to remove any debris.
Then, the specimens were etched with 37% phosphoric acid for 1 minute. Etched slabs
were washed in distilled water for 10 minutes under stirring and then air
dried. Two different in vitro remineralization procedures have been performed.The first in vitro remineralization procedure utilizes a 10 wt% CHA in the range from 100
to 150μm granules slurries constituted of both 20 nm or 100 nm sized
nanocrystals. CHA nanocrystals aqueous slurries were applied for 10 minutes on the
surfaces of the enamel slabs at room temperature with 100% relative humidity and
then removed by water washing and air dried.The second in vitro remineralization process utilizes toothpaste containing
both fluoride ions and CHA 0.5–3.0μm sized clusters constituted by
intergrowth of 100 nm sized
nanocrystals.Comparable etched enamel slabs were
divided into 3-groups of treatment using, respectively, fluoride or CHA-based
toothpaste and only water (control). Each slab was brushed three times a day
for a period of 15 days. The intervals between brushing sessions were at least
5 hours. Every brushing session has been performed for 30 secons by an electric
toothbrush using constant pressure and a bean sized toothpaste aliquot wetted
with tap water, closely resembling the in vivo usual tooth brushing procedure.
After every treatment, the single enamel slab was washed with tap water using a
cleaned toothbrush in order to remove residual toothpaste.


