International Journal of Nanomedicine

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O riginal R esearc hOpen Access Full Text Article

Different corrosive effects on hydroxyapatite
nanocrystals and amine fluoride-basedmouthwashes on dental titanium brackets:
a comparative in vitro study
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
16 January 2013
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Marco Lelli 1
Olivia Marchisio 2
Ismaela Foltran 1
Annamaria Genovesi 2
Giulia Montebugnoli 1
Massimo Marcaccio 1
Ugo Covani 2
Norberto Roveri 1
Alma Mater Studiorum, University of
Bologna, Department of Chemistry,
Bologna, Italy; 2University of Pisa,
Istituto Stomatologico Tirreno, Lidodi Camaiore, Lucca, Italy
1

Abstract: Titanium plates treated in vitro with a mouthwash containing amine fluoride(100 ppm F−) and another containing zinc-substituted carbonate–hydroxyapatite have been
analyzed by scanning electron microscopy and atomic force microscopy to evaluate the modification of the surface roughness induced by treatment with these two different ­mouthwashes.
The treatment with F−-based mouthwash produces a roughness characterized by higher
peaks and deeper valleys in the streaks on the titanium bracket surface compared with thoseobserved in the reference polished titanium plates. This effect causes a mechanical weakness
in the metallic dental implant causing bacterial growth and therefore promotes infection andprosthesis contamination. However, the in vitro treatment with a mouthwash containing zincsubstituted carbonate–hydroxyapatite reduced the surface roughness by filling the streaks
with an apatitic phase. This treatment counteracts the surface oxidative process that can affectthe mechanical behavior of the titanium dental implant, which inhibits the bacterial growthcontaminating prostheses.
Keywords: mouthwash, titanium brackets, corrosion, hydroxyapatite, aminic fluoride

Introduction

Correspondence: Norberto Roveri
Department of Chemistry “G Ciamician”,
Alma Mater Studiorum University of
Bologna,Via Selmi 2, I-40126 Bologna, Italy
Tel +39 05 1209 9486
Fax +39 05 1209 9593
Email norberto.roveri@unibo.it

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http://dx.doi.org/10.2147/IJN.S35245

The use of titanium in dental implant materials is based on the low thermal conductivityof this metal, besides the fact that it doesn’t release toxic ions and has an appropriate
elasticity module, which helps bone to heal when in close contact with the titanium
implant surface.1
Titanium is highly ductile and resistant to cyclic forces in the oral environment.
These cyclic forces, known as fatigue, are produced by masticatory movements, which
are capable of causing internal microcracks in titanium-based implants.2 Titanium
constantly undergoes mechanical and thermal stresses in an aggressive host oral
environment.
Mouthwashes and toothpastes containing fluoride ions are among the mostimportant products in the oral care market. In fact, fluoride has been considered
the main method to reduce the enamel dissolution and prevent caries. Fluoride ionshave the ability to interact with the hydroxyapatite crystals forming fluoridated­hydroxyapatite (Ca10(PO4)6(OH)F) or fluorapatite (Ca10(PO4)6F2). These minerals havegreater lattice energy, higher crystallinity, and better resistance to dissolution thanhydroxyapatite. Fluoride also increases the seeding rate of apatite crystals.1

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which permits unrestricted noncommercial use, provided the original work is properly cited.

