Materials Technology

Advanced Performance Materials

ISSN: 1066-7857 (Print) 1753-5557 (Online) Journal homepage: http://www.tandfonline.com/loi/ymte20

Enhancing biocompatibility of Co-Cr alloy implants

via electrical discharge process

Amit Mahajan & Sarabjeet Singh Sidhu

To cite this article: Amit Mahajan & Sarabjeet Singh Sidhu (2018): Enhancing biocompatibility
of Co-Cr alloy implants via electrical discharge process, Materials Technology, DOI:
10.1080/10667857.2018.1475144

To link to this article: https://doi.org/10.1080/10667857.2018.1475144

Published online: 07 Jun 2018.

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MATERIALS TECHNOLOGY
https://doi.org/10.1080/10667857.2018.1475144

Enhancing biocompatibility of Co-Cr alloy implants via electrical discharge

process
Amit Mahajana and Sarabjeet Singh Sidhub
a
Research Scholar, IKGPTU, Kapurthala, INDIA; bDepartment of Mechanical Engineering, Beant College of Engineering &Technology,
Gurdaspur, INDIA

ABSTRACT ARTICLE HISTORY

With increasing popularity of Electrical Discharge Machining (EDM) method upon part man- Received 23 March 2018
ufacturing precision with tailorable surface properties; the assessment of EDM process was Accepted 3 May 2018
therefore carried out concerning the surface alteration of medical grade Co-Cr alloy bioma- KEYWORDS
terials. The samples surface were EDMed at different spark energy levels by varying current Co-Cr alloy implants; EDM;
and On-time to investigate the surface characteristics, and it's biological responses. MTT assay surface modification;
and cytocompatibility test were conducted to examine the in-vitro biocompatibility confirm- biocompatablity
ing the higher competent cell viability and proficient RBC lysis for the surface treated with
spark energy ranging from 960J–1500J. Here the experimental results revealed that applica-
tion of low-current (5 A) and high On-time (200 µs) favors the synthesis of high-quality
biocompatible substrates. Thus, surface modification of Co-Cr implants using EDM can be
considered as a promising method for improving surface properties essential for biomedical
applications.

Introduction to all sorts of metallic materials independent of their

mechanical properties. Thus, EDM is the valuable
Today, the medical field routinely demands bioma-
process for designing the biological materials
terials in the province of tissue engineering, drug
implants and improved morphology favoring
delivery, and medical imaging. These biomaterials
ingrowths of cells [5].
have been specially designed with inflated stability,
EDM process involves controlled spark energy
appropriate pharmacokinetics, and effectual cell pro-
between the tool electrode and workpiece after creating
liferation [1]. It has long been recognized that for
the ionization zone in the dielectric medium. The high-
biomaterials, surface chemistry and microstructure
frequency electric sparks from the electrodes increase
are the most critical facets in tissue implant relation
the temperature of the specimen region, which causes
that has a profound impact on the implants biocom-
metallurgical transformation and affects the surface
patibility and osseointegration [2]. The implant fail-
integrity the most [6]. Thus, understanding the influ-
ure has been associated with the lack of
ence of machining process parameters leads to the
functionalities in terms of inflammatory response,
significant improvement in its potential for the medic-
bacterial infection, wear, and corrosion as well as
inal domain. The characteristics of EDM process was
the loosening of implants which lead to revised
investigated on the Titanium alloy (Ti-6Al-4V) which
surgery. With the aim to alleviate the implant inade-
was intended to enhance its biological performance
quacy, surface engineering of biomaterials plays a
[7].They concluded that the EDMed surface promotes
critical role to establish the long-term interaction
the cell growth and substrate adhesion, but it also
between the implants and human body environment
reduces the fatigue performance which results in clin-
[3]. In this regards, for effective interlocking of
ical failure. Furthermore, the diminished fatigue
tissues and implants the dimensional accuracy and
strength of machined surface can be enhanced with
surface properties of implants are the significant
the application of subsequent acid etching and shot
factors for the successful surgery. The newest study
peening treatment [8]. These three steps of surface
highlighted that these complex parameters for
treatment processes promote the osteoblast cell growth
enhanced bioactivity could be stimulated by non-
and develop the superimposed surface topography.
conventional machining methods, in which
The surface chemistry depicted that nanostructured
Electrical Discharge Machining (EDM) propagated
oxide layer on the surface, can be developed by EDM
excellent results [4]. This machining method pro-
process which promotes the cell proliferation and
vides an advantage of its application and precision
enhanced the biocompatibility of the alloy [9].

