javadi2019

Materials Science & Engineering C 99 (2019) 620–630
Contents lists available at ScienceDirect
Materials Science & Engineering C
journal homepage: www.elsevier.com/locate/msec
Surface engineering of titanium-based implants using electrospraying and
dip coating methods
T
Akbar Javadia, Atefeh Solouka, , Masoumeh Haghbin Nazarpakb, , Fatemeh Bagheric
⁎
⁎
a
Department of Biomedical Engineering, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran
New Technologies Research Center (NTRC), Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran
c
Department of Biotechnology, Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, Iran
b
ARTICLE INFO
ABSTRACT
Keywords:
Titanium implants
Surface modification
Electrospraying
Dip coating
Titanium and its alloys due to their low density, good mechanical and biological properties are of the most
common orthopedic metals. One of the main challenges regarding to titanium implants is their loosening after
long term implantation in patient's body. Many methods such as alteration in surface topography with focus on
improving osseointegration or biocompatibility in overall are supposed to overcome this issue. In this research,
titanium surface topography is altered via electrospraying a solution of titanium salt, carrier polymer (polyvinylpyrrolidone) and solvents. The dip coated samples in the same solution are prepared and investigated as
control. The electrosprayed or dip coated samples were pyrolysised in furnace at 500 °C to remove polymeric
components. Then the stabilized microstructures on the surfaces were evaluated via scanning electron microscopy (SEM), water contact angle (WCA) measurement, X-ray diffraction (XRD) and atomic force microscope
(AFM). Also, in order to study the bioactivity of modified samples, they were immersed in simulated body fluid
(SBF) and their precipitates were studied. The cellular investigations were done by studying the cell morphology,
MTT and alkaline phosphatase (ALP) activity assays. The results showed improvement in bioactivity and cellular
response for DP3 and SP15 more than other samples implying the promising potential of these two approaches
for titanium implant surface modification.
1. Introduction
[6,7]. Chouirfa et al. studied the effect of different solutions to decrease
bacterial infections on the Titanium surface according two main parts:
surface modification and coatings (chemical or physical) [8]. Janson
et al. also provided antibacterial activity by soaking the surfaces of
Titanium samples in hydrogen peroxide [9]. Loosening is a consequence of implant poor integration with the surrounding bone tissue
remains as one of the major obstacles for permanent orthopedic implants [5]. Also, nanoscale surface topography and biomaterial roughness are considered as crucial as surface chemical composition for tissue
acceptance and cell viability [10].
Hamlekhan et al. have reported that surface modification and presence of nanoscale features on the surface of implants can enhance the
growth of osteoblast bone-forming cells. The rationale behind this is the
similarity of nanometer features with the osteoblast biological environment [11]. Different surface modification procedures are studied
to improve the performance of medical implants. Some of them are
laser surface modification [12], thermal oxidation [13], sand blasting
[14], sol-gel coating [15], physical vapor deposition (PVD) procedures
[16], chemical vapor deposition (CVD) [17], plasma spray [18] and the
Titanium (Ti) and its alloys are widely used in orthopedic implant
materials because of their appropriate properties, such as relatively low
modulus, good fatigue strength, formability, machinability, corrosion
resistance, and biocompatibility. Ti and its alloys like other biomaterials must meet all the essential criteria, especially the physical, chemical, and mechanical properties, to have a desirable function in the
human body. Designing biomaterials is often challenging to fulfill all
functional requirements, therefore fabricating a material with adequate
and acceptable bulk properties along with additional steps to modify
surface is a common approach [1,2]. So, various surface modification
processes in micro and nanoscale are studied and investigated [3,4].
Biomaterial surface plays an important role in the body response. In
the case of titanium based implants, the normal manufacturing steps
usually create a non-uniform and rather poorly defined oxidized and
contaminated surface layer. In order to take these “native” surfaces for
biomedical applications surface modification seems essential [5]. Infection and loosening are two major challenges of orthopedic implants
⁎
Corresponding authors at: Amirkabir University of Technology (Tehran Polytechnic), Iran.