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The in vivo treatment using fluoride toothpastes may leadto a partial substitution of the hydroxylic groups with fluoride
ions in the native enamel hydroxyapatite.3,4The bone-promoting activity of F− has been extensively
documented in the literature.16 Titanium fluoride forms a stablelayer on hydroxyapatite surfaces where titanium shares theoxygen atoms of phosphate, which becomes covalently boundto the hydroxyapatite surface. Fluoride is essential in this reaction because it allows phosphate ions to react directly withtitanium to form a firm and stable connection.1 The oxygen
and phosphate in the tissue fluids may replace the fluoride
and the phosphate becomes covalently bound to the titanium
surface. Such a reaction may induce a bone formation where
the phosphate in the bone is firmly (covalently) bound to thetitanium implant. This process may catalyze bone formationand facilitate deposition of particularly well-mineralized bone
tissue close to the implant surface.5,6
Oxidative processes can thicken and condense thetitanium oxide layer on the surface, which improves the
anticorrosion stability of the underlying titanium.7,8 On
the other hand, reductive agents, such as fluoride (F−), may
have the opposite effect and attack this layer.8 The corrosion
process is caused by electrochemical reactions. Titanium
is known to present high resistance to corrosion against
attack by solutions containing strong mineral acids, such ashydrochloric acid or sulfuric acid. However, when titanium
is placed in contact with a fluorinated medium at pH below
3.5, its titanium oxide layer is damaged and degraded. Thisphenomenon is macroscopically visible by the loss of surface
brightness.9,10 The fluorides may also cause corrosion, which
increases roughness on the surfaces exposed to the oral cavity,
making it easier for microorganisms to grow where they areprotected from oral hygiene mechanisms (brushing, swallowing, and flow of crevicular fluid).2,11,12
Anodic polarization and immersion tests indicated thatthe titanium corrosion in solutions containing F− depends onthe concentration of hydrofluoric acid (HF). The passivation
film on titanium was destroyed when the HF concentrationin the solution was .30 ppm.
A rough surface favors plaque accumulation13 in the periimplant crevices of the gingiva, which is an undesirable effectin this very sensitive region of the implant. Accordingly, toavoid pathogenic plaque accumulation,14 the neck (abutment)of an implant must be polished. It is very important to ensurethat these surfaces receive continuous care.
Many studies shown the high risk for fluoride to produce
fluorosis, especially in children, and bone diseases inthe elderly.15–21 The European Food Safety Authority (EFSA)

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scientific panel has advised that there is a risk of fluoride
intake from ingested oral care products and toothpastes thatcan be fluoridated with a maximum level of 1500 mg/Kg.
The EFSA considers that the maximum fluoride intake shouldbe 0.1 mg fluoride/kg/day in children aged 1–8 years whichis equivalent to 1.5 and 2.5 mg fluoride per day in children
aged 1–3 years and 4–8 years, respectively.22,23
Innovative commercial mouthwashes and toothpastes
in the last decade have replaced fluoride with biomimetic
hydroxyapatite nanocrystals as a remineralizing agent toavoid the dangerous effects of fluoride in human health,which have been ascribed to the amount of fluoride ingested
daily.16
In the inorganic phase of bone and dental hard tissues,hydroxyapatite [Ca10(PO4)6(OH)2] (HA) has been widely
studied as a bone filler, prosthetic coating, and implantablebiomaterial in dentistry and orthopedic surgery because of its
biocompatibility, bioactivity, and osteoconductivity.17
Biological HA is not stoichiometric according to theideal formula Ca10(PO4)6(OH)2, but at low quantities Ca2+ is
replaced by other ions such as Na+, K+, Zn2+, Mg2+, Sr2+, whilePO43− and OH− can be partially substituted by other anions
like CO32−, HPO42−, P2O74−, SiO44−.17,24−26
Carbonate is the prevalent foreign anion present into bonehydroxyapatite structure and represents about 4–8 wt%.27,28
The substitution of CO32− groups into the PO43− sites (type B
carbonate apatite) is prevalent in children, while the carbonate replacement to OH− groups (type A carbonate apatite)
increases with the age of the individual.29
Biomimetic carbonate–hydroxyapatite nanocrystals(CHA) have been synthesized nearly stoichiometrically inbulk Ca/P molar ratio of about 1.7 and contains 4 ± 1 wt%
of carbonate ions that replaced prevalently phosphate groups.
Biomimetic CHA nanocrystals have been synthesized from20 nm to 100 nm (Figure 1) with both acicular and plate-likemorphology to deliver drugs30–33 with a surface infused withamino acids and proteins.26,34–36
Biomimetic CHA (Figure 2) can aggregate in microsized
crystal clusters that have a nanostructured surface area of
about 80 m2/g and can be utilized as mineralized agents in
toothpastes and mouthwashes.37
Only recently, a chemical–physical experimental
approach has been followed to investigate in vitro37,38 andin vivo3,4 the capability of CHA nanostructured crystals to
produce in vivo biomimetic mineral deposition on enameland dentin surfaces through daily use.11
Demineralized enamel and dentine slabs have beentreated in vitro with synthetic biomimetic hydroxyapatite

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Corrosive effects on dental titanium brackets

The aim of this work is to evaluate the action of ananostructured CHA-based mouthwash on titanium implants,
in comparison with a F−-based mouthwash.
In this work, we have performed an in vitro analysis on
the effect of F− and CHA on the surface of titanium brackets,
using scanning electron microscopy (SEM) and atomic
force microscopy (AFM), which allows a direct quantitative
characterization of the surface roughness.

