Arabian Journal of Chemistry (2018) 11, 959–969

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

In vitro and in vivo characterization of mineralized

hydroxyapatite/polycaprolactone-graphene oxide
based bioactive multifunctional coating on Ti alloy
for bone implant applications
Nagaraj Murugan a,b, Chandran Murugan c, Ashok K. Sundramoorthy a,b,*

a
Department of Chemistry, SRM Institute of Science and Technology, Kattankulathur 603203, Tamil Nadu, India
b
SRM Research Institute, SRM Institute of Science and Technology, Kattankulathur 603203, Tamil Nadu, India
c
Department of Zoology, Periyar University, Salem 636011, Tamil Nadu, India

Received 2 December 2017; accepted 27 March 2018

Available online 5 April 2018

KEYWORDS Abstract Hydroxyapatite (HAP) is a form of naturally occurring calcium apatite present in bone
Polycaprolactone; and tooth enamel. It is an important biomaterial with diverse biomedical applications such as a sur-
Graphene oxide; face coating for metallic orthopedic implants. Synthesized pristine HAP has poor mechanical prop-
Hydroxyapatite; erties, inferior wear resistance and has limits for directly used in bone tissue engineering
Bone implants; applications. To address these limitations, we synthesized a suitable orthopedic implant hybrid
Antibacterial activity; material (M-HAP/PCL/GO) by using positively charged calcium ions of mineralized HAP
MG63 osteoblasts cells; (M-HAP) combined with Polycaprolactone-negatively charged graphene oxide (PCL-GO). The
Surface coating successfully synthesized M-HAP/PCL/GO composite was comprehensively characterized by
Fourier-transform infrared spectroscopy (FT-IR), powder X-ray diffraction (XRD) and
field-emission scanning electron microscopy (FE-SEM). The micro-hardness technique was used
to determine the mechanical strength of M-HAP (315 ± 4 Hv), M-HAP/GO (370 ± 3 Hv) and
M-HAP/PCL/GO (455 ± 5 Hv). M-HAP/PCL/GO was also tested for its anti-bactericidal impact
against Staphylococcus aureus and Escherichia coli. MG63 osteoblast cells cultured on the M-HAP/
PCL/GO composite (10 mg/mL) coated sample, displayed outstanding viability after 3 and 5 days
of incubation at pH 7.4, which indicated that the composite is suitable material for bone implants
and induces the cell proliferation. It was also tested in vivo in Wistar rats and was observably
beneficial bone formation within 28 days post-implant operation. These tests proved that the

* Corresponding author at: Department of Chemistry, SRM Institute of Science and Technology, Kattankulathur 603203, Tamil Nadu, India.
E-mail address: ashokkumar.sun@ktr.srmuniv.ac.in (A.K. Sundramoorthy).
Peer review under responsibility of King Saud University.