E-mail addresses: [email protected] (A. Solouk), [email protected] (M. Haghbin Nazarpak).
https://doi.org/10.1016/j.msec.2019.01.027
Received 13 August 2018; Received in revised form 23 December 2018; Accepted 7 January 2019
Available online 25 January 2019
0928-4931/ © 2019 Published by Elsevier B.V.
Materials Science & Engineering C 99 (2019) 620–630
A. Javadi et al.
polymer (polyvinylpyrrolidone), appropriate solvents, and secondly dip
coating using the same solution. Then, the physical, chemical and
biological properties of these altered surfaces are investigated and
compared with unmodified Ti surface.
Table 1
Electrospraying optimum parameters.
Feeding rate
Needle to substrate distance (d)
Applied voltage
100 mm
150 mm
10 kV
15 kV
0.5 mL/h
0.5 mL/h
2. Materials and methods
2.1. Materials and reagents
Table 2
Dip coating optimum parameters.
Solution volume to surface area ratio (mL/mm2)
3/100
3/100
Polyvinylpyrrolidon (PVP, Mw = 1,300,000 g/mol) as the carrying
polymer, Ti(IV) n-butoxide (TBut) as the alkoxide precursor, and all the
solvents such as acetic acid (AcA), N,N dimethylformamide (DMF,
≥99) and Isopropanol were purchased from Merck company. A mixture
of N,N dimethylformamide and Isopropanol was used in 1:1 mass ratio.
All reagents were used without further purification. Commercially pure
titanium with a purity of 99.9% was used as substrate.
Simulated body fluid (SBF) for evaluating the bioactivity of specimens, was provided with mixing NaCl, NaHCO3, KCl, K2HPO4.3H2O,
MgCl2.6H2O, HCl (1 M), CaCl2, Na2SO4, (CH2OH)3CNH2 (Tris), all obtained from Merck company.
All Titanium samples (0.1 mm thick plates, 99.7% purity [ATI CPTi]) were cleaned with 15 min ultra-sonication in distilled water then
washed three times with distilled water and air dried [23].
Dip coating time (h)
2
3
Table 3
Coding the specimens.
Specimens
Description
CTRL
SP10
SP15
DP2
DP3
Control specimen without any surface modification
Electrosprayed (V = 10 kV, d = 100 mm, f = 0.5 mL/h)
Electrosprayed (V = 15 kV, d = 150 mm, f = 0.5 mL/h)
Dip coating specimen (2 h)
Dip coating specimen (3 h)
2.2. Preparation of solution
procedures that result in titanium dioxide structures on titanium surface [19]. Two of these surface modifications created titanium dioxide
nanotubes on the surface via anodizing method [20] and titanium dioxide nanofibers on the surface via electrospinning [21,22]. One simple
and cost effective approach to form microfibers/microparticles on the
Titanium surface is using Titanium salts and electrospinning/electrospraying techniques.
Ti(IV) n-butoxide salt has been used to create nanofibers structure
on Ti surface via electrospinning method for biomedical applications
[23]. Recent studies about the electrospraying of TiO2 nanoparticles on
the Ti surface have been done for some other applications such as light
sensitive solar cells [24] and as coating membrane for aluminum substrate [25] but to the best of our knowledge there is no report in using
mentioned Ti salt in order to cover the Ti implant surface with microparticles.
In this research, titanium surface topography is altered via two
approaches, firstly electrospraying a solution of titanium salt, carrier
Solvents with the weight percentage of 43% (Isopropanol), 43%
DMF and 2% AcA were mixed together. Next, PVP (8 wt%) was added
and the solution stirred for 4 h with stirring speed of 400 rpm and then
the titanium salt (TBut) (4 wt%) was added and stirred with speed of
250 rpm for 14 h. At the end, a milky solution was obtained for both
electrospraying and dip coating test [22,23].
2.3. Preparing the specimens
In this research, two methods (electrospraying and dip coating)
were used to create the microstructured morphology on titanium
samples. The electrospraying/electrospinning apparatus parameters
were used to prepare the TiO2 microfibers before [23], by changing the
parameters and evaluating the results and surface morphologies, two
optimal groups of electrospraying parameters were obtained (Table 1).