Materials and method
In vitro test
Figure 1 TEM image of biomimetic CHA nanocrystals.
Abbreviations: CHA, carbonate–hydroxyapatite; TEM, transmission electron
microscopy.

nanocrystals for a few minutes and the surfaces chemicallyand physically characterized. CHA induces surface remineralization, which forms a biomimetic apatite coating on
enamel and dentin surfaces. Enamel remineralization occurs
quickly due to the specific chemical–physical characteristics of innovative nanostructured hydroxyapatite particles,
which closely resemble the natural hydroxyapatite present
in enamel.39 Therefore, our experimental results suggest thatthe deterioration of teeth can be prevented.
CHA synthesized with tailored biomimetic characteristics for composition, structure, size, and morphology canchemically bind themselves on the surfaces of hard tissues
in teeth, filling in the scratches, and producing a boundbiomimetic apatitic coating to protect the enamel surface
structure.3,40,41
There are some commercial products for oral care, like
toothpastes and mouthwashes, based on CHA, which comeinto contact with titanium prostheses.

Figure 2 TEM image of aggregated biomimetic CHA nanocrystals.
Abbreviations: CHA, carbonate–hydroxyapatite; TEM, transmission electron
microscopy.

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We considered four types of titanium resulting from different processes: polished, blasted, machined, and acidetched. All samples were titanium grade plates 3 mm thickand 6 mm diameter. In vitro tests were carried out using
six samples for each type of titanium. Polished titanium
brackets were tested at room temperature in nanostructured
microcrystal CHA-based mouthwash for comparison toan amine fluoride F−-based mouthwash with a pH ranging
between 4.3 and 5.5. The stoichiometric composition of
biomimetic CHA, which represents the active component
in the used mouthwash, is Ca(10-x) Znx(PO4)(6-y)(CO3)y (OH)2
where x is 0.1 and y is 0.4.
Each plate sample was dipped in mouthwash for
60 ­seconds, twice a day for 30 days to simulate a proper oral
hygiene rinse. After each treatment, the titanium plates were
washed under running water to replicate in vivo the usual
teeth rinse procedure.

Morphological characterization
SEM observations were carried out by means of a CarlZeiss EVO, 40 XVP (Oberkochen, Germany) equippedwith an energy dispersive detector (EDAX) Inca 250(Oxford, UL), using secondary electrons at 25 KV and­different magnifications.
AFM was carried out directly on the titanium platesamples’ substrates. AFM imaging was performed on a
Digital Instruments Nanoscope IIIa Multimode SPM (Digital Instruments, Santa Barbara, CA, USA). The advanced
analog and digital circuit designs and innovative softwareand hardware provide superior multitasking control forscanning probe microscopes (SPMs).42,43 The samples incontact mode using a J scanner and silicon nitride tips
(200 lm long with nominal spring constant 0.06 N/m).

Results
The surface morphologies of different polished, blasted,machined, and acid-etched titanium plates have been charac-

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Lelli et al

Figure 3 SEM images of the different polished, blasted, machined, and acid-etched titanium disk surfaces used in preliminary selection: (A) polished titanium surface,(B) blasted titanium surface, (C) machine-treated titanium surface, and (D) etched titanium surface.
Abbreviation: SEM, scanning electron microscopy.

terized by SEM and the images are reported in Figure 3A–D
respectively.
Figure 3 shows that blasted, machine-treated, and acidetched titanium plates have an inhomogeneous surface, while
the polished plate surface (Figure 3A) has low grooves andthus a smooth surface (Figure 3C). Polished titanium plates
that have a homogeneous surface with a very small roughness
appear to be the more suitable titanium plates to be utilized
in this study.
Comparable samples of polished titanium plates have been
surface characterized by SEM before and after a daily in vitro
treatment with two different mouthwashes. A mouthwash containing an amine fluoride (100 ppm F−) and another containing
zinc-substituted CHA have been utilized to investigate theirdifferent effects on the titanium plate surface.
After the in vitro treatment with the two different
mouthwashes, SEM analysis of the titanium plates showed
an appreciable difference between the plates treated withamine F−-based mouthwash (Figure 4A) and those treated310