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1878-5352 Ó 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
960
M-HAP/PCL/GO composite can be considered as a prospective candidate for future bone implant
applications.
Ó 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Titanium (Ti-6Al-4V) alloys are considered as a good implant
material for dental and orthopedic applications due to their
biocompatibility, excellent corrosion resistance, mechanical
properties and lower elastic modulus (Ananth et al., 2015;
Chellappa and Vijayalakshmi, 2017; Otsuka et al., 2016); how-
ever, Ti alloys surfaces have low bioactivity which in turn can
facilitate bacterial contamination and reduce corrosion resis-
tance property. Additionally, the implant-to-bone binding
properties need to be taken into account. Some research has
been done to look at various surface modification strategies
to improve the bone bonding ability of Ti implants (Wu
et al., 2014). Ti alloy implants are usually coated with hydrox-
yapatite (HAP) due to its structural similarity with inorganic
minerals of human bones. The HAP coating can provide sev-
eral advantages such as promoting osteoblast proliferation, tis-
sue adhesion, improved bioactivity and biocompatibility, and
bone tissue integration in hard tissue engineering (Kesteven
et al., 2015; Liu et al., 2014b; Lu et al., 2004). Ti alloys with
HAP coating (HAP/Ti alloys) have shown remarkable efficacy
in dental, joint replacement and orthopedic surgery to full-fill
bone defects and improved implant amalgamation with the
host bone.
Furthermore, by introducing various trace metal ions onto
HAP/Ti alloys, it is possible to improve their biological prop-
erties. The incorporation of various metal ions such as stron-
tium (Sr2+), magnesium (Mg2+), manganese (Mn2+), silver
(Ag2+), zinc (Zn2+), cerium (Ce3+), lanthanum (La3+) and
yttrium (Y3+) in substituted-HAP coatings on metallic sub-
strates has been studied (Bakin et al., 2016; Cox et al., 2014;
Deliormanlı 2016; Gopi et al., 2015; Murugan et al., 2017).
Among these cations, Mg2+ is naturally found in human bone,
attributing to the bone cell proliferation and bone cell repair-
ing mechanism. Mg2+ doped HAP exhibited excellent corro-
sion resistance with high biocompatibility in various
orthopedic applications. Zn2+/HAP coated Ti alloys effec-
tively stimulate new bone formation, fibroblastic proliferation
and bone restoration in both in vitro and in vivo application.
These studies suggest that the Zn2+ improves the antimicro-
bial efficacy and biological safety of the implanted materials
(Ito et al., 2002; Li et al., 2009).
It has also been shown that HAP loaded with multi-ions
can induce effective bone growth (Gayathri et al., 2018;
Liangzhi et al., 2016; Manoj et al., 2015; Wallach 1990). Addi-
tional studies also demonstrated that multi-ion/HAP had low
mechanical property, low tensile strength, low fracture tough-
ness and inferior wear resistance which limits their application
in large bone defects (Baradaran et al., 2014; Liu et al., 2013;
Prajatelistia et al., 2013; Rodrigues et al., 2016; Shin et al.,
2015). The mechanical property of multi-ions/HAP can be
appreciably increased by incorporating new reinforcing mate-
rials such as ceramics, polymers and graphene (Balani et al.,
2007; Elangomannan et al., 2017; Liu et al., 2014a; Núñez
N. Murugan et al.
et al., 2014). For example, the addition of biodegradable poly-
caprolactone (PCL)-graphene oxide (GO) to multi-ion/HAP
could improve many aspects of the coating such as their bio-
compatibility, toughness, the fracture energy, mechanical
property and degradation rate (Izquierdo et al., 2008; Rezaei
and Mohammadi 2013; Roh et al., 2017).
Based on the information from other studies, the multi-ion/
HAP coating surface properties may be improved by the addi-
tion of GO. GO is a potential reinforcing material with one
atom thickness and high flexibility (Stankovich et al., 2006;
Wang et al., 2015). Additionally, GO has good dispersibility
in organic solvents, water and different matrixes (Cheng
et al., 2017; Zhang et al., 2013). Low quantities of GO in
biopolymers has been found to increase cell adhesion by inter-
acting with proteins such as fibronectin and integrins on cell
surface showing potential for improved cell–material interac-
tions. GO contains oxygen-containing groups which con-
tribute to the hydrophilicity of the composite. In addition,
their excellent biological activity induce corrosion resistance
in simulated body fluid (SBF) solution, high rate of osteoblasts
proliferation and improve the wound healing properties after
implantation (Mukhopadhyay and Gupta 2011; Wan et al.,
2011; Yang et al., 2010).