It should be noted that in electrospraying method, titanium plates were
Fig. 1. SEM micrographs of CTRL specimen at different magnifications, a) 5000×, b) 10,000×.
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Fig. 2. SEM micrographs of electrosprayed specimens a) SP10 (5000×), b) SP10 (10,000×), c) SP15 (5000×), d) SP15 (10,000×).
used as the substrate. The specimens prepared by electrospraying, were
then exposed to air under the fume hood in order to evaporate the
solvents.
The second method was dip coating the titanium plates in the same
solution which was used in electrospraying. Two main parameters of
this method is dip coating time and solution volume/surface area ratio.
This ratio was kept constant and four different dip coating times selected and the surface morphologies were evaluated, then two optimal
parameters were used to prepare the specimens for carrying on the tests
(Table 2).
After preparing the specimens with two different methods, heat
treatment was done for three main purposes:
the PVP was cleaned from the surface. Specimens coding is presented in
Table 3.
2.4. Characterization of the surfaces
To study the morphology of the surfaces before and after modification and also after immersing in the simulated body fluid (SBF), the
scanning electron microscopy was used. The solution used for the surface modification process included a polymer (PVP) and 3 different
solvents, in addition to titanium salt. In order to assure removing these
four materials during the heat treatment, X-ray diffraction peaks of the
specimens with wavelength of 1.54187 Å, working voltage 40 kV and
working current 30 mA were studied. Wettability is one of the important factors affecting the cell adhesion; so that more wettability of
the surface, more adhesion of the osseous cells to the surface [27,29]. In
order to investigate the wettability of the specimens, water contact
angles (WCA) were measured. In this method, a drop of water was put
on the surface with a specified needle and then imaging with a camera
was done. Then the drop contact angle was measured with image-j
software. Then atomic force microscope (AFM) was used to study the
surface morphology and roughness with high precision. AFM microscope evaluates the surface by the means of a sharp needle at the free
end of a lever. The forces between the needle and surface, results in
deviation of the lever and a detector measures the amount of deviation
1- Burning the PVP and getting a pure structure of TiO2 on the surface.
2- Achieving the TiO2 anatase crystal structure.
3- Strengthening the coating/substrate adhesion.
It should be noted that the reason of choosing the anatase structure
for TiO2 is its desirable physical and biological properties compared to
rutile or amorphous structure [26]. The heat treatment program was as
follows; heating the specimens from room up to 500 °C at a heating rate
of 2 °C/min at air atmosphere, and soaked for 2 h and then cooled back
to room temperature with the cooling rate of 2 °C/min [23]. After
taking the specimens out of the furnace, the ash, resulting from burning
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Fig. 3. SEM micrographs of dip coating specimens a) DP2 (5000×), b) DP2 (10,000×), c) DP3 (5000×), d) DP3 (10,000×).
Fig. 4. X-ray diffraction spectra of samples a) SP15, b) DP3.
during the surface scanning [30]. In this project a noncontact AFM
microscope with a silicon needle, 150 kHz frequency and 2 Hz scanning
speed was used to study the surface properties of the titanium specimens, before and after modification by both methods and then the results were investigated with Q- port software.
2.5. Bioactivity investigation
The sample in vitro bioactivity was investigated by their apatiteforming ability after immersing in simulated body fluid (SBF). This
solution was prepared with a protocol suggested by Kokubo et al. [28].
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Fig. 5. Topographic pictures of AFM microscope a) CTRL, b) SP10, c) SP15, d) DP2 and e) DP3.
to decreasing the concentration of salts in SBF solution (because of
precipitation on the specimens), the SBF solution was substituted every
2 days with a fresh one.
Table 4
Average roughness numbers for electrospraying and dip coating specimens.