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with the CHA-based mouthwash (Figure 4C). In fact, thesurface roughness present on titanium surface increases
after the treatment with amine F −-based mouthwash
compared to that observed on the reference titanium plate(Figure 3A).
Contrarily, the CHA-based mouthwash seems to covervalleys and grooves present on the titanium surface, which
appreciably reduces the roughness present on the reference
titanium plates.
The EDAX elemental analysis performed on the surfaceof the titanium after treatment with a fluoride rinse showed
only one peak related to titanium (Figure 4B). If the EDAX
was performed after treatment with CHA-based mouthwash,the same survey grade showed peaks attributable to the calcium and phosphate ions present in the right stoichiometricmolar ratio of hydroxyapatite, 1.7 (Figure 4D).
For more detailed results, the titanium brackets treatedwith the two different mouthwashes were investigated by
AFM microscopy.

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Corrosive effects on dental titanium brackets

A

B
Ti

Ti

2

2.5

3.5

3

4

4.5

5

40 µm

D

C

Ti

Ca

P
2

2.5

3

Ti

Ca

3.5

4

4.5

5

Figure 4 SEM images of titanium surfaces after mouthwash treatments: (A) titanium surface treated F−-based mouthwash, (B) elementary analysis with EDAX probe ontitanium surface treated with F--based mouthwash, (C) titanium surface treated with CHA-based mouthwash; (D) elementary analysis with EDAX probe on titanium surface
treated with CHA-based mouthwash.
Abbreviations: CHA, carbonate–hydroxyapatite; EDAX, energy dispersive detector; F−, fluoride; SEM, scanning electron microscopy.

A

10 µm

400.0 nmA

8

0.0 nm

100 µm
1.2 µm75

0.0 µm
6

50

10 µm

100 µm

4
8

2
6

50

10 µm

4

100 µm
75

8

6

2

25

75

25

50

4

25

2

B

400.0 nmB

0.0 nm

1.2 µm10

6100 µm
100 µm

8

4

75

6

2

4

50

25
50

2

2

C

0.0 µm

10 µm
10 µm

8

4

25
75

6

8

100 µm

10 µm

C 1.2 µm
400.0 nm

100 µm

0.0 µm10 µm0.0 nm

75

8
10 µm

100 µm
6

8

50
75

4
6

2

25

50

4
2
2

4

6

8

10 µm

Figure 5 AFM analysis on titanium disks with 10 × 10 µm section: (A) reference
titanium plate, (B) titanium plate treated with F−-based mouthwash, (C) titanium
plate treated with CHA-based mouthwash.
Abbreviations: AFM, atomic force microscopy; CHA, carbonate–hydroxyapatite;F−, fluoride.

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25
25

50

75

100 µmFigure 6 AFM analysis on titanium disks with 100 × 100 µm section: (A) reference
titanium plate, (B) titanium plate treated by F−-based mouthwash, (C) titanium plate
treated by CHA-based mouthwash.
Abbreviations: AFM, atomic force microscopy; CHA, carbonate–hydroxyapatite;F−, fluoride.

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Titanium plates treated with the two different mouthwashes
were investigated using titanium sections of different
dimensions. We have examined titanium plate sections of
10 × 10 µm and 100 × 100 µm wide. Figures 5 and 6 show
AFM images obtained on the surface of 10 × 10 µm and100 × 100 µm sections, respectively. In fact, in the 10 × 10 µm
samples, AFM measures the surface ­nanoroughness withhigh accuracy, while in the 100 × 100 µm samples, it ispossible to evaluate the roughness extension with highaccuracy.
In Figures 5A and 6A, the AFM images of the titanium
reference surface are reported, in Figures 5B and 6B, theAFM images of F− mouthwash-treated titanium surface
are reported, while in Figures 5C and 6C, the AFM images
show the effect of CHA mouthwash on the titanium surface.
The numerical analysis carried out on the AFM images isreported in Tables 1 and 2. The root mean square (Rq),highest peak (Rp), and deepest valley (Rv) were measured
on 10 × 10 µm sections of reference titanium surface
and after treatment with mouthwashes containing F− andCHA (Table 1). The Rq, Rp, and Rv were measured on
100 × 100 µm sections of reference titanium surface andafter treatment with the mouthwashes containing F− andCHA (Table 2).