In this study, we synthesized a novel multi-ion (Mg2+ and
2+
Zn ) loaded HAP (M-HAP) surface coating topped with a
PCL-GO layer, which we will refer to as M-HAP/PCL/GO.
M-HAP/PCL/GO exhibits superior biocompatibility, mechan-
ical and antibacterial properties as a coating on Ti alloys. The
PCL component can be easily bond with the either epoxide or
AOH group on the basal plane or the ACOOH group and car-
bonyl moieties on edges of GO. The hybrid material (M-HAP/
PCL/GO) significantly enhanced the osteoblast cells forma-
tion, cell-material interaction and antimicrobial activity. Fur-
ther, the M-HAP/PCL/GO composite showed porous-like
structure which contributes a good foundation for the growth
and integration of bone. This is demonstrated in the electrode-
position of novel multifunctional M-HAP/PCL/GO composite
on Ti alloy and the in vitro and in vivo application. This novel
combination has potential use for different tissue engineering
and soft bone tissue replacements.
2. Experimental
2.1. Materials
Materials include Titanium (Ti) alloy (99.9% purity), poly-
caprolactone (PCL), calcium chloride dihydrate (CaCl2
2H2O),
graphite, potassium permanganate (KMnO4), magnesium chlo-
ride hexahydrate (MgCl2
6H2O), zinc chloride hexahydrate
(ZnCl2
6H2O), ethanol (C2H6O), sulfuric acid (H2SO4), phos-
phoric acid (H3PO4), hydrogen peroxide (H2O2), dipotassium
hydrogen phosphate (K2HPO4), hydrochloric acid (HCl),
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
tetrazodium (MTT), streptomycin, and penicillin were pur-
In vitro and in vivo characterization of mineralized hydroxyapatite/polycaprolactone-graphene oxide
chased from Sigma-Aldrich, India. All chemicals were of ana-
lytical grade and used without further purification. Aqueous
solutions were prepared using deionized water from a Milli-Q
purification system.
2.2. Synthesis of graphene oxide (GO)
The GO was synthesized from graphite by using a simplified
Hummers method (Lim et al., 2011). Briefly, 60 mL of con.
H2SO4 and 7 mL H3PO4 were added to a beaker containing
0.5 g of expanded graphite solution. Afterward, 3 g of KMnO4
was slowly added into the mixture solution under continuous
stirring at room temperature. Complete oxidation of the gra-
phite was ensured after 3 days with a constant stirring. The
suspension was cooled and diluted with 200 mL of ice (cold)
water. Then, 30% H2O2 was added until the gas evolution
reaction ceased to remove any KMnO4. Observations of this
reaction include a plethora of bubbles and suspension chang-
ing color from a dark brownish to a yellow color. The suspen-
sion was then purified and diluted with ultrapure deionized
water and centrifugation at 10,000 rpm for 15 min. The precip-
itate was collected and dried for 5 h in hot air-oven to get the
GO powder (Baradaran et al., 2014).
2.3. Specimen preparation
Titanium alloy (Ti-6Al-4V) (purity = 99.9%) were cut into
small pieces of 10  105 mm3 size and inserted in epoxy resin
by leaving an area of 1 cm2 for exposure to the solution. The
specimens were polished and roughened through electrodepo-
sition process by using various grades of silicon carbide paper
(SiC) ranging from 180 to 2000 grit. After polishing, all the
specimens were cleaned by ethanol and acetone. Finally, the
specimens were rinsed with the ultrapure deionized water
and dried in flowing air for future use.
2.4. Preparation of M-HAP electrolyte
To obtain M-HAP, 0.1 M MgCl2
6H2O, 0.1 M ZnCl2
6H2O
and 0.3 M CaCl2
2H2O were dissolved in ultrapure water to
get a mixture solution in the ratio of 8:1:1, respectively. Next,
0.3 M K2HPO4 solution was added drop wise into the reaction
mixture under constant magnetic stirring for 5 h at room
temperature (25 °C) using thermostat to produce the target
(Ca + Mg+Zn) and P ratio of 1:67. Finally, the pH of the
product was adjusted to 4.7 by using either HCl or NH4 OH.
2.5. Preparation of M-HAP/GO electrolyte
To prepare M-HAP/GO electrolyte, GO (1 wt%) powder was
suspended in 2 mL of ultrapure distilled water. Then, the
solution was sonicated for 15 min to prepare a stable aqueous
dispersion. After that, the GO dispersion was added into the
aqueous solution of M-HAP with vigorous stirring at 25 °C.
The pH of the electrolyte was maintained at 4.7.
2.6. Preparation of M-HAP/PCL/GO electrolyte
For the preparation of M-HAP/PCL/GO electrolyte, the
different concentrations of PCL (5, 10 and 15 wt%) were
961
dissolved in chloroform under vigorous stirring for 5 h to get
a clear solution. Then, it was added drop wise into the
M-HAP/GO electrolyte under magnetic stirring for 5 h at
25 °C. The pH of the electrolyte was also maintained at 4.7
and used for the electrodeposition process.
2.7. Electrodeposition of M-HAP/GO and M-HAP/PCL/GO
composite on Ti alloy