## 3. Results

### 3.1. Synthesis and Characterization of Biomimetic Carbonate-Hydroxyapatite Nanocrystals

Biomimetic carbonate-hydroxyapatite nanocrystals have been synthesized
with a nearly stoichiometric in bulk Ca/P molar ratio of about 1.6-1.7 and containing 4  ± 1 wt% of carbonate ions replacing
prevalently phosphate groups. CHA
nanocrystals have been
synthesized both about 100 nm and
20 nm sized with an acicular
and plate morphology,
respectively. TEM
images of synthetic 20 nm sized CHA
nanocrystals showing the plate-shaped morphology and synthetic 100 nm sized CHA nanocrystals showing the acicular morphology are
reported in Figures1(a) and 1(b), respectively.TEM images of (a) synthetic plate-shaped 20 nm sized CHA
nanocrystals (scale bar = 100 nm), (b) synthetic plate-acicular 100 nm sized CHA nanocrystals (scale bars  = 200 nm).
(a)(b)CHA nanocrystals can aggregate in
microsized crystal clusters, whose dimensions increase
prolonging maturation time in mother solution at constant temperature
and stirring [33].Powder X-ray diffraction patterns of
plate shaped about 20 nm sized CHA nanocrystals and acicular shaped about 100 nm sized CHA nanocrystals
(see Figures2(b) and 2(c), resp.) show
characteristic diffraction maxima of an apatite single phase (JCPDS
01-074-0565).Figure 2
X-ray
diffraction patterns of (a) natural carbonate-hydroxyapatite
from deproteined dentine, (b) synthetic plate shaped 20 nm sized CHA nanocrystals, (c) synthetic plate-acicular shaped 100 nm sized CHA nanocrystals, and (d) natural carbonate-hydroxyapatite
from enamel.These X-ray diffraction patterns are
compared with those collected for natural carbonate hydroxyapatite
from deproteined dentine and enamel reported
in Figures2(a) and 2(d), respectively. The broadening of the diffraction maxima
present in the X-ray diffraction patterns reported in Figures 2(a), 2(b), and 2(c) indicates
a relatively low degree of crystallinity, which was quantified according to
previous [35]. The degree of crystallinity of synthesized about 20 nm sized CHA
nanocrystals with plate
morphology and
synthesized about 100 nm sized CHA nanocrystals with acicular morphology is 30% and 50%,
respectively. The crystallinity degree of about 20 nm sized CHA nanocrystals is
very close to that one determined from the X-ray diffraction pattern of deproteined
dentine natural carbonate-hydroxyapatite (28%) reported
in Figure 2(a). Furthermore, the crystallinity degree of natural hydroxyapatite of deproteined enamel, reported in Figure 2(d),
is 70%. The crystal domain sizes along
the c direction have been calculated by Scherrer's formula using the 2θ= 26° (002) diffraction peak of the X-ray diffraction
patterns reported in Figures 2(a), 2(b), and 2(c). The calculated crystal domain sizes for
20 nm sized CHA nanocrystals and deproteined dentine are 250 Å  and 213 Å, respectively. These results obtained by X-ray diffraction investigation reveal that
the crystal structures of the synthesized CHA nanocrystals are very close to
that observed for natural dentine.The same similarity can be observed
from the comparison of the FTIR spectra of synthesized CHA nanocrystals and
natural apatite of deproteined dentine reported in Figures3(a) and 3(b),
respectively. In these spectra, the characteristic absorption bands of phosphate
and carbonate groups are clearly resolved. The absorption band at 1468 cm−1 is related to the carbonate group substitution to the phosphate one, while the
shoulder at 1545 cm−1 can be considered the contribution of the
carbonate group substituting the hydroxyl group in the apatite structure. This
finding reveals that synthesized CHA nanocrystals not only contain a similar
carbonate amount, but also underline that the carbonate substitution to the
phosphate and/or hydroxyl group is very similar in the synthetic and
biological crystals.Figure 3
(a) FTIR spectra of synthetic 20 nm sized CHA nanocrystals, (b) natural
carbonate-hydroxyapatite from deproteined dentine.A surface characterization of the
synthetic carbonate-hydroxyapatite
nanocrystals has been carried out in order to highlight their surface chemical-physical
characteristic which directly interfaces and reacts with exposed dental
tissues. The ATR spectra (data not shown) of the synthetic about 20 nm and 100 nm sized CHA nanocrystals reveal a 4% and 3 wt% surface carbonate, respectively. The consistent amount of surface of carbonate percentage
present in synthetic CHA is appreciably higher than the surface carbonate percentage
present in enamel and dentine about 2 wt%.Specific surface area of 100m2g−1 and 80 m2g−1 has been determined for 20 nm sized CHA nanocrystals
with plate morphology and synthesized 100 nm sized CHA nanocrystals
with acicular morphology, respectively. These specific surface
area values obtained for synthetic nanocrystals are only slightly lower than
the 110 m2g−1 obtained for biological nanocrystals.The surface Ca/P molar ratio
determined by XPS analysis for CHA nanocrystals and CHA crystals microclusters
does not reveal appreciably differences and result significantly lower than Ca/P
molar ratio determined by ICP analysis in bulk indicating a surface calcium
deficiency probably due to surface disorder. In fact, the Ca/P molar ratios of
1.7 determined in bulk for synthetic CHA nanocrystals reduce to a value of 1.4-1.5 when determined on the crystals surface by XPS analysis (see Table1). XPS
analysis of spectral features of the O 1s region of the synthetic 100 nm sized CHA nanocrystals (see Figure 4(c)) shows a definite O 1s shape fitted by three components: a first, very intense, peak at lower BE
attributed to oxygen in phosphate group, a second peak due to OH of the
carbonate-hydroxyapatite, and a final peak at very high BE, which can be
attributed to trapped undissociated water and carbonates.Table 1
XPS analysis of CHA nanocrystals, enamel tooth treated with ortophosphoric
acid before and after treatment
with either CHA 20 or 100 nm sized nanocrystals and after brushing with
toothpaste containing booth CHA or fluoride.
SampleO (%)C (%)Ca (%)P (%)N (%)Si (%)Na (%)F (%)Ca/P20 nm CHA nanocrystals50.417.619.412.6————1.5100 nm CHA nanocrystals48.418.019.613.9————1.4Tooth treated with ortophosphoric acid30.749.45.45.35.91.91.4—1.1Tooth treated with 20 nm CHA nanocrystals43.327.313.410.12.3—1.9—1.3Tooth treated with 100 nm CHA nanocrystals43.726.014.811.21.0—1.5—1.3CHA containing toothpaste30.751.95.54.71.65.6——1.2Fluoride containing toothpaste20.963.34.11.22.04.9—3.63.4Figure 4
XPS
analysis of spectral features of (a) the O 1s region of the enamel
demineralized by ortophosphoric acid 37% for 1 minute,
(b) enamel remineralizated by a treatment with synthetic microclusters of CHA
nanocrystals 100 nm sized for 10 minutes, and (c) synthetic microclusters of CHA
nanocrystals 100 nm sized.


