Materials Research Express

PAPER

Effect of acid etching temperature on surface physiochemical properties

and cytocompatibility of Ti6Al4V ELI alloy
To cite this article: Pankaj Chauhan et al 2019 Mater. Res. Express 6 105412

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 Mater. Res. Express 6 (2019) 105412 https://doi.org/10.1088/2053-1591/ab3ac5

PAPER

Effect of acid etching temperature on surface physiochemical

RECEIVED
26 June 2019
properties and cytocompatibility of Ti6Al4V ELI alloy
REVISED
28 July 2019
ACCEPTED FOR PUBLICATION
Pankaj Chauhan1,2, Veena Koul2 and Naresh Bhatnagar1
13 August 2019 1
Mechanical Engineering Department, Indian Institute of Technology Delhi, India
2
PUBLISHED Centre for Biomedical Engineering, Indian Institute of Technology Delhi, India
28 August 2019
E-mail: nareshb@mech.iitd.ac.in

Keywords: topography, Ti6Al4V ELI, hydrophillicity, dental implant, acid etching, roughness

Abstract
Acid etching is one of the most commonly used surface modification process for the present day
marketed dental implants. Literature related to acid etching of titanium alloy is very limited. In the
present research work, the effect of acid temperature (HCl and H2SO4) as an etchant solution was
studied and evaluated on Ti6Al4V ELI grade material surface by physiochemical properties such as
topography, surface compositions, roughness, hydrophilicity and cytocompatibility. Surface topo-
graphy of alloy at optimized temperature (60 °C–80 °C) was found to be dual porous while
commercially pure titanium was non porous at the same parameters. With the increase in etching
temperature from 40 °C to 100 °C, there was a decrease in surface roughness and an increase in
hydrophilicity. Cell proliferation was found to be significantly higher at 60 °C and 80 °C etched
surfaces. The result of this study suggests that topography, surface chemistry and cytocompatibility of
an alloy surface is significantly affected by the temperature of acids used in etching. Moreover, leaching
of Al and V ions from the alloy surfaces is also a major concern for long term performance of dental
and orthopaedic implants, so modification of an alloy surface should be optimized meticulously for
these medical applications.

1. Introduction

Titanium and its alloys have a good combination of properties which are required for any dental or orthopaedic
implant applications [1]. Manufacturing of most of the available commercial dental implants is done either by
commercially pure titanium (Cp-Ti) or Ti6Al4V ELI alloys. Surface modification of titanium is required to make
the surface bioactive and enhance the osseointegration process. Acid etching is one of the most commonly
employed procedure for the surface modification of dental implants as it improves the bioactivity of the surface
[2]. Bioactivity of any implant surface depends on the roughness, micro-topography and overall developed
interfacial surface area, surface chemistry, chemical composition and hydrophilicity of the surface [3–5]. Cp-Ti
have better corrosion resistance and biocompatibility, while its alloys have more strength required for bone
implant application [6].
The acid etched surface allows better cell adhesion compared to implants without surface treatment as
supported by previous studies [7–9]. Park et al also demonstrated that titanium surfaces after double acid
etching treatment lead to increased fibrin and osteogenic cells attachment [10]. Higher removal torque was
reported by Nelson et al [11] on acid etched surfaces implants as compared to machined implants. Implant
surface physiochemical properties like surface topography, roughness do play an essential role in the initial bone
cell response and thus the overall osseointegration process [12–14]
Most of the commercial dental implants available in the current market are made up of either Cp-Ti or its
alloy depending on the clinical requirements. For example, in situations where smaller diameter implant is
needed due to space or bone deficiency or in other situations where too high forces are anticipated on an implant
as in case of bruxism, an implant with higher strength is indicated. The present research work was focused on

© 2019 IOP Publishing Ltd

Mater. Res. Express 6 (2019) 105412 P Chauhan et al

Figure 1. Photograph of (A) dental implant (B) disc samples.

Figure 2. Schematics of customized sandblaster.