Roughness (Rt) (nm)
–
427.8
305.2
345.9
554.7
±
±
±
±
11.9
6.26
1.51
5.76
Roughness (Ra) (nm)
Specimen
–
81.3 ± 2.2
70.1 ± 1.5
70.8 ± 1.4
105.7 ± 1.8
CTRL
SP10
SP15
DP2
DP3
2.6. Cellular investigation
(1)
2.6.1. Mesenchymal stem cells (MSCs) isolation
Rabbit bone marrow was harvested from the tibia of 3–4 months old
New Zealand white rabbits with the procedure were approved by the
Ethics Committee of Tarbiat Modares University (Tehran, Iran). Bone
marrow was mixed with DMEM (Dulbecco's Modified Eagle's Medium)
containing 100 IU/mL penicillin, 100 IU/mL streptomycin and 15% FBS
(Fetal Bovine Serum) and incubated at 37 °C and 5% CO2. We then
incubated the cultures until confluency and replaced the medium every
other day. Passaged-3 cells were used for following experiments [32].
After dip coating of the specimens in SBF, the samples were air
dried. SEM images, EDX mapping of Ca and P, Ca/P ratio, and the
amount of increased weight was provided. It should be noted that due
2.6.2. Three-dimensional cultures
First, the specimens were cut into small size of 5 × 5 mm and
sterilized by 70% ethanol for 10 min. Then, 15,000 passaged-3 MSCs
In this method, the required volume of the solution was calculated by
Eq. (1). At this equation, Vs is the required volume for dip coating (mL)
and Sa is the area of the surface in contact with solution (mm2)
[26,30,31]:
Vs = Sa /10
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Fig. 6. Water drop contact angle test results, (n = 3) a) CTRL (82.6 ± 0.4), b) SP10 (54.2 ± 1.7), c) SP15 (25.3 ± 1.0), d) DP2 (36.9 ± 0.8) and e) DP3
(13.4 ± 0.4).
were suspended in 20 μl DMEM medium and placed on top surfaces of
the specimens. Before the cultures were provided with medium, they
were preincubated at 37 °C for 30 min for cell attachment. The cultures
were then provided with DMEM medium containing 15% FBS, 10 IU/
mL penicillin, and 10 IU streptomycin and incubated in an atmosphere
of 5% CO2 and temperature of 37 °C.
2.6.3. Cell morphology on the surfaces
Morphology of MSCs cultured on the surfaces was assessed using
SEM images. After 3 days, the samples were washed with PBS and fixed
with 2.5% glutaraldehyde. The samples were then dehydrated in a
concentration gradient of ethanol solutions (30%, 50%, 70%, 80%,
90%, and 100%), coated with gold and visualized at scanning electron
microscope.
2.6.4. MTT assay
The attachment and viability of the cultured MSCs on the different
surfaces was evaluated using MTT (3–(4, 5-dimethylthiazol-2-yl) 2, 5diphenyltetrazolium bromide) (Sigma, USA) assay. Succinctly, 3 days
after culture, the fresh medium containing MTT solution (5 mg/mL in
PBS) was added in a 5:1 ratio. After incubation at 37 °C for 2 h, the
medium was removed and the precipitate was dissolved in dimethyl
Fig. 7. Weight increase for 1 and 7 days after immersion in SBF.
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Fig. 8. SEM pictures of a) CTRL (250×), b) CTRL (1000×), c) SP15 (250×), d) SP15 (1000×), e) DP3 (250×) and f) DP3 (1000×) specimens after 1 day immersion
in SBF.
sulfoxide (DMSO). Absorbance was measured at the wavelength of
545 nm and cell viability was calculated as the percent value relative to
the untreated surface [33].
dip coated. All of these specimens were observed by SEM microscopy
under two magnifications, 5000× and 10,000×. According to Fig. 1,
the surface of the specimens was covered with passive natural oxide
layers before modification. Also, this surface had micron size pores
without any ordered morphology.
As can be seen in Fig. 2, modified surface of SP15 had higher density
of TiO2 particles covering its surface compared with SP10.
Surface modified specimens by dip coating (DP) method also are
shown in Fig. 3. At first glance, the amount of particles (or the layer of
particles) created on the surface for DPs looks more than SP specimens.