Discussion
A morphological characterization was performed on thesurface of polished, titanium brackets after in vitro treatment
with mouthwashes containing amine F− and CHA. Titanium
plates treated with the two different mouthwashes wereanalyzed by SEM and AFM using titanium sections of two
different dimensions (10 × 10 μm and 100 × 100 μm) for awell-defined highly accurate evaluation of the modificationof surface nanoroughness and its extension (respect the value
Rq, Rp and Rv reported in table 2) induced by the treatment
with the two different mouthwashes.
Table 1 AFM analysis of different titanium disks, 10 × 10 μm
section
10 × 10 μm sections

Root meansquare (Rq)

Highestpeak (Rp)

Deepestvalley (Rv)

Polished titanium disk
(reference)
F--based mouthwash
treated disk
CHA-based mouthwash
treated disk

93 nm

350 nm-140 nm

92 nm

460 nm-230 nm

56 nm

300 nm-120 nm

Abbreviations: AFM, atomic force microscopy; CHA, carbonate–hydroxyapatite;F−, fluoride.

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Table 2 AFM analysis of different titanium disks, 100 × 100 μm
section
100 × 100 μm

Root meansquare (Rq)

Highestpeak (Rp)

Deepestvalley (Rv)

Polished titanium disk
(reference)
F--based mouthwash
treated disk
CHA-based mouthwash
treated disk

280 nm

700 nm-740 nm

460 nm

840 nm-940 nm

235 nm

620 nm-520 nm

Abbreviations: AFM, atomic force microscopy; CHA, carbonate–hydroxyapatite;F−, fluoride.

The treatment with the F−-based mouthwash induces aroughness characterized by streaking with higher peaks anddeeper valleys on the titanium bracket surface comparedwith those observed in the reference polished titanium plates.
Contrarily, the treatment with the CHA-based mouthwash
induces a roughness characterized by streaking withsmoothed peaks and less deep valleys on the titanium bracket
surface compared with those observed in the reference
polished titanium disc. These findings can be explainedby the hydroxyapatite deposition filling the streaks on thetitanium surface and therefore reducing surface roughness.
The EDAX analysis revealed the presence of calcium andphosphorus in a molar ratio of 1.7, which was characteristicof the CHA present in the mouthwash.
The increased roughness induced by the in vitro
treatment with the mouthwash containing amine F −(Tables 1 and 2) modify the mechanical behavior of thetitanium brackets, which promotes a mechanical weaknessof the metallic dental implant. The increased depth of thestreaks encourage bacterial growth promoting infection
and prosthesis contamination and mobility. 44 Contrarily,
the in vitro treatment with the CHA-based mouthwash
reduces the surface roughness by filling in the streaks(Tables 1 and 2). This hydroxyapatite deposition protects
against the surface oxidative process, which can damage
the mechanical behavior of the titanium brackets. The
depositation of hydroxyapatite coating on titanium surface
is able to prevent the bacterial growth contaminating theprosthesis. Furthermore, when bacterial plaque, by its
acidity, solubilizes the zinc-substituted CHA present on
the titanium implant.40,41,45 On one hand, our data showsthat the presence of F− in mouthwashes should be avoided
to prevent any degradation of the metallic dental implantand subsequent contamination with bacteria. On the other
hand, the CHA-based mouthwash prevents the degradationof the metallic dental implants and the formation of bacterial
film.

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Conclusion
The morphological characterization of the surface of polishedtitanium brackets after in vitro treatments with a mouthwash
containing an amine F− and another containing CHA showedthat the presence of F− in a mouthwash induces mechanical weakness in the metallic dental implant and bacterial
contamination. The presence of zinc-substituted CHA in amouthwash prevents the deterioration of the metallic dental
implant and contamination with bacteria.

Acknowledgment
We thank the RBAP114AMK, RINAME Project, “Reteintegrata per la nano medicina” (funds for selected research
topics), the Chemical Center Srl, and the Interuniversity
Consortium for Research on Chemistry of Metals in
Biological Systems (CIRCMSB) for the financial support.

Disclosure
The authors report no conflicts of interest in this work andno payment has been received for the preparation of thismanuscript.

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