ASTM grade 23 (Ti6Al4V ELI) titanium alloy because of its combination of mechanical properties like more
fatigue life as well as biocompatibility which are very important for dental and orthopaedic implant applications
[15]. Acid etching behaviour of Ti6Al4V alloy surface is faster than Cp-Ti [16]. The temperature of acid is a
major etching parameter which can affect the surface topography and other physiochemical properties and
hence biocompatibility. Due to biphasic composition, the effect of acid etching on surface topography and other
physiochemical properties of Ti6Al4V needs to be explored in detail as the surface of Ti6Al4V might be sensitive
to the alteration in the acid etching parameters. Moreover, the leaching of Al and V ions from the alloy surfaces is
also a major concern since several years [17, 18] hence the modification of alloy surface need be optimized for
dental and orthopaedic applications.
The main purpose of the present research work was to study the effect of temperature of etchant on the
physiochemical properties and cytocompatibility of alloy surface.

2. Materials and methods

Screw shape dental implants of 4.5 mm diameter and discs of 5 mm diameter and 2 mm height (figure 1) were
machined from 5 mm diameter bar (Ti6Al4V ELI alloy from Carpenter Technology Corporation, USA) on a 10
axis CNC turn-mill (Tornos make). Sandblasting of all experimental samples were done in a customized
sandblaster (figure 2). Customized sandblaster consists of two blasting nozzles mounted at a fixed distance from
the fixture for implants and the diameter of nozzles are fixed as it affect the speed of blasting media. Sandblaster
is customized in such a way that at a time 6 implants can be placed inside the blasting chamber on rotating
platform and the implant fixture itself also rotate at a fixed rpm so that a uniform blasting can done on all surface
of implants thus reducing the overall blasting time. The pressure and duration of blasting can be set to obtain the
desired surface roughness. To obtain an average surface roughness of 1.5–2 micron range, sandblasting was done
at 5bar pressure for 10 s with large grit alumina particles (250–400 μm size). Acid etching was done in a two steps
process, first in HF at room temperature (15% w/w) to remove the oxide layer and then in a mixture of H2S04
(97% w/w) and HCl (37% w/w) at 4 different temperatures. Threaded dental implants were used in pilot
experiments to select the duration of acid etching so that the profile of threads can be preserved. In the present
study, first etching was done in HF for 30 s duration and second etching was done for a duration of 5 min at
40 °C, 60 °C, 80 °C and 100 °C temperature and thus obtained 4 experimental groups (along with two control
groups, i.e. machined and sandblasted).

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Surface topography was characterized by Scanning Electron Microscope (Zeiss EVO 50) at different
magnifications. Roughness parameters measurement were done by 3D optical surface profilometer (KLE
Tencor). Chemical composition of the experimental surfaces was quantified by EDX (RONTEC’s QuanTax
200). X-ray diffraction (XRD) patterns of all experimental surfaces and machined Ti6Al4V ELI alloy surface was
analyzed with an x-ray diffractometer (Rigaku Ultima IV, US).Surface hydrophilicity was determined by
measuring static contact angle. Contact angle were measured by Image J software (LB-ASDA drop shape
analysis).
Human osteoblast sarcoma cell lines (MG-63) were used to study the cell adhesion and proliferation on
different experimental surfaces. Procurement of cell lie was done from NCCS Pune, India. Cell lines were
cultured in DMEM media with 10% FBS and 1% Penicillin and Streptomycin antibiotics. Commercially
available MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) from Sigma was used to study
the proliferation of MG 63 cells on different titanium surfaces. Cells were seeded on titanium disc with
5000 cells cm−2 density and cultured for 3 days. After 3 days disc were rinsed with PBS and incubated with MTT
reagent for 4 h in an incubator. There is a formation of purple colour formazan crystal due to cleavage of
tetrazolium ring by the mitochondrial dehydrogenase of live cells. After incubation, DMSO was added to
dissolve the formazan crystals, and the optical density of the resulting purple solution was measured
spectrophotometrically at 574 nm wavelength.
Scanning Electron microscopy and fluorescent microscopy were used to study the relative cell viability,
adhesion and proliferation on different surfaces. For cell morphology evaluation by SEM, disc samples were
fixed with 4% formaldehyde after 24 h of culture and then sequentially dehydrated in a series of alcohol
(50%–100%). After gold sputter coating SEM was used to evaluate cell adhesion and proliferation on the disc
surface. Live and dead cells after 24 h of cell culture, were seen under the fluorescent microscope following
staining with Calcein green and Propidium Iodide [19].
After 72 h of cell culture, disc samples were washed with the PBS and then fixed with 4% formaldehyde for
20 min. Again samples were washed with PBS and blocked with the 1% BSA solution for 1 h, then disc samples
were again washed with PBS and stained with Rhodamine-Phalloidin for 20 min and DAPI for 5 min. Cell
morphology was seen in a the fluorescent microscope.