CONTACT Sarabjeet Singh Sidhu sarabjeetsidhu@yahoo.com Department of Mechanical Engineering, Beant College of Engineering
&Technology, Gurdaspur 143521, INDIA
© 2018 Informa UK Limited, trading as Taylor & Francis Group
2 A. MAHAJAN AND S. S. SIDHU

Otsuka et al. addressed progression of implant surface

by improving the crystallinity and chemistry of recast
layer [10]. The recent research demonstrated that the
surface modification by powder mixed electrical dis-
charge machining induces the better surface quality
and is also considered as the most promising approach
to enhance the mechanical properties as well as for
superior osseointegration surface [11]. Another most
preferred metallic biomaterial for permanent joints is
Co-Cr alloy which is known for its high corrosion and
wear resistances. To alleviate the limitation of Co-Cr
alloy in clinical domain numerous surface modifica-
tion methods have been proposed. Examples include
plasma treatment [12], nono-sized self assembled
monolayer coating [13], laser treatment [14], ultravio-
Figure 1. SEM image and pictorial view (insert) of femoral
let photofunctionalization treatment [15] etc.
component of knee joint; Ra = 0.49 µm.
However, to date, little work has been demonstrated
on the influence of EDM process parameters on surface
modification of medical grade Co-Cr alloy. process parameters faclitates different spark energy
This study is devoted to elucidating the impact of on the machining zone. The spark energy is culmina-
EDM process on the surface characterization of med- tion of three parameters (such as V, I, Pon) and is
ical grade cobalt chromium (Co-Cr) implant. calculated as follows: E ¼ V  I  Pon whereas
Furthermore, the in-vitro cytocompatibility and hemo- E = Energy (J); V = Voltage (V); I = Current (A);
compatibility tests were performed on the machined Pon = On-Time (µs).
region of the specimens for investigating the biological Four levels of current and pulse-on time were
behavior of the altered surfaces. chosen, and the full factorial experimental array was
designed to analyze the impact of spark energy on the
workpiece surface as shown in Table 3.
Materials and methods
Material and processing surface treatment
In this study we used medical grade Co-Cr-Mo alloy,
which is utilized for knee joints. The material was Table 2. Parameter descriptions and values.
provided by DePuy Synthes Company (Raynham, Parameter, units Values
Current (I), A 1, 5, 10, 16
Massachusetts, United States) and chemical composi- On-Time (Pon), µs 20, 60, 150, 200
tion is shown in Table 1. The pictorial view and micro-
structure are shown in Figure 1.
The specimen was treated using finely grained gra-
phite electrode (particles sized ~5µm) in (make: Table 3. Experimental array and surface roughness response.
OSCARMAX, Taiwan) die sinking EDM with negative Trial No. I (A) Pon (µs) Ra (µm)
polarity (i.e., the work piece (-) and tool electrode (+)) 1 1 20 1.15
at constant pulse Pause time (20µs). The working gap 2 1 60 0.48
3 1 150 0.49
voltage between the electrodes was preset at 140 V 4 1 200 0.48
which allowed the efficient side flushing conditions. 5 5 20 1.23
The machining was conducted in the presence of 6 5 60 1.21
7 5 150 0.89
dielectric medium to ensure the process stability. In 8 5 200 1.40
this process, the maximum level of current and On- 9 10 20 1.31
10 10 60 0.42
time (i.e. time span of peak current during machining) 11 10 150 0.49
was set at 16A and 200 µs respectively, as beyond this, 12 10 200 1.16
13 16 20 1.06
unstable arcing on the work piece, results in surface 14 16 60 2.39
damage. Table 2 indicates the process parameters and 15 16 150 2.47
16 16 200 1.42
levels used in the experiments. The combination of

Table 1. Chemical composition of chromium cobalt (Co-Cr-Mo) alloy.

Cr Mo Si Mn Ni C Fe Ti P Co
Base Material 28.5% 6% 0.7% 0.5% 0.25% 0.22% 0.2% 0.01% 0.02% Remainder