### 3.2. In Vitro Enamel Surface Remineralization by Biomimetic CHA Nanocrystals

SEM analysis allows investigating
the morphology of both demineralized enamel and the features observed after
remineralization procedures induced by biomimetic CHA nanocrystals in vitro application.The demineralization procedure by
ortophosphoric acid 37% for 1 minute removes the aprismatic enamel, and exposed
hydroxyapatite prisms became evident. Figure5(a)
shows demineralization of both interprismatic and prismatic enamel structures.SEM image of enamel surface after application of 37% ortophosphoric
acid for 1 minutes. The etching treatment removed aprismatic layer and exposed
prismatic and interprismatic hydroxyapatite structures (a), remineralized
enamel surface after treatment by synthetic CHA
micro-clusters constituted of nanocrystals 20 nm (b) and 100 nm sized (c) and
synthetic CHA micro-clusters.
(a)(b)(c)After treatment for 10 minutes by
aqueous slurry of both synthetic 20 nm and 100 nm sized CHA nanocrystals, the surface of the demineralized enamel appears
covered by the CHA phase (see Figures5(b) and 5(c), resp.) arranged in a thick
and homogeneous apatitic layer.XPS analysis of spectral features of
the O 1s region of the enamel
demineralized by ortophosphoric acid 37% for 1 minute shows a single broad
band, in which it is difficult to determine precisely and to quantify the
binding energy (BE) and, therefore, the kind of the surface oxygen components
(see Figure4(a)). On the contrary, the enamel remineralized by synthetic 100 nm sized
CHA nanocrystals for 10 minutes shows a definite O 1s shape fitted by three components at different binding energy
(see Figure 4(b)). These components correspond to the same three ones used to fit O 1s shape recorded for synthetic 100 nm
sized CHA nanocrystals (see Figure 4(c)), the first at lower BE very intense peak is
attributed to oxygen in phosphate group, the second peak is due to OH of the
carbonate-hydroxyapatite, and a final peak at high BE, can be attributed to
trapped undissociated water and carbonate groups. These results unequivocally
confirm the presence of synthetic CHA at the surface of the treated enamel and
the consequent validation of the enamel remineralization. The same finding is
pointed out by the ATR spectrum of enamel treated for 10 minutes by synthetic
100 nm sized CHA nanocrystals, showing appreciable higher intensity of the
characteristic absorption bands of carbonate ions (1420–1460 and 1680 cm−1)
in respect of the same absorption bands present in the demineralized enamel ATR
spectrum. No differences are appreciable in the phosphate ions bands (1000–1100
and 530–580 cm−1). ATR spectrum (data not shown) of remineralized
enamel reveals that surface apatite is richer in carbonate than natural one,
such as synthetic 100 nm sized CHA nanocrystals.


### 3.3. In Vitro Enamel Surface Remineralization by Toothpaste Containing Either Fluoride or Biomimetic CHA Nanocrystals Microclusters