3. Results

3.1. Surface topography

SEM images of each experimental groups are shown in figures 3 and 4 at 2000X and 10000X magnifications
respectively. Etching of Ti6Al4V alloy with HF resulted in selective etching of alpha phase of alloy surface and
the second etching with H2SO4+HCl at 40 °C temperature was unable to etch both alpha and beta phases
uniformly. White spots as shown in figure 4(A) (1.5 to 3 micron size) depicts the presence of un-etched beta
phase on the surface. However, initiation of porous microstructure formation was observed over the alpha
phase. The size of the pores was in the range of 180 to 300 nm. When the temperature of second acid etching was
increased to 60 °C, there was a decrease in the overall presence of beta phase crystals (figure 3(B)) from the
surface and the evolution of interconnected porous geometry on surface due to merging of smaller pores into
larger ones was also found (figure 4(B)) and the size of the pores were increased in 0.2–1 μm range. At 80 °C acid
etch condition, beta phase crystals were barely visible and pore formation was uniform on both the alpha and
beta phases (figure 3(C)). At this etched surface (figure 4(C)) dual size pores are visible (larger pore of 5–10 μm
range and smaller pore of 0.5–2 μm range). Figure 4(D) shows the topography of surface etched at 100 °C. Here
beta phase appears again on the surface and the surface has grooves and ridges type of topography instead of
being porous. Figures 3(A)–(D) shows the SEM images of all etched surfaces at lower magnification (2000X)
depicting the relative presence of β phases as white spots on 40 °C, 60 °C and 100 °C etched samples and
uniformly porous surface devoid of unetched β phase at 80 °C etched surface. Figure 5 shows the surface
topography of Grade 4 Cp-Ti and Ti6Al4V ELI grade alloy etched at 80 °C for 5 min duration. The etched
surface of Cp-Ti in figure 5(A) shows etching at grain boundaries and nonporous microstructure; however, at
the same etching parameter alloy surface depict porous topography, thereby indicating a difference in surface
topography of two different titanium surfaces.

3.2. Surface chemical composition

Figure 6(A) shows the bar graph of the elemental composition of different experimental surfaces. Highest
carbon content was seen at the blasted surface and least Carbon is seen at 80 °C etched conditions. The machined
surface contains contaminated oxide layer and blasting process also increased the surface contaminations. Acid
etching removes this oxide layer contaminated with hydrocarbons and therefore, it has less C content on etched
conditions. Aluminium content was found to be higher on the blasted samples as compared to etched

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Figure 3. SEM images showing surface topography (white spots depicting unetched beta phase) of titanium surface etched at different
temperatures - (A) 40 °C, (B) 60 °C, (C) 80 °C, (D) 100 °C (2000× magnification).

Figure 4. SEM images showing surface topography of titanium surface etched at different temperatures - (A) 40 °C, (B ) 60 °C, (C)
80 °C, (D) 100 °C ( 10000X magnification).

conditions. With an increase in temperature, there was a visible decrease in aluminium concentration. Acid
etching thus removes any remaining embedded blasting alumina as well. Vanadium concentration was highest
at 40 °C etched condition and least at 80 °C etched condition.
XRD graph of both the surfaces are shown in figure 7. Diffraction peaks of 60 °C acid 80 °C etched sample are
nearly similar to machined surface. The intensity of βTi 110 peak at 38.43° theta angle and βTi 211 peak at 70.85°
theta angle are higher at 40 °C and 100 °C etched surfaces as compared to as received machined surface and
60 °C and 80 °C acid etched surfaces [20]. However αTi 101 peak intensity at 40° theta angle are relatively

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Figure 5. Surface Topography of (A) Commercially pure titanium showing etching at grain boundaries and (B) Titanium alloy (Grade
5 ELI) showing dual porous topography. Both etched at 80 °C for 5 min duration and 5000X magnification.

Figure 6. A. Bar graph of elemental composition of machined and experimental etched surfaces B. Roughness (Ra) and C. Contact
angle of different control and experimental surfaces.