Surface roughness test
The surface roughness was measured at three different
locations on the machined zone in terms of the arith-
metic average of absolute value (Ra µm – Avearge
deviation of roughness profile from the mean line)
using surface profilometer (Mitutoyo-SJ-400). In this
test, the measuring probe estimated the erosion or
deposition on the machined surface.
In-vitro biocompatibility test
To evaluate the EDMed surface biological test, in-
vitro hemolysis and cytocompatibility test were per-
formed. Originally, the moist heat sterilization tech-
nique was utilized to remove the foreign particles and
obliteration of micro organisms from the samples.
The process was carried out in an autoclave
(Narang Scientific works, New Delhi, India) in
which samples were sterilized at 121°C for 20 minutes
to confront microbes or spores from substrates. After
that, the experiments were conducted in triplicate,
and three specimens were used in each
experimentation.
Hemolysis test
In-vitro hemolysis study was carried out on the samples
in the form of circular discs (diameter 8mm, thickness
3mm) treated by EDM process parameters according to
selected design matrix (Table 2). These samples were kept
in intimate contact with the blood pool and thus may lead
to RBC (Red blood cells) lysis. For the hemocompatibility
test, healthy human blood was collected in heparinized
microcentrifuge tubes. The collected blood was then sub-
jected to 2500 rpm (4°C, 5 min) centrifugation to separate
RBCs. Further, the supernatant consisting of plasma and
a buffy layer of leukocytes were removed and washed
three times with isotonic 7.4 pH phosphate-buffered
saline (PBS) to obtain a pellet of RBCs. A stock of 1%
RBC suspension was utilized in this study.
Briefly, all the samples were placed in the 12 well
plate to expose the modified portion outwards to be
in contact of the RBCs. A volume equivalent to 1 ml
of stock RBC suspension (1%) was incorporated into
each well. Triton XTM-100 was utilized as a positive
control which caused 100% hemolysis of RBCs, and
isotonic PBS pH 7.4 was considered as a negative
control as it promoted negligible hemolysis of RBCs.
The samples were incubated at 37°C for one hour,
and then the RBC suspension in contact with samples
was withdrawn from the plate and collected in micro-
centrifuge tubes, followed by centrifugation at
2500 rpm for 5 min at 4°C. The supernatant was
collected, and its absorbance was measured using
the spectrophotometric method at 540 nm [16], and
% hemolysis was calculated using the formula:
MATERIALS TECHNOLOGY 3
%hemolysis ¼ ABs
ABp x100, ABs is the absorbance of
testing samples and ABp absorbance of positive
control.
The RBC pellet obtained after one hour of incuba-
tion was redispersed and incubated with 0.5% glutar-
aldehyde in PBS at 4°C for one and half hours to fix the
RBCs. Centrifugation at 2000 rpm and 4°C was subse-
quently carried out to remove traces of free glutaralde-
hyde. RBC pellet thus obtained was further washed
thrice with distilled water. Final dispersion of RBCs
was then visualized under SEM (S- 3400, Hitachi Ltd.,
Japan) at an acceleration voltage of 10 kV. After placing
on the carbon tape, samples were sputter coated (E-
1010, Hitachi Ltd., Japan) with gold-palladium alloy to
make the surface conductive and were observed at
different magnifications [17].
Cytocompatibility test:
Cytocompatibility of samples was
determined using MTT[3-(4,5-Dimethylthiazol-2-yl)-2,
5-Diphenyltetrazolium Bromide] [C18H16BrN5S] assay
in MCF-7 (Michigan Cancer Foundation-7, human
breast adenocarcinoma) and HeLa cell lines by the fol-
lowing reported protocol. Briefly, cells were grown in
Minimum Essential Medium Eagle (MEM, Sigma),
accompanied with Earle’s salts, L-glutamine, nonessential
amino acids, sodium bicarbonate, sodium pyruvate, 10%
FBS, 100 U ml−1penicillin, and 100 mg ml−1 streptomycin
(PAA LaboratoriesGmbH, Austria). These cells were
maintained under 5% CO2 atmosphere at 37°C. It fol-
lowed the cells harvesting by using 0.25% w/v trypsin –
Ethylenediaminetetraacetic acid (EDTA) solution
(Sigma, USA). Then these were subcultured in 24-well
culture plate(Costars, Corning Inc., NY, USA) at a den-
sity of 10,000 cells per well-containing the circular disc
samples(8mm in diameter and 3 mm thickness). After
48 h of incubation, the substrates were removed and
further placed in fresh 24 well culture plates [26]. After
that, the extent of the viability of the cells is indicated by
conversion of MTT to purple colored formazan by meta-
bolically active cells and further compared with negative
control (coverslip) and positive control (untreated sam-
ple). The cells were then solubilized with Dimethyl sulf-
oxide (DMSO) and optical density of the released;
solubilized formazan reagent measured at 540 nm spec-
trophotometrically [27]. The following equation evalu-
ABs
ated the cell viability:%cell viability ABc x100, where ABs
is the absorbance of the testing samples and ABc is the
absorbance of the control.
Statistical Analysis:
In the first stage, analysis of variance (ANOVA)
was employed to identify the significant process para-
meters affecting surface roughness. In the second
phase, the biological response on the machined sur-
face was evaluated and presented as mean  S.D
(Standard Deviation) at 95% confidence. The differ-
ences were considered significant when P  0.05.
4 A. MAHAJAN AND S. S. SIDHU
X-ray diffraction (XRD) technique
The study of phase formation and material trans-
ferred on the machined surface were investigated
by XRD using Cu-Kα X-ray radiation (λ=1.5406Å,
scan rate of 1°/min range-140°) with generator set-
ting of 40 mA and 45 KV (PANalyticalX’Pert Pro
MPD, The Netherland).
Scanning electron microscopy (SEM)
The morphology of samples after EDM was investi-
gated using SEM (JSM-6610 LV Joel, Japan). This
technique was also used to check the RBC (Red
Blood Cells) response (hemocompatibility) on the
EDM treated and untreated sample.
Results and discussion
Analysis of EDMed surface: The surface treatment of Co-
Cr sample was conducted according to selected design
layout as shown in Table 3.The surface finish of the
specimen is corresponding to the craters and material
deposition. The response was expressed in the last col-
umn of Table 3 that is the mean of data measured at five
different location of each sample. The surface roughness
measured on different experimental trials and were ana-
lyzed, and the ANOVA results shows that current level is
the significant (i.e. p < 0.05) process parameter. Thus the
highly finished surface (Ra = 0.42 µs) can be achieved at
current of 10 A with the On-time at 60 µs. Figure 2
represents the variation of the surface roughness with
EDM process parameters.
It was observed that the machining at a current of
1 A and higher On-time (i.e. 60 µs, 150 µs and200 µs)
the material was not extracted from the specimen.
Thus, the negligible machining or surface alteration
zone in Trail 2 to Trial 4 was observed. This is due to
the fact that, a small amperage level (i.e., 1 A) coupled
with the substantial gap between the performance
parameters viz. pulse On- time (60 µs, 150 µs
and200 µs) and pause time(20 µs) leads to the low
frequency of spark energy formation . Furthermore,
Figure 2. EDM process parameter effect on surface
roughness.
Figure 3. Percentage hemolysis of EDM treated samples (All
values expressed as Mean±SD, at n = 3).
these samples (Trial 2 to Trial 4) were considered as
untreated surface and the biological tests had not
been performed on these samples.
Effect of the EDM treated samples on Hemolysis:
The extent of hemolysis for different samples can be
seen in Figure 3, where, positive control (Triton
XTM-100) gave 100% hemolysis and negative control
(Isotonic PBS, pH 7.4) 4.53 ± 2.61%. Amongst all the
samples, the untreated sample showed significantly
higher hemolysis as compared to negative control
(p < 0.001). It was also observed with sample 6
showing hemolysis of 30.86 ± 4.97%. Similarly, sam-
ple 1, 5, 7, 10, 13, and 15 also had significantly higher
hemolysis as compared to negative control (p < 0.05).
However, there was the insignificant difference
(p > 0.05) in hemolysis between negative control
and sample 8, 9, 11, 12, 14 and 16 depicting their
higher hemocompatibility incurred on them by sur-
face modification.
Similar results were also observed in RBC mor-
phology by SEM images as illustrated in Figure 4.
Sample 1, 5, 7, 10, 13, and 15 groups RBCs
showed aggregated, clumped, distorted images as
compared to substrates 8, 9, 11, 12, 14 and 16
which had their morphology intact similar to
negative control images. Also, it was indicated
that, with contact to RBCs samples 1, 5, 7, 10,
13, and 15 also showed blurred background which
is due to protein agglomeration on the RBC sur-
face which was negligible or absent in other sam-
ples and negative control. Whereas, sample 8 was
found to be significantly less hemolysis percentage
as compared to untreated samples. These results
in accordance to % hemolysis are indicative of
improved hemocompatibility of EDM treated
samples as compared to the untreated sample.
Some treatments do not yield fruitful results, but
the investigation leads to the identification of
some useful modification parameters to improve
hemocompatibility.