SEM analysis allowed us to
investigate the morphology of both demineralized enamel and the features
observed after a remineralization process which utilizes in vitro application
of toothpaste containing either fluoride or CHA microclusters constituted of 100 nm in size nanocrystals.The surfaces of the teeth treated
with fluoride (see Figure6(b)) were not consistently changed respect to that of
demineralization by ortophosphoric acid (see Figure 6(c)). Actually both the interprismatic
and prismatic enamel structures appear still evident. On the contrary after
treatment of the enamel slabs with a toothpaste containing synthesized CHA microclusters constituted of 100 nm sized nanocrystals, the interprismatic
and prismatic enamel structures appear to be completely hidden by a thick
homogeneous apatitic layer (see Figure 6(a)).SEM images of enamel after brushing treatment with (a) CHA toothpaste
containing, (b) fluoride containing toothpaste, and (c) ortophosphoric acid application.
(a)(b)(c)The XRD patterns collected on the
surface of enamel slabs after treatment with CHA or fluoride toothpaste and
water are reported in Figures7(b), 7(c), and 7(d), respectively, and compared with the XRD
pattern (see Figure 7(a)) of CHA microclusters constituted of 100 nm sized
nanocrystals utilized to prepare the used CHA toothpaste. The XRD diffraction
maxima recorded on the surface of enamel slabs treated with fluoride containing
toothpaste appear slightly more sharpened than those obtained on the enamel etched
slabs brushed only with water. This observation reveals an increased
crystallinity degree probably due to a partial structural conversion of
hydroxyapatite into fluoride substituted hydroxyapatite. On the contrary, the
XRD pattern obtained on the surface of enamel slabs brushed with CHA containing
toothpaste shows the broadened diffraction maxima characteristic of the
synthetic biomimetic CHA, revealing its presence on the enamel surface. The CHA
not removed by brushing procedures suggests the formation of chemical bonds
between the synthetic CHA microclusters constituted of 100 nm sized nanocrystals
and natural enamel apatite crystals. These bonds allow the formation of a
persistent CHA coating on the enamel surface whose morphology was detected by
SEM analysis.Figure 7
(a) XRD pattern of synthetic CHA, (b) enamel after brushing treatment with CHA containing toothpaste, (c) enamel after brushing treatment with fluoride containing toothpaste, (d) enamel after brushing treatment with water. *indicates Al holder diffraction maxima.The surface Ca/P molar ratio
determined by XPS analysis for demineralized enamel slabs before and after in
vitro remineralization by brushing whit toothpaste containing either fluoride
or CHA are compared in Table1. In this table, a comparison with the Ca/P molar
ratio of CHA microclusters constituted of 100 nm sized nanocrystals also
present. The enamel surface Ca/P molar ratio changes before and after the
brushing treatment with toothpaste containing fluoride, assuming a value of
3.4 very different from the apatite stoichiometric one (Ca/P  = 1.7). This finding
reveals that the only structural modification of enamel hydroxyapatite induced
by fluoride is restricted to a partial hydroxyl groups replacement by fluoride
ions without affecting appreciably the Ca and phosphate structural network. On
the contrary, enamel slabs treated with the toothpaste containing synthesised CHA
microclusters of 100 nm sized nanocrystals exhibit a surface Ca/P molar ratio
very similar to that one of the synthetic CHA.This coating is highly less
crystalline than native enamel apatite, and consists of a new apatitic mineral
deposition which progressively fills the scratches
and pits. On the contrary, the surface remineralization observed on the
specimens treated with fluoride containing toothpaste is mainly based on chemical-physical enamel apatite
surface modifications rather than a formation of a
new mineral deposition.The CHA biomimetic
coating formation is a remineralization process corresponding to a new apatite
deposition in the demineralized
area of enamel surface.


