|Título/s:||Manufacture of nanosized apatite coatings on titanium with different surface treatments using a supersaturated calcification solution|
|Autor/es:||Paz Ramos, Adrian; Ybarra, Gabriel O.; Pazos, Leonardo M.; Parodi, María B.; Rodríguez, Laura; López Hernández, Mónica; González Ruíz, Jesús E.|
|Palabras clave:||Recubrimientos superficiales; Titanio; Procesos superficiales; Calcio; Biomateriales; Cerámica; Materiales cerámicos; Fosfato de calcio; Nanopartículas|
| Ver+/- |
Quim. Nova, Vol. 39, No. 10, 1159-1164, 2016
MANUFACTURE OF NANOSIZED APATITE COATINGS ON TITANIUM WITH DIFFERENT SURFACE
TREATMENTS USING A SUPERSATURATED CALCIFICATION SOLUTION
Adrian Paz Ramosa, Gabriel O. Ybarrab, Leonardo M. Pazosb, María B. Parodib, Laura Rodríguezb, Mónica López
Hernándezc and Jesús E. González Ruízd,*
aDepartment of Chemistry, Université de Montréal, Succursale Centre-Ville, Montréal – Québec, Canada
bNational Institute of Industrial Technology, General Paz Avenue 5445, San Martin – Buenos Aires, Argentina
cNational Center for Scientific Research, 25 Avenue and 158st, 11300 – Havana, Cuba.
dDepartment of Ceramic and Metallic Materials, Biomaterials Center, Universidad Avenue and G, 10400 – Havana, Cuba.
Recebido em 16/02/2016, aceito em 24/06/2016, publicado na web em 30/08/2016
The biomimetic method is used for the deposition of calcium phosphate coatings (Ca – P) on the surface of different biomaterials.
However, the application of this method requires long exposure times in order to obtain a suitable layer thickness for its use in
medical devices. In this paper, we present a fast approach to obtain apatite coatings on titanium, using a combination of supersaturated
calcification solution (SCS) with chemical modification of the titanium surface. Also, it was evaluated the effect of four different
surface treatments on the apatite deposition rate. Commercially pure titanium plates were activated by chemical or thermochemical
treatments. Then, the activated samples were immersed in a solution with high content of calcium and phosphate ions at 37 °C for
24 h, mimicking the physiological conditions. The coatings were studied by Fourier transform infrared spectroscopy (FTIR), X-ray
diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). The use of SCS solutions
allowed the formation of crystalline hydroxyapatite coatings within a period of 24 h with a thickness between 1 and 5.3 µm. Besides,
precipitates of hydroxyapatite nanoparticles with a globular configuration, forming aggregates with submicrometer size, were found
in SCS solutions.
Keywords: Coatings, thin films, biomaterials, ceramics.
Titanium and its alloys are among the most widely used metallic
materials in the manufacture of implants due to its excellent mecha-
nical properties, low specific weight, high corrosion resistance and
excellent biocompatibility.1-6 However, its osseointegration is slow,6
affecting the life quality of patients. Several works have shown that
changes in the topography, surface energy and phase composition on
the titanium surface can significantly influence its rate of osseoin-
tegration.7-11 The biomimetic method has been widely used in the
past decades because it is possible to obtain coatings with a phase
composition analogous to the inorganic component of the bone.12 In
addition, this technique does not require special facilities and high
temperatures for its implementation, it enables the treatment of porous
surfaces and also the possibility to incorporate bioactive agents and
proteins.13-15 Unfortunately, the length of the processes has limited
the extension of this method to the treatment of dental implants and
prostheses made of titanium and its alloys.16
The behavior of calcium phosphate coatings in vivo depends
largely on its crystallinity, elemental and phase composition.17,18
The biomimetic method allows to modify those parameters from
the ionic composition of the solution, the ion content of the pre-
cursor employed and their relationship. Several works described
the formation of apatite coatings, which are completely or partially
constituted by octacalcium phosphate (OCP), dicalcium phosphate
(DCP), hydroxyapatite (HA), hydroxycarbonate apatite (HCA) or
carbonated apatite (CA).19-24 Also, calcium phosphate coatings with
different degree of crystallinity were reported.25
Among the alternatives to shorten the exposure time are the