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Figure 7. XRD Diffraction Pattern of control and experimental surfaces.

reduced on 40 °C and 100 °C etched surfaces as compared to 60 °C and 80 °C acid etched surfaces. Hence XRD
cross confirm the SEM finding that 40 °C and 100 °C etched surfaces have more β phases exposed on the surface.
XRD results also shows that there are no TiH2 peaks on all experimental acid etched surfaces.

3.3. Surface roughness and hydrophilicity

There was an overall decrease in the Ra value after acid etching as shown in figure 6(B). At 100 °C etched condition,
least roughness was observed which can be attributed to faster etching rate. Figure 6 shows the roughness profile of
experimental and controlled surfaces. In figure 8(A) machined surface profile is random and roughness is in nano
range (200–400 nm). Blasted sample has a profile of crater and peak in micron the range (1.7–1.9 μm) due to
surface attrition by blasting particles. In figure 8(C), there is a superimposition of nano roughness profile on the
peak and crater profiles due to initiation of the etching process. Figure 8(E) shows the more regular micron and
submicron range profile peaks at 80 °C etched surface which is due to ensuing porous topography.
There was a continuous decrease in static contact angle values after sandblasting and acid etching as shown in
figure 6(C). Machined disc surface has the highest water contact angle (average value 100.5°) while 80 °C etched
surface has 64° water contact angle. Disc surface etched at 100 °C has 56° average static water contact angle.

3.4. Biocompatibility studies

MG-63 cells are sensitive to the physiochemical properties of the titanium surface. MTT assay was used to
measure the viability and proliferation of MG-63 cells on the control and experimental implant surfaces.
Figure 9 shows the relative optical density at 574 nm (MTT assay) of different experimental surfaces cultured
with cells for 24 h. The statistically significant difference in cell proliferation was observed by MTT assay on
machined surface versus 60 °C and 80 °C etched surfaces, and also on 40 °C etched surfaces versus 60 °C and
80 °C etched surfaces. Figures 10(A)–(D) shows SEM images of cells on different titanium disc surfaces at 500X
magnification. There is good adhesion of cells on all experimental and control surfaces showing polygonal to
elongated morphology. Enhanced proliferation can be observed in figure 8 on all etched surface as compared to
the machined surface. More numbers of cells are adhered on 60 °C and 80 °C etched surfaces (figures 10(C) and
(D)) as compared to the other etched surfaces. Figures 11(A)–(E) shows the fluorescent images of live and dead
after 24 h of cell culture on different surfaces. Calcein stains live cells green and Propidium Iodide stains dead
cells red. All surfaces show good cell viability, suggesting surface to be not damaging the integrity of the cells.
However, a few dead cells observed on machined and 40 °C surfaces. Fluorescence microscope images of cells on

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Figure 8. Profile graph of control and experimental surfaces under optical profilometer (A) Machined (B) Blasted (C) 40 °C (D) 60 °C
(E) 80 °C (F) 100 °C.

Figure 9. Viability of MG-63 cells on different temperature etched surfaces after 24 h of culture time, evaluated by the MTT assay.

the different experimental sample surfaces, stained with DAPI and Rhodamine-phalloidin are shown in
figures 12(A)–(E). Actin filaments of cells are stained red by Rhodamine-phalloidin and nucleus of the cell are
stained blue in colour by DAPI. There is elongated morphology of cells on all machined and experimental
surfaces. 60 °C and 80 °C etched surfaces (figures 12(C) and (D)) shows relatively more spread morphology and
more numbers of cells as compared to other surfaces.

4. Discussion

The topography of titanium alloy was found different at the 40 °C, 60 °C, 80 °C and 100 °C etching temperatures.
There was continuous evolution and enhancement of porous structure with an increase in temperature up to 80 °C
and at this temperature uniform porous microstructure was found but again at 100 °C etched conditions, this
porous structure disappeared fully. This topography difference can be explained on the basis that α and β phases in
a titanium alloy have different etch rate [16] and etchant under the study at 60 °C–80 °C temperature range are
sufficient to etch uniformly both the phases without losing the porous microstructure. However, at the same

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Figure 10. SEM images of MG-63 cells cultured for 24 h. (A) Machined, (B) 40 °C, (C) 60 °C, (D) 80 °C, (E) 100 °C etched conditions
of titanium alloy.