## 3.1. Synthesis and Characterization of Biomimetic Carbonate-Hydroxyapatite Nanocrystals

Biomimetic carbonate-hydroxyapatite nanocrystals have been synthesized
with a nearly stoichiometric in bulk Ca/P molar ratio of about 1.6-1.7 and containing 4  ± 1 wt% of carbonate ions replacing
prevalently phosphate groups. CHA
nanocrystals have been
synthesized both about 100 nm and
20 nm sized with an acicular
and plate morphology,
respectively. TEM
images of synthetic 20 nm sized CHA
nanocrystals showing the plate-shaped morphology and synthetic 100 nm sized CHA nanocrystals showing the acicular morphology are
reported in Figures1(a) and 1(b), respectively.TEM images of (a) synthetic plate-shaped 20 nm sized CHA
nanocrystals (scale bar = 100 nm), (b) synthetic plate-acicular 100 nm sized CHA nanocrystals (scale bars  = 200 nm).
(a)(b)CHA nanocrystals can aggregate in
microsized crystal clusters, whose dimensions increase
prolonging maturation time in mother solution at constant temperature
and stirring [33].Powder X-ray diffraction patterns of
plate shaped about 20 nm sized CHA nanocrystals and acicular shaped about 100 nm sized CHA nanocrystals
(see Figures2(b) and 2(c), resp.) show
characteristic diffraction maxima of an apatite single phase (JCPDS
01-074-0565).Figure 2
X-ray
diffraction patterns of (a) natural carbonate-hydroxyapatite
from deproteined dentine, (b) synthetic plate shaped 20 nm sized CHA nanocrystals, (c) synthetic plate-acicular shaped 100 nm sized CHA nanocrystals, and (d) natural carbonate-hydroxyapatite
from enamel.These X-ray diffraction patterns are
compared with those collected for natural carbonate hydroxyapatite
from deproteined dentine and enamel reported
in Figures2(a) and 2(d), respectively. The broadening of the diffraction maxima
present in the X-ray diffraction patterns reported in Figures 2(a), 2(b), and 2(c) indicates
a relatively low degree of crystallinity, which was quantified according to
previous [35]. The degree of crystallinity of synthesized about 20 nm sized CHA
nanocrystals with plate
morphology and
synthesized about 100 nm sized CHA nanocrystals with acicular morphology is 30% and 50%,
respectively. The crystallinity degree of about 20 nm sized CHA nanocrystals is
very close to that one determined from the X-ray diffraction pattern of deproteined
dentine natural carbonate-hydroxyapatite (28%) reported
in Figure 2(a). Furthermore, the crystallinity degree of natural hydroxyapatite of deproteined enamel, reported in Figure 2(d),
is 70%. The crystal domain sizes along
the c direction have been calculated by Scherrer's formula using the 2θ= 26° (002) diffraction peak of the X-ray diffraction
patterns reported in Figures 2(a), 2(b), and 2(c). The calculated crystal domain sizes for
20 nm sized CHA nanocrystals and deproteined dentine are 250 Å  and 213 Å, respectively. These results obtained by X-ray diffraction investigation reveal that
the crystal structures of the synthesized CHA nanocrystals are very close to
that observed for natural dentine.The same similarity can be observed
from the comparison of the FTIR spectra of synthesized CHA nanocrystals and
natural apatite of deproteined dentine reported in Figures3(a) and 3(b),
respectively. In these spectra, the characteristic absorption bands of phosphate
and carbonate groups are clearly resolved. The absorption band at 1468 cm−1 is related to the carbonate group substitution to the phosphate one, while the
shoulder at 1545 cm−1 can be considered the contribution of the
carbonate group substituting the hydroxyl group in the apatite structure. This
finding reveals that synthesized CHA nanocrystals not only contain a similar
carbonate amount, but also underline that the carbonate substitution to the
phosphate and/or hydroxyl group is very similar in the synthetic and
biological crystals.Figure 3
(a) FTIR spectra of synthetic 20 nm sized CHA nanocrystals, (b) natural
carbonate-hydroxyapatite from deproteined dentine.A surface characterization of the
synthetic carbonate-hydroxyapatite
nanocrystals has been carried out in order to highlight their surface chemical-physical
characteristic which directly interfaces and reacts with exposed dental
tissues. The ATR spectra (data not shown) of the synthetic about 20 nm and 100 nm sized CHA nanocrystals reveal a 4% and 3 wt% surface carbonate, respectively. The consistent amount of surface of carbonate percentage
present in synthetic CHA is appreciably higher than the surface carbonate percentage
present in enamel and dentine about 2 wt%.Specific surface area of 100m2g−1 and 80 m2g−1 has been determined for 20 nm sized CHA nanocrystals
with plate morphology and synthesized 100 nm sized CHA nanocrystals
with acicular morphology, respectively. These specific surface
area values obtained for synthetic nanocrystals are only slightly lower than
the 110 m2g−1 obtained for biological nanocrystals.The surface Ca/P molar ratio
determined by XPS analysis for CHA nanocrystals and CHA crystals microclusters
does not reveal appreciably differences and result significantly lower than Ca/P
molar ratio determined by ICP analysis in bulk indicating a surface calcium
deficiency probably due to surface disorder. In fact, the Ca/P molar ratios of
1.7 determined in bulk for synthetic CHA nanocrystals reduce to a value of 1.4-1.5 when determined on the crystals surface by XPS analysis (see Table1). XPS
analysis of spectral features of the O 1s region of the synthetic 100 nm sized CHA nanocrystals (see Figure 4(c)) shows a definite O 1s shape fitted by three components: a first, very intense, peak at lower BE
attributed to oxygen in phosphate group, a second peak due to OH of the
carbonate-hydroxyapatite, and a final peak at very high BE, which can be
attributed to trapped undissociated water and carbonates.Table 1
XPS analysis of CHA nanocrystals, enamel tooth treated with ortophosphoric
acid before and after treatment
with either CHA 20 or 100 nm sized nanocrystals and after brushing with
toothpaste containing booth CHA or fluoride.
SampleO (%)C (%)Ca (%)P (%)N (%)Si (%)Na (%)F (%)Ca/P20 nm CHA nanocrystals50.417.619.412.6————1.5100 nm CHA nanocrystals48.418.019.613.9————1.4Tooth treated with ortophosphoric acid30.749.45.45.35.91.91.4—1.1Tooth treated with 20 nm CHA nanocrystals43.327.313.410.12.3—1.9—1.3Tooth treated with 100 nm CHA nanocrystals43.726.014.811.21.0—1.5—1.3CHA containing toothpaste30.751.95.54.71.65.6——1.2Fluoride containing toothpaste20.963.34.11.22.04.9—3.63.4Figure 4
XPS
analysis of spectral features of (a) the O 1s region of the enamel
demineralized by ortophosphoric acid 37% for 1 minute,
(b) enamel remineralizated by a treatment with synthetic microclusters of CHA
nanocrystals 100 nm sized for 10 minutes, and (c) synthetic microclusters of CHA
nanocrystals 100 nm sized.