Figure 4. Comparative images of RBC morphology of different samples (Conductive carbon tape substrate; Samples from Trail 2,
Trial 3, and Trial 4 are represented as untreated surface).
MCF-7
120
100
80
% viability
60
40
20
0 hemocompatibility assay. Thus the results depicted
Figure 5. Percentage viability of MCF-7 cells upon different
samples.
Effect of the EDM treated samples on
Cytocompatibility: The biocompatibility of EDM trea-
ted and untreated samples were evaluated by comput-
ing the % cellular viability, as shown in Figure 5. The
viability of MCF-7 cell line on Cr-Co specimens was
found to be significantly less as compared with negative
control cells (cell grown over coverslip). Furthermore,
the cell viability in the case of EDM treated samples 5,
12 and 15 was found to be insignificant as compared
with the positive control (untreated Cr-Co sample).
Whereas, the EDM treated samples 1, 6, 7, 8, 9, 10, 11,
13, 14 and 16 demonstrated higher viability as
MATERIALS TECHNOLOGY 5
compared to positive control and was found to be in
the range of 39% to 54%. However, significantly higher
cells viability (~80%) was observed in the case of sample
8 as compared to positive control. The MCF-7 cells
viability of sample 8 was sorted out as 2.05 times greater
than untreated sample. Whereas, the specimens 12 and
15 have nearly equal cells viability with the untreated
sample. The outcomes obtained were observed to be
inline with the blood interaction found via in-vitro
improved biocompatibility and safety of EDM treated
samples and can be efficiently used for the orthopedic
implants.
The microstructures of these EDMed treated specimens
are presented in Figure 6. Figure 6(a) depicts the surface
texture associated with 10 A current and 60µs as On-
time (i.e., Trial 10).The topography demonstrates the
least formation of peaks and valleys on the surface, i.e.,
least roughness (Ra = 0.42µm). It was due to the fact
that the spark energy frequency and flushing mechan-
ism effectively synchronized for the removal the debris
from the machining zone. However, the increase in
current to 16 A (i.e., Trial 14) leads to higher energy
of spark resulting in the formation of high ridges
around the craters, thus highly irregular surface was
observed Figure 6(b). The samples obtained from
Trial 8, Trial 11, and Trial 14 revealed the improved
biocompatibility. Further, it is affirmed that the surface
finish (micro-roughness; depicted in Table 3) measured
6 A. MAHAJAN AND S. S. SIDHU