use of solutions with high content of calcium and phosphate ions
and the incorporation of magnesium salts into these solutions.19,26-28
Furthermore, it has been shown that the surface roughness, chemical
composition and phases present in the surfaces can enhance the rate
of apatite coating formation. Acid etching and their combination
with heat treatments (thermochemical treatments) are used for the
surface modification of titanium, because they modify its elemental
and phase composition, but also the topography and wettability.29-31
In addition, this treatment improves the in vitro and in vivo behavior
of titanium and its alloys.9,29,32 Within the chemical procedures used
to activate the surface of titanium and its alloys are treatments that
enhance the formation of oxides or hydroxides,30,31,33 and others which
enable the formation of titanates (treatments in sodium hydroxide or
The aim of this study is to determine the effect of four treatments
of surface activation applied to the commercially pure titanium on
the deposition of apatite coatings in a supersaturated calcification
Twenty plates of commercially pure titanium (Ti – cp) of 15 ×
5 × 1 mm in size were polished using silicon carbide (SiC) paper of
120, 240, 400 and 600 grit. After polishing, the samples were cleaned
in an ultrasonic bath with distilled water and then activated by four
chemical and (or) thermochemical treatments (Table 1).
Ca-P coating formation
The reagent grade chemicals CaCl2, NaH2PO4 and NaHCO3
were successively dissolved in 500 mL distilled water to prepare a
Ramos et al.1160 Quim. Nova
supersaturated calcification solution (SCS). The masses of salts and
ion concentrations of SCS were equal to those employed by Li et al.
(Table 2).21 After the activation treatment, the titanium plates were
tied with a cotton thread and immersed vertically into 500 mL of
SCS solution at 37 ºC for 24 h, with a stirring rate of 80 rev. min-1. A
Ti plate representing each surface treatment was immersed into the
solution in each experimental run. After exposure, the samples were
carefully washed with distilled water and dried at 60 ºC for 2 h. At
the end of the deposition process, the solution was centrifuged and
filtered. Finally, the precipitates were collected and dried at 60 oC
for 2 h. All experiments have been repeated at least 3 times in order
to ensure the data reproducibility.
The pH value was measured during the solutions preparation
and at the coating process with a Pracitronic MV870 pHmeter. The
functional groups of the coatings were determined by Fourier transfor-
med infrared (FTIR) spectroscopy on a Nicolet Magna 550 Series II.
Besides, the coatings were investigated using a Philips PW1730 X-ray
diffractometer (XRD) with a CuKα target. The phase identification
was made using the PCPDFWIN database, v. 2.4 ICDD PDF-2/2003.
The size and morphology of the particles obtained in solution
were characterized by transmission electron microscopy (TEM), in
a Philips CM10 microscope, using an acceleration voltage of 80 kV.
The dimensions of the particles were determined by digital image
analysis using the software ImageJ.
The morphology of the coatings was characterized by scanning
electron microscopy (SEM) using a Philips SEM 505 microscope
for low magnification and a Zeiss microscope FESEM Supra 40 for
increases greater than 5000x. In addition, it was identified their ele-
mental composition using an energy dispersive X-ray spectroscopy
(EDX) coupled to SEM. The statistical processing of results was
performed in Statgraphics software, using a multi-sample compari-
son test (Multiple Range Tests) and analysis of variance (ANOVA).
RESULTS AND DISCUSSION
Figure 1 shows the behavior of the average pH value of the
solution, which was characterized by four different stages. In the
first stage (I in Figure 1), it was observed a significant increase in
pH values up to a maximum at around one hour of exposure (6.44 ±
0.06). Subsequently, it was observed a rapid decrease in pH values of
5.8 ± 0.06, between 1.5 and 3 h (stage II, Figure 1). Then, between
3 and 5.5 h, the values decreased slowly (stage III) and finally the
reduction was even slower to reach a value of 5.58 ± 0.09 at 24 h
(stage IV). It is important to notice that during stage II, it was observed
the formation of precipitates, consisting of tiny white particles on the
surface of the samples and in the solutions.