## 3.2. In Vitro Enamel Surface Remineralization by Biomimetic CHA Nanocrystals

SEM analysis allows investigating
the morphology of both demineralized enamel and the features observed after
remineralization procedures induced by biomimetic CHA nanocrystals in vitro application.The demineralization procedure by
ortophosphoric acid 37% for 1 minute removes the aprismatic enamel, and exposed
hydroxyapatite prisms became evident. Figure5(a)
shows demineralization of both interprismatic and prismatic enamel structures.SEM image of enamel surface after application of 37% ortophosphoric
acid for 1 minutes. The etching treatment removed aprismatic layer and exposed
prismatic and interprismatic hydroxyapatite structures (a), remineralized
enamel surface after treatment by synthetic CHA
micro-clusters constituted of nanocrystals 20 nm (b) and 100 nm sized (c) and
synthetic CHA micro-clusters.
(a)(b)(c)After treatment for 10 minutes by
aqueous slurry of both synthetic 20 nm and 100 nm sized CHA nanocrystals, the surface of the demineralized enamel appears
covered by the CHA phase (see Figures5(b) and 5(c), resp.) arranged in a thick
and homogeneous apatitic layer.XPS analysis of spectral features of
the O 1s region of the enamel
demineralized by ortophosphoric acid 37% for 1 minute shows a single broad
band, in which it is difficult to determine precisely and to quantify the
binding energy (BE) and, therefore, the kind of the surface oxygen components
(see Figure4(a)). On the contrary, the enamel remineralized by synthetic 100 nm sized
CHA nanocrystals for 10 minutes shows a definite O 1s shape fitted by three components at different binding energy
(see Figure 4(b)). These components correspond to the same three ones used to fit O 1s shape recorded for synthetic 100 nm
sized CHA nanocrystals (see Figure 4(c)), the first at lower BE very intense peak is
attributed to oxygen in phosphate group, the second peak is due to OH of the
carbonate-hydroxyapatite, and a final peak at high BE, can be attributed to
trapped undissociated water and carbonate groups. These results unequivocally
confirm the presence of synthetic CHA at the surface of the treated enamel and
the consequent validation of the enamel remineralization. The same finding is
pointed out by the ATR spectrum of enamel treated for 10 minutes by synthetic
100 nm sized CHA nanocrystals, showing appreciable higher intensity of the
characteristic absorption bands of carbonate ions (1420–1460 and 1680 cm−1)
in respect of the same absorption bands present in the demineralized enamel ATR
spectrum. No differences are appreciable in the phosphate ions bands (1000–1100
and 530–580 cm−1). ATR spectrum (data not shown) of remineralized
enamel reveals that surface apatite is richer in carbonate than natural one,
such as synthetic 100 nm sized CHA nanocrystals.