Figure 6. Morphology of machined surface obtained at different sets of parameters (a) I = 10 A; Pon = 60µs (b) I = 16 A;
Pon = 60µs.

for these samples was not directly proportional to the
biological performance. However, the macro-roughness
(distinguish by the human eye) [7] of these machined
surfaces is more favorable surface conditions for the
surrounding tissues.
XRD analysis: The X-ray diffractometer (XRD)
pattern of untreated in comparison of EDMed surface
is depicted in Figure 7. All specimens consisted basic
compounds of Cr, Fe, Mo and Co such as
CoFe3, CrSi2, Co3.31Mo3.31Mn3.31. Additionally, the
surface treated with EDM spark energy resulted in
the formation of tetragonal structure Phosphide
(Cr2.4 Ni0.6 P1) and cubic structured carbides and
oxides (Cr3O) on the surface. It was detected that
EDMed surface morphology and chemical alterations
supports the cell viability; however, the formation of
Phosphides and oxides displayed good biocompatible
substrate. The oxide film formation protects intergra-
nular and corrosion attacks and also contribute
towards excellent biocompatibility [18].These phase
formation on the surface was observed when the
process parameters generates the spark energy in
the ranges of 960 J – 1500 J (Figure 8)
The SEM images and EDX investigation of the
highly bio-favorable sample (i.e., Trial 8) was carried
out. Figure 9 illustrated surface morphology and che-
mical composition of the machined surface
(E = 1000 J). The micro-porosity (~3 to ~5 µm), as
well as resolidified droplets, were observed on the
surface. The presence of microporous [19,20] and
droplets facilitated the more suitable surface for the
adhesion and cell proliferation. It was deduced from

Figure 7. XRD pattern of Co-Cr specimen (a) Untreated surface (b) Trail 6: EDMed surface (mark (hash): # corresponds to carbides
phase) (c) Trial 8: EDMed surface (mark (Star): *corresponds to phosphide phase) (d) Trial 11: EDMed surface (mark (Dollar):$
corresponds to oxide phase).
 MATERIALS TECHNOLOGY 7

performance. Nevertheless, the research can further

be extended to estimate the after machining impact in
terms of residual stress, microhardness, etc.

Acknowledgments
The authors thank IKG Punjab Technical University,
Kapurthala for its support to this research work.

Disclosure statement
No potential conflict of interest was reported by the
authors.
Figure 8. Illustration of spark energy vs. Trials and bio-favor-
able spark energy zone.
References
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Conclusion
The surface of the Co-Cr alloy knee joint specimen was
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observed that the EDMed samples at the spark energy
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