The electrodeposition of M-HAP/GO and M-HAP/PCL/GO
composite coating on Ti alloys at various concentrations were
performed by using three-electrode system with an electro-
chemical workstation (Model: CHI 760C). The platinum (Pt)
disk, Ti alloys (the specimen) and saturated calomel electrodes
(SCE) were used as the counter, working and reference elec-
trode respectively. The electrodeposition was performed at
1.5 V for duration of 30 min at room temperature as shown
in Scheme 1. After the deposition, the coated samples were
rinsed with ultrapure water and dried under air for future use.
2.8. Mechanical characterization
The adhesion strength of the M-HAP/GO and M-HAP/PCL/
GO (with different weight percentage of PCL) composite film
coatings adhered to Ti alloy was determined by the pull-out
test. Using the international standard machine (Instron 5565)
and the ASTM F 1044-05 standard, a minimum of five inden-
tations were made on each sample to measure and the average
value was recorded. All the coated specimens were kept in oven
at 100 °C for about 60 min prior to testing and the fixtures
were subjected to the pull-out test at a crosshead speed of
1 mm/min.
The microhardness (Hv) of M-HAP, M-HAP/PCL and
M-HAP/PCL/GO composite materials was performed by
Vickers micro-hardness tester system (Akashi AAV-500
series). The applied load was 0.49 N with an indentation time
of 10 s, five replicates in each group was performed per sample
and the average microhardness strength was recorded.
Scheme 1 Schematic representation of electrolyte preparation
and electrodeposition of M-HAP/PCL/GO composite coating on
Ti alloy.
962
2.9. Characterization methods
Fourier-transform Infrared spectrophotometer (FT-IR;
NICOLET380) was used to analyze the functional groups pre-
sent on the composite materials using the KBr pellet technique.
The crystalline structures and phase compositions of the M-
HAP/PCL/GO composite was carried out by X-ray diffraction
spectroscopy (XRD-6000, Shimadzu, Japan). The structural
morphologies of the composites and coated films were assessed
by using field-emission scanning electron microscope (FE-
SEM 1430 VPLEO, UK) at an acceleration voltage of 15 kV.
2.10. Assessment of antimicrobial activity
The two experimental bacterial strains were obtained and then
maintained at the Department of Microbiology, University of
Madras, Chennai, India. The antimicrobial activities of M-
HAP, M-HAP/GO and M-HAP/PCL/GO composites were
tested with two bacterial strains: Escherichia coli and Staphylo-
coccus aureus by agar disc diffusion method. For the disc dif-
fusion technique, nutrient agar was poured into petriplates and
allowed to solidify. After solidification, the overnight incu-
bated bacterial culture was brought out and introduced to
the agar by using an inoculation loop. After inoculation, the
bacterial containing petriplates were covered and kept in an
incubator at 37 °C for 24 h. After incubation, the M-HAP,
M-HAP/GO and M-HAP/PCL/GO composites with different
concentration (25 mL, 50 mL, 75 mL, 100 mL and 125 mL) discs
of filter paper were dipped in the solutions and then placed
onto the bacteria culture. Again, the plates were kept over-
night in incubator at 37 °C for 24 h. The zone of inhibition
was quantified using a ruler and expressed in millimeters
(mm) around the disc. Digital images were also captured to
assist in the evaluation of the observable antibacterial activity
of the composites. This procedure was followed in triplicate
and results recorded.
2.11. Cell viability and cell proliferation assay
The study determines the cell viability and proliferation effects
of M-HAP, M-HAP/PCL and M-HAP/PCL/GO composites
on osteoblast cells. Human osteoblast cells (MG63) were
obtained from National Centre for Cell Sciences (NCCS),
Pune, India. The MG63 cells were maintained in Dulbecco’s
Modified Eagle Medium (DMEM) media supplemented with
1% L-glutamine, 10% fetal bovine serum (FBS), 1% strepto-
mycin and 1% penicillin at 37 °C in 5% CO2 humidified incu-
bator. Culture Media was changed every 2 days and the cells
were subcultured by trypsinization. The cell viability study
was performed with a MTT assay (Murugan et al., 2017). In
brief, MG63 osteoblast cells were added to a 96-well plate
(1  105 cells/each well) in 90% DMEM medium for a fixed
time at 37 °C (5% CO2). After being incubated for 24 h,
M-HAP, M-HAP/PCL and M-HAP/PCL/GO (10 mg) were
added and incubated for 1 day, 3 days and 5 days, respectively.
Then, 3 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte
trazolium bromide (MTT, 100 lL) solution was poured and
permitted to incubate for 3 h. Finally, the produced formazan
was diluted in 200 lL of dimethyl sulfoxide (DMSO) and