## 3.3. In Vitro Enamel Surface Remineralization by Toothpaste Containing Either Fluoride or Biomimetic CHA Nanocrystals Microclusters

SEM analysis allowed us to
investigate the morphology of both demineralized enamel and the features
observed after a remineralization process which utilizes in vitro application
of toothpaste containing either fluoride or CHA microclusters constituted of 100 nm in size nanocrystals.The surfaces of the teeth treated
with fluoride (see Figure6(b)) were not consistently changed respect to that of
demineralization by ortophosphoric acid (see Figure 6(c)). Actually both the interprismatic
and prismatic enamel structures appear still evident. On the contrary after
treatment of the enamel slabs with a toothpaste containing synthesized CHA microclusters constituted of 100 nm sized nanocrystals, the interprismatic
and prismatic enamel structures appear to be completely hidden by a thick
homogeneous apatitic layer (see Figure 6(a)).SEM images of enamel after brushing treatment with (a) CHA toothpaste
containing, (b) fluoride containing toothpaste, and (c) ortophosphoric acid application.
(a)(b)(c)The XRD patterns collected on the
surface of enamel slabs after treatment with CHA or fluoride toothpaste and
water are reported in Figures7(b), 7(c), and 7(d), respectively, and compared with the XRD
pattern (see Figure 7(a)) of CHA microclusters constituted of 100 nm sized
nanocrystals utilized to prepare the used CHA toothpaste. The XRD diffraction
maxima recorded on the surface of enamel slabs treated with fluoride containing
toothpaste appear slightly more sharpened than those obtained on the enamel etched
slabs brushed only with water. This observation reveals an increased
crystallinity degree probably due to a partial structural conversion of
hydroxyapatite into fluoride substituted hydroxyapatite. On the contrary, the
XRD pattern obtained on the surface of enamel slabs brushed with CHA containing
toothpaste shows the broadened diffraction maxima characteristic of the
synthetic biomimetic CHA, revealing its presence on the enamel surface. The CHA
not removed by brushing procedures suggests the formation of chemical bonds
between the synthetic CHA microclusters constituted of 100 nm sized nanocrystals
and natural enamel apatite crystals. These bonds allow the formation of a
persistent CHA coating on the enamel surface whose morphology was detected by
SEM analysis.Figure 7
(a) XRD pattern of synthetic CHA, (b) enamel after brushing treatment with CHA containing toothpaste, (c) enamel after brushing treatment with fluoride containing toothpaste, (d) enamel after brushing treatment with water. *indicates Al holder diffraction maxima.The surface Ca/P molar ratio
determined by XPS analysis for demineralized enamel slabs before and after in
vitro remineralization by brushing whit toothpaste containing either fluoride
or CHA are compared in Table1. In this table, a comparison with the Ca/P molar
ratio of CHA microclusters constituted of 100 nm sized nanocrystals also
present. The enamel surface Ca/P molar ratio changes before and after the
brushing treatment with toothpaste containing fluoride, assuming a value of
3.4 very different from the apatite stoichiometric one (Ca/P  = 1.7). This finding
reveals that the only structural modification of enamel hydroxyapatite induced
by fluoride is restricted to a partial hydroxyl groups replacement by fluoride
ions without affecting appreciably the Ca and phosphate structural network. On
the contrary, enamel slabs treated with the toothpaste containing synthesised CHA
microclusters of 100 nm sized nanocrystals exhibit a surface Ca/P molar ratio
very similar to that one of the synthetic CHA.This coating is highly less
crystalline than native enamel apatite, and consists of a new apatitic mineral
deposition which progressively fills the scratches
and pits. On the contrary, the surface remineralization observed on the
specimens treated with fluoride containing toothpaste is mainly based on chemical-physical enamel apatite
surface modifications rather than a formation of a
new mineral deposition.The CHA biomimetic
coating formation is a remineralization process corresponding to a new apatite
deposition in the demineralized
area of enamel surface.