Moreover, TiO2 particles on the surface of DP samples could join together in better way and cover the large natural pores on titanium
surface. Comparing SEM images of DP2 and DP3, it can be realized that
increasing immersion time could results in precipitation of more particles on the surface.
In order to characterize the TiO2 phase and being sure of removing
the solvents and carrier polymer from the surface after heat treatment,
the prepared specimens were analyzed with X-ray diffraction which is
shown in Fig. 4. Only Ti and TiO2-anatase peaks were observed in the X-
2.6.5. Alkaline phosphatase (ALP) assay
To investigate osteogenic differentiation, alkaline phosphatase
(ALP) activity, was measured using ALP kit (BioVision, USA). Briefly,
cell seeded specimens in osteogenic media were washed with PBS and
lyses with lysis buffer. The cell lysate (200 μL) was then mixed with
300 μL of p-nitrophenyl phosphate (pNPP) substrate solution. After
incubation at 37 °C for 45 min, 100 μL of stop solution was added to the
above to stop the reaction and the absorption at 405 nm was measured
spectrophotometrically [34].
3. Results and discussion
3.1. Surface characterization
There were three different specimens: control, electro-sprayed and
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Fig. 9. SEM pictures of a) CTRL (250×), b) CTRL (1000×), c) SP15 (250×), d) SP15 (1000×), e) DP3 (250×) and f) DP3 (1000×) specimens after 7 days
immersion in SBF.
ray spectra of both SP and DP samples. In fact, other four additive used
during dip coating or electrospraying (isopropanol, DMF, acetic acid
and PVP) escaped during the heat treatment in the furnace because of
their lower evaporation temperature (i.e. 82.6 °C, 152 °C, 118 °C and
150–180 °C, respectively) [35].
In order to investigate the surface topography, AFM analysis was
performed on all samples and results are shown in Fig. 5 and Table 4. As
it was expected, untreated Ti surface had nearly smooth surface and Qport software could not report any roughness number for it. It is known
that roughness number can be explained with parameters such as Ra
and Rt. Ra is the difference between average of valleys and peaks; and
Rt is the difference between highest peak and deepest valley on the
surface [36]. Comparing SP10 and SP15 results showed smoother surface for SP15 (Ra = 70.1 nm) with more covered pores as the consequence of monolayer formation and better surface coverage with TiO2
particles which confirmed SEM results. In the case of dip coated samples, DP3 has rougher surface (Ra = 105.7 nm) than DP2
(Ra = 70.8 nm) as the immersion time was increasing. In all four
modification treatments, the obtained surface showed higher roughness
rather than smooth control but SP15 and DP3 had highest surface
roughness, 70.1 and 105.7 nm, respectively.
This microstructure may be advantageous for biomedical applications, because it can augment bone intergrowth and speed up interfacial
bonding between the implant and the natural bones. It is because of the
rapid growth of HAp crystals due to the high Ca content [37–39].
Water contact angle measurements of all samples are shown in
Fig. 6. It is known that decreasing the WCA indicates increasing in
water tendency to spread on the surface then wettability of the surface
also increased [40]. Wettability is affected by both chemical composition and topography of the surface. Since in the present study chemical
composition of all specimens was the same, the difference in WCA is
related to modifications of the surface topography. According to the
Fig. 6, the WCA of control specimen is about 82.6° which is significantly
decreased after performing both surface modification processes (electrospraying and dip coating) to 54.2°, 25.3°, 36.9° and 13.4° for SP10,
SP15, DP2 and DP3, respectively. These results are in agreement with
AFM data and show the effect of enhancing surface roughness on increasing wettability. In all modified samples the surfaces are coarser
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environment is an important factor to assay metallic implant osteointegration. In the current study, it is evaluated by soaking the
samples in SBF in two time intervals (i.e. 1 and 7 days) as presented in
Fig. 7. Due to the high corrosion resistance of titanium salt, the
bioactivity can be measured by the mass change in the samples before
and after immersion. As expected from AFM and WCA results, SP15 and
DP3 showed the most significant weight increase in both time intervals.