The study by TEM of the particles formed in solution showed
a globular configuration and an average diameter of 29 ± 5 nm
(Figure 2). In addition, the nanoparticles formed a cluster with irre-
gular shape and size generally less than 1 µm (Figure 2).
FTIR results of the precipitates showed the main groups of
hydroxyapatite (Figure 3). At 1040 and 1091 cm-1 was observed the
most important band of hydroxyapatite, the antisymmetric vibration
of calcium phosphates υ3as (PO43-). At 952 cm-1 was revealed the
symmetrical vibration, while at 561 and 606 cm-1 was observed the
antisymmetric vibration υ4as (PO43-). Also, at 3400 cm-1 was observed
a broad band of water, produced by the absorption of humidity by the
samples during the deposition process. In addition, it was observed
a band at 635 cm-1, corresponding to the out-of-plane bending (ρ)
of the structural OH- group and a signal located at 1643 cm-1, from
water adsorption (Figure 3).
The morphology of the activated titanium surface using the four
variants is presented in Figure 4 A, E, I and M. As a result of acid
treatment, it was obtained a surface with several micropores, with size
between 3 and 15 µm. Subsequent treatments in hydrogen peroxide
and (or) thermochemical did not significantly affect the topography
of the surfaces (Figure 4 E, I). On the samples with alkali treatment
(AEAT) were observed not only the pores generated by acid etching
(Figure 4 M), but also additional nanopores evenly distributed across
the surface (not shown).
The topography of titanium samples after treatment in SCS
showed significant variations (Figure 4 B, F, J and N). The pores
and ridges formed during the activation treatments disappeared in
all cases. Higher-magnification micrographs of the samples soaked
in SCS showed coatings with dense and microporous zones (Figure
4 C, G, O, D, H and P). An exception of this behavior was observed
in the samples under thermochemical treatment (AEPTT), in which a
majority of dense coating was formed (Figure 4 K and L). This last co-
ating was constituted by nanoparticles with a globular configuration,
Table 1. Treatments used for the surface activation
Treatment Treatment details
Acid etching (AE) Acid etching in HF 2.75 M - HNO3 3.94 M, v/v 1:1
at room temperature for 2 min.
Oxidation 1 (AEP) Acid etching + treatment in H2O2 8.8 M - HCl 0.1 M
v/v 1:1 at 80 oC for 30 min.
Oxidation 2 (AEPTT) Acid etching + oxidation 1 + thermochemical treat-
ment at 400 oC for 1 h in air.
Oxidation 3 (AEAT) Acid etching + treatment in 10 M NaOH at 80 °C
for 24 h.
Table 2. Masses of salts and ion concentration in 500 mL of supersaturated
calcification solution (SCS)
Masses of salts (mg) in 500 mL of SCS
CaCl2 NaH2PO4 NaHCO3
555 150 63
Ion concentration (mM)
Na+ Ca2+ Cl- H2PO4- HCO3-
4 10 20 2.5 1.5
Figure 1. pH of the solution versus exposition time
Manufacture of nanosized apatite coatings on titanium with different surface treatments 1161Vol. 39, No. 10
with a size between 11 and 82 nm and an average diameter of 34 ± 15
nm. In that area, it was observed that the nanoparticles formed some
clusters with a maximum length of 245 ± 126 nm. Furthermore, it
could be found some plate-like shape particles on the coating with a
perpendicular orientation to the surface and a length of 170 ± 84 nm.
The samples activated by AE, AEP and AEAT variants exhibited
a combination of dense and microporous zones (Figure 4 C, G, O, D,
H and P). Those microporous zones were constituted by several plate-
-like shape particles, oriented perpendicularly to the surfaces. The
length of the particles changed depending on the activation treatment
(AE = 133 ± 54 nm, AEP = 358 ± 149 nm and AEAT = 378 ± 197
nm), while the maximum size of the pores was in the submicron scale.