## 4. Discussion

Enamel
is the hardest and most mineralized tissue of human body. It is structured in
order to resist to mechanical injuries, abrasion, and chemical attack. Differently
from all the other mineralized tissues, it lacks proteins even if they are
essential to its formation. Actually, matrix proteins are cleaved by proteinases secreted by
the ameloblasts during tooth formation; hence, the matrix proteins of enamel
are not incorporated into enamel prisms [38]. Degradation and resumption of
enamel matrix proteins is the reason why enamel prisms, once formed, cannot be
remodelled. After enamel prism formation, only the amount of hydroxyapatite
within the prisms may decrease in consequence of chemical changes into the oral
environment.Acid
attack is one of the major causes of enamel hydroxyapatite loss. It may occur
even in young age as a consequence of plaque metabolism or simply due to food
and beverage intakes [2–4].In the
present paper, synthetic
carbonate-hydroxyapatite biomimetic nanocrystals have been
chemical-physical characterized
and investigated regarding the possibility to obtain an in vitro remineralization of the altered enamel surfaces.The
experimental model used in the present investigation was the demineralization
by means of ortophosphoric acid of sound enamel of the interproximal surfaces
and its subsequent remineralization.This
model was chosen because the demineralizing effect of ortophosphoric acid is
well known in terms of chemical and microhistological features because it is
an essential part of the procedures of adhesive restoration applications.The SEM
observations showed that the enamel is characterized by both amorphous and
prismatic hydroxyapatite and by an irregular surface even. The use of ortophosphoric
acid causes an exposure of prism and a loss of both interprismatic and
prismatic substances.The treatment of
demineralized enamel only for ten minutes, by synthetic CHA nanocrystals, induces
a consistent enamel remineralization through the formation of a surface
carbonate-hydroxyapatite coating. This coating is due to the
chemical bond of the synthetic CHA nanocrystals
biomimetic for composition, structure, size, and morphology on the surface prismatic hydroxyapatite enamel. It can be supposed that the
application of CHA nanocrystals 20 nm sized allows a better mineralization in
the lower surface fissure, because the interprismatic and prismatic enamel
structures appear totally hidden. Using an equal time of treatment, the
application of CHA nanocrystals 100 nm sized produces the formation of a
homogeneous coating which hides interprismatic and prismatic enamel structures
when examined with SEM.In this paper, an in vitro enamel surface remineralization
by brushing with toothpaste containing either fluoride or biomimetic CHA
nanocrystals microclusters has been also carried out. The XRD patterns, SEM
images, and XPS spectra collected on the surface of enamel slabs before and
after treatment with CHA or fluoride toothpaste underline the possibility to
obtain an enamel remineralization through the formation of a surface apatite
coating which covers the enamel structure avoiding the most probably health
dangerous fluoride effects, to contrast the mechanical abrasion and acid
attacks to which tooth enamel is exposed. The documented CHA biomimetic coating
formation which is less crystalline than enamel natural apatite represents an
enamel repair process corresponding to an apatite deposition inside the
demineralized area of enamel surface and may be considered an innovative
approach to contrast enamel demineralization.



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*Source: 746383-2009-04-05.xml*