Figure 11. Fluorescent microscope images of MG-63 cells cultured for 24 h. duration on (A) Machined, (B) 40 °C, (C) 60 °C, (D)
80 °C, (E) 100 °C, etched conditions of titanium alloy.

parametric etching conditions, Cp-Ti has a nonporous topography when compared to alloy surface. This
topographical difference can be attributed to difference in the microstructure of the alloy and Cp-Ti and higher
corrosion resistance of Cp-Ti to acids [1]. Ti6Al4V alloy is composed of submicron sized non-equiaxed α and β
grains. Due to the heterogeneous composition of Ti6Al4V ELI alloy, the etched surface has a dual porous surface
with peaks in β phases and valleys in α phases.
Surface elemental composition was also found to be dependent on the temperature of the etchant. Acid
etching removes carbon contaminated oxide layer and there was a continuous decrease in overall carbon
percentage as the temperature of etching was increased. However, these surfaces are highly porous hence more
prone to carbon contaminations from the atmosphere. Hence the final surface modification step of the medical
implant was done in a controlled environment (clean room class 1000) and post-modification packaging was
done in an inert atmosphere. With an increase in etching temperature, the oxygen content of the outer layer was
also found to increase, suggesting a thicker oxide layer. Several authors have demonstrated that a titanium
dioxide layer enhances corrosion resistance, osteoblast differentiation, and bone growth [21–24]. EDS analysis
also cross confirmed the SEM microscopy finding, that there was a relative decrease in Vanadium percentage on
the surface at 80 °C and 60 °C etched surfaces as compared to the machined, 60 °C and 100 °C etched surfaces.
Further studies are required to characterize the biocompatibility and long-term performance of beta titanium
alloys since this Al and V elements are considered as cytotoxic.
Roughness values of all experimental etching group were between 1–2 μm (Ra). Increase in etching temperature
resulted in a decrease in surface roughness resulting from the rapid etching of peaks created by sandblasting.
Although all surface possesses roughness in the range of 1–2 μm. Several studies have demonstrated that surface
roughness is within the 1–2 μm range are beneficial for the biochemical anchorage of oral implants [25, 26].

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Mater. Res. Express 6 (2019) 105412 P Chauhan et al

Figure 12. Fluorescent microscope images of MG-63 cells cultured for 72 h showing cell morphology, nucleus stained with DAPI and
actin filament stained with rhodamine phalloidin. (A) Machined, (B) 40 °C, (C) 60 °C, (D) 80 °C, (E) 100 °C, etched conditions of
titanium alloy.

An improvement in the hydrophilicity of the surface with the increase in temperature of etchant was also
demonstrated. Sandblasting leads to an increase in surface roughness and acid etching removes hydrocarbon
contaminants from the surface. Both of these phenomena is responsible for decreased water contact angle value.
The increased cells proliferation at these surfaces can be attributed to the enhanced physiochemical
properties of these surface as compared to the controlled machined surface [27]. At 40 °C etched conditions,
there was a slight decrease in cell proliferation and at 100 °C etched surfaces, there was a slight increase in cell
proliferation as compared to control machined surface. Although the difference was not statistically significant,
the same trend was also observed in SEM and fluorescent images. Topography at 40 °C and 100 °C etched
surfaces were not porous and have more exposed beta phases on the surfaces as shown in figure 2(A) and D,
which might have leached out in the culture media and might be responsible for the decrease in the viability of
osteoblast cells [18, 28]. However, further studies need to be done to explore the effect of etching on ions
leaching in case of titanium and its alloys and more detailed cell studies and in vivo studies are also required.

5. Conclusions

It is demonstrated that cytocompatibility of titanium surface is enhanced by topography, roughness, surface

chemical composition and surface hydrophilicity, hence, for faster osseointegration of dental and orthopaedic
implants, these properties should be optimized for an alloy. The acid etching procedure is critical for the alloy
surface as the surface composition of the alloy is non-uniform and the etch rate is also higher than Cp-Ti. Due to

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biphasic composition, the surface is quite sensitive to the alteration in the acid etching parameters and
temperature of acid is an important parameter which affects the topography and other physiochemical
properties.

Acknowledgments

The authors are indebted to the generous funding of the CSIR NMITLI Project RP02756 for the ‘Design and
Development of Indigenous Dental Implants’ at IIT Delhi.

ORCID iDs

Naresh Bhatnagar https://orcid.org/0000-0002-9959-4341

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