The elemental composition analysis by EDX showed that the
coatings are constituted of calcium, phosphorus, oxygen, and also
revealed the titanium from the substrate (Figure 5). Additionally, in the
samples activated by AEAT variant was observed a greater intensity
in the characteristic peaks of calcium and phosphorus compared with
the other samples (Figure 5). Besides, the spectra do not reveal the
presence of other elements.
The FTIR spectra of the coatings showed the signals correspon-
ding to hydroxyapatite (Figure 6). The comparison between the FTIR
spectra of coating and precipitates shows that there is no difference
on the presence and location of the signals, which indicates that the
compositions of both materials are presumably the same.
The XRD patterns corroborate the formation of the apatite
coatings. The reflection peaks with interplanar distances of 3.46,
2.79, 2.67 and 1.84 Å (2θ = 25.74, 32.11, 33.77 and 49.39o) were
assigned to crystalline apatite (Ca10(PO4)6(OH)2) (Figure 7). Also, at
the interplanar distances of 2.57, 2.36 and 2.25 Å (2θ = 34.97, 38.20
and 40.03o) the titanium substrate was revealed.
Cross-section micrographs of the deposits corroborate that all
samples were uniformly coated and that the layer thicknesses were
between 1 and 5.3 µm (Figure 8). The greater coating thicknesses were
obtained using both the AEPTT and AEAT treatments. In addition,
it was observed that the greatest thicknesses are generally found in
the porous areas, generated during the acid treatment (Figure 8).
The pH curves of the SCS solution have four well differentiated
stages (Figure 1). The pH increase in the first stage is related to the
reaction of H+ ions with HCO3- (generated by the dissociation of
NaHCO3), according to equation 1.21 During this period, it must
be generated some apatite nuclei, which enhances the film growth
according to the mechanism described by Barrere et al.19 In the limit
of stages 1 and 2, after reaching the maximum pH value, there is a
sharp inflection in the curve (Figure 1). This phenomenon is caused
by the combination of high ionic strength with a high pH value, which
promotes the supersaturation of the solution. This causes a strong
reaction between Ca2+ ions and OH- (equation 2) and the apatite
precipitation in the solution and on the activated titanium surface. A
similar behavior is reported when using solutions with an ionic con-
centration five times superior to the simulated body fluid (SBF x 5).19,25
In addition, the rapid consumption of OH- during stage 2 causes the
rapid drop of pH values (Figure 1). At this stage Habibovic reported
a significant increase in thickness of the calcium phosphate layer.20
H+ + HCO3- → CO2 + H2O (1)
10 Ca2+ + 6 PO43- + 2 OH- → Ca10(PO4)6(OH)2 (2)
The inflection of the pH curve may be explained by the precipita-
tion of HA, according to equation (2). Due to the decreased amount
of OH- ions in the solution, a pH drop was observed. The tendency
observed in stages 3 and 4 can be related to the film growth. In those
stages the pH values decrease becomes slower until the end of the
process. This behavior should be linked to the slow consumption of
Ca2+ and PO43- ions in the solution as a result of the coating process and
the formation of particles in the solution during the previous stages.
The approach employed allows the formation of hydroxyapatite
nanoparticles in the solution and on the titanium surface activated
Figure 2. TEM image and particle size distribution of the precipitates formed
in SCS solution
Figure 3. FTIR spectrum of the precipitates formed in SCS solution
Ramos et al.1162 Quim. Nova
Figure 4. SEM images of the surface modification on titanium (A, E, I, M) and apatite layers obtained on titanium (B, C, D, F, G, H, J, K, L, N, O, P) after
soaking in SCS for 24 h
by chemical and thermochemical methods. The morphology of the
nanoparticles obtained in the solution (globular) differs from the shape
of short rods obtained by Han et al.37 The combination of nanoparticle
size (34 ± 15 nm) with the globular shape can result in a high specific
surface area, an issue that promotes their bioreactivity.