Therefore, their SEM images in two mentioned time periods and EDX
analysis were presented in Figs. 8, 9, and 10, respectively. The surface
coverage with a precipitated layer is evident and EDX investigations
revealed that the layers are composed of calcium and phosphorous.
Furthermore, the Ca/P ratio for control, SP15 and DP3 in day 1 were
1.59, 1.60, 1.58 and 1.60, 1.67 and 1.64 after passing 7 days. From the
literature, it has been mentioned that Ca/P ratio for hydroxyapatite
composition is about 1.67 [40]. The formation of hydroxyapatite layer
was started by immersing the titanium samples in SBF and allowing the
hydroxyapatite particles to coat the surface [41]. Comparing the results
of days 1 to 7 showed increasing trend by enhancing soaking time
which is in agreement with Eslami et al. [42]. Moreover, the Ca/P ratio
of both SP15 and DP3 after 7 days immersion in SBF are very close to
hydroxyapatite (i.e. 1.67) This effect plays a key role on bone ingrowth
and on the implant fixation bone-like apatite layer, which was also in
agreement with Kokubo and Takadama's research [29]. From these
findings, it can be concluded that the Ca/P ratio of both SP15 and DP3
after 7 days immersion in SBF are very close to hydroxyapatite.
3.3. Cellular investigation
The number of viable cells on modified surfaces in compare with
control group was quantified using MTT assay (Fig. 11). According to
results, the viable cells showed significant increase in contact with both
DP3 and SP15. No significant difference has been found in the cell vitality comparison between the DP2 and SP10 groups and control.
The interaction of samples with Mesenchymal stem cells (MSCs) was
investigated after 3 days as presented in Fig. 12. It can be seen that in
all samples the MSCs cells has proper cells attachment but in comparison with unmodified samples, modified titanium samples show more
cell flatting and ECM secretion of adhered cells is observable, especially
for DP3 and SP15 that the cells formed nearly a monolayer on their
surfaces. As reported by other researchers, the cell activity on implants
is affected by surface properties such as morphology, roughness, chemical composition, and wettability (surface free energy) [43]. So the
results of MTT and cell adhesion confirmed AFM, wettability and
Fig. 10. Ca and P weight percent on the surface of specimens after immersion in
the SBF solution for 1 and 7 days.
than control one (Fig. 5) which may cause smaller WCA. It can be
concluded that Ti-Salt precipitate is more hydrophilic comparing to
Titanium oxide.
3.2. Bioactivity
The ability to encourage apatite growth in a physiological
Fig. 11. Cell viability of MSCs on the specimens after 3 days.
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a
b
c
d
e
Fig. 12. SEM images of cell morphology on the surface of a) CTRL, b) DP2, c) DP3, d) SP10 and e) SP15 specimens.
with the control (p < 0.01). The influence of surface roughness on
differentiation of MSC is reported previously [44,45].
4. Conclusion
In this study, titanium surface modification was reported via two
different coating methods under four conditions. SEM and AFM results
showed that unmodified smooth and porous surface of Ti completely
changed to rough microstructured surface especially for DP3 and SP15
samples. XRD results revealed that only Ti and TiO2-anatase phase were
formed on the surface of both SP and DP samples. WCA of control
specimen was about 82.6° which significantly dropped to 25.3° and
13.4° for SP15 and DP3, respectively. Also, the bioactivity of modified
samples was studied after immersion in SBF, and the apatite growth
ability along with weight increase were found predominantly in SP15
and DP3. MSCs were also cultured on the surface of all samples but the
cell proliferation and ALP activity for DP3 and SP15 were again more
than other samples. These findings may imply the potential of electrospraying and dip coating methods to promote bone growth and
matrix mineralization rate on the surface of modified titanium implant
as a result of surface chemical and structural alterations in samples.
Fig. 13. ALP activity results on day 14.
bioactivity assays findings.
At last, differentiation of MSCs to osteoblastic phenotype was
evaluated by alkaline phosphatase (ALP) activity. ALP activity was
evaluated in three replicates on day 14 and reported in Fig. 13. ALP
activity significantly increased in SP15 and DP3 samples in comparison
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