The morphology and dimensions of the nanoparticles obtained
in solution and on the coatings formed on the surface activated
by AEPTT variant were similar. However, the high-magnification
micrographs of dense areas (AE, AEP and AEAT) do not clearly
reveal the formation of nanoparticles in the surface (Figure 4 C, D,
G, H, O and P). The formation of nanoparticles with plate-like shape
agrees with the results of different works using supersaturated cal-
cification solution (SCS) and some variants of SBF.38 The presence
of these two morphologies in HA nanoparticles can be explained by
the intervention of two nucleation mechanisms in their formation.
Particles showing a globular shape are generated by a heterogeneous
Manufacture of nanosized apatite coatings on titanium with different surface treatments 1163Vol. 39, No. 10
nucleation rate while those with a plate-like shape are formed by a
homogeneous nucleation. Both processes probably occurred at the
end of precipitation (end of stage 2, Figure 1) as a result of low ionic
concentration in the solution.
Micrographs of apatite coatings deposited on the four surfaces
studied showed no cracks (Figure 4 A, E, I and M), a common defect
reported in coatings formed in solutions with high concentrations of
calcium and phosphate ions.19,26,27 The absence of cracks in the coat-
ing should have a positive impact on their mechanical performance.
FTIR analysis reveals that the coatings obtained on titanium and
the nanoparticles formed in solution must be constituted by apatite
crystals, a result confirmed by XRD. In addition, EDX tests confirm
that the coatings are formed by calcium, phosphorus and oxygen. The
presence of the maximum intensity peak at 2 = 25.74 o is attributed
to a preferential orientation in the c-axis (diffraction plane 002) of
Figure 5. EDX spectra of the surface of the apatite layers obtained on titanium
soaked in SCS for24 h
Figure 6. FTIR spectra of apatite layers obtained on titanium soaked in
SCS for 24 h
Figure 7. XRD patterns of titanium substrate and apatite coating formed on
titanium (AEPTT) after soaking in SCS for 24 h
Figure 8. Cross-section micrographs (SEM) of the coatings obtained on
titanium soaked in SCS for 24 h
Ramos et al.1164 Quim. Nova
the crystals and it was previously reported by Li and Muller.21,39 The
sharp peaks observed in the XRD pattern confirm that the coatings
are formed of HA with a crystalline structure. This result must be
related to the low content of NaHCO3 used in the solution.
Obtaining nanostructured crystalline hydroxyapatite should
have positive influence on the stability and mechanical properties of
the developed coating in vivo. It is known that the cellular response
depends on the chemical composition of the substrate, but also on
their solubility, crystallinity and microstructure. Previous studies17,40
have shown an increase in cell amount and mineralized extracellular
matrix on crystalline substrates, compared with amorphous Ca-P
coatings. This fact was related with the cell-surface interaction. In
contrast with crystalline Ca-P coatings, amorphous coatings should
be constantly dissolving and re-precipitating, which can decrease
the number of cells attached on the surface, and ultimately decrease
the bone formation.17 The low dissolution rate of crystalline Ca-P
coatings might also reduce the risk of implant loosening. Finally,
the nanotopography of the obtained coatings should promote the cell
response and enhance the osteogenic activity,41 which could positively
contribute to the implant success.
Nanostructured apatite coatings were obtained on titanium plates,
activated by four different treatments. The coatings completely cover
the treated surfaces in 24 hours. In addition, they were constituted of
crystalline hydroxyapatite, showing a combination of dense and high
microporosity areas. All layer thicknesses were found between 1 and
5.3 µm, while the greater layer thickness was obtained with the acid
etching and subsequent alkali treatment (AEAT). Furthermore, it was
observed the formation of hydroxyapatite nanoparticles with a glob-
ular configuration in the solution. The application of the biomimetic
method under the above-mentioned conditions allows the formation
of a suitable layer of bone-like apatite in a very short time. This fact
should extend the use of this method in the coating of implantable
devices, with the aim to increase their bioactivity.
The authors wish to express their thanks to the Chancellery and
the Ministry of Science and Technology of the Republic of Argentina
for his contribution to this work through projects FOAR 5714 and
CU/08/13. They also thank the National Institute of Industrial
Technology (Argentina) and the Center of Biomaterials of the
Polytechnic University of Valencia (Spain) for their assistance in
conducting the tests.
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