viewed using an Enzyme-Linked Immunosorbent Assay
(ELISA) analyzer (SPR-960) at 540 nm. Percentage (%) of cell
N. Murugan et al.
proliferation and cell viability were measured with respect to
control at 1 day, 3 days and 5 days. All experiment was per-
formed in triplicates for each set of samples. The cell viability
was calculated by the following formula;
ð%Þ Cell viability ¼ Test=Control  100
2.12. In vivo study and surgical procedure
To evaluate the tissue and cell behavior effects of M-HAP/
PCL and M-HAP/PCL/GO composites coated Ti alloys, the
composites were implanted into the femur bone in Wistar rats
and then observed by histological study. All the animal exper-
imental procedures were approved by the Institutional Animal
Ethical Committee (IAEC) meeting held at Periyar University,
Salem, India (PU/IAEC/085/PO/OC/07/CPCSEA/ZOO
C/02/2016–2019). Six male Wistar rats (weight 250–300 g) were
used for the experimental study. The rats were housed under
normal conditions at 20 °C with a dark cycle (12 h) and light
cycle (12 h). The Wistar rats were randomly divided into two
groups: M-HAP/PCL coated (group I, n = 3), and M-HAP/
PCL/GO (group II, n = 3). All of the Wistar rats were individ-
ually anesthetized by given intravenous injections of xylazine
(1.5 mg/kg) and ketamine (15 mg/kg) and were provided with
a combination of 20% v/v isoflurane and propylene glycol
for inhalation. Afterward, the target location in the femur
region was shaved and wiped with iodine. Then, the muscular
regions were disconnected at implant region over the femur to
expose the periosteum. Next, a 2 mm hole at the femur bone
was made using a drilling machine, using sufficient irrigation
with saline solution to minimize temperature increase in the
bone. Following surgery, the penicillin antibiotic was given
to all the experimental Wistar rats for two post-operative days.
The Wistar rats were placed aside on nurturing during surgical
period (14 and 28 days). After 14 and 28 days of implantation,
the rats were sacrificed and the implant materials were care-
fully gathered for the histological observations.
For histological examinations, the implants with rat bones
were cut into a thickness of 2 mm. The sliced sections were
fixed in 20% formalin and decalcified inside 10% acetic acid
solution for 4 days. Afterward, the decalcified sample sections
were embedded in paraffin and a 70 nm sections were taken by
microtome. Then, the thick sections were stained with Mallory
and hematoxylin with eosin staining solution and observed
under the light microscopy.
2.13. Statistical analysis
All the experimental values were shown the mean ± standard
deviation (SD). One-way analysis of variance (ANOVA) with
p* < 0.05 as the level of significance was utilized for statistical
analysis. All of the experimental groups were performed in
triplicate.
3. Results and discussion
3.1. FT-IR analysis
FT-IR spectra of M-HAP, M-HAP/PCL and M-HAP/PCL/
GO composite coated Ti alloy were measured. As shown in
Fig. 1a, the characteristic peaks at 485 cm1, 560 cm1, 610
In vitro and in vivo characterization of mineralized hydroxyapatite/polycaprolactone-graphene oxide
Fig. 1 FT-IR spectra of (a) M-HAP (b) M-HAP/PCL and (c)
M-HAP/PCL/GO composite coated Ti alloys.
cm1 and 959 cm1 indicated the appearance of PO3 4 groups
in M-HAP coating; additionally, the absorption bands at 3485
cm1 point out the stretching vibration of hydroxyl (OH)
groups. All the M-HAP peaks showed a slight shift when com-
pared with the peaks obtained for HAP (Murugan et al., 2015).
As shown in Fig. 1b, the spectra of M-HAP/PCL, the strong
absorption band located at 3480 cm1 is due to the presence
of OH group, whereas, the signal peaks at 484 cm1, 563
cm1, 607 cm1, 957 cm1 and 1094 cm1 clearly confirmed
the presence of PO3
4 groups in M-HAP/PCL. On the other
hand, the major peak observed at 1245 cm1 represented the
asymmetric C-O stretching of carbonyl groups, besides corre-
sponding to amorphous and crystalline nature of PCL
Fig. 2 XRD pattern of (a) M-HAP (b) M-HAP/GO and (c) M-HAP/PCL/GO composite coated Ti alloys.
963
(Rezaei and Mohammadi 2013). The M-HAP/PCL/GO com-
posite coating in the Fig. 1c confirmed the presence of the sig-
nal peak at 3488 cm1 indicating the OHstretching vibrations.
The appearance of additional absorption peaks at 490 cm1,
565 cm1, 604 cm1, 960 cm1 and 1092 cm1 correspond to
the (PO34 ) groups in M-HAP/PCL/GO. The new absorption
peak of carboxyl groups (C‚O) in GO at 1620 cm1 was also
distinct. These findings indicated the successful formation of
M-HAP/PCL/GO composite coating on Ti alloy.
3.1.1. XRD pattern
Fig. 2a–c shows the XRD spectra of M-HAP, M-HAP/GO
and M-HAP/PCL/GO coated films. As shown in Fig. 2a, the
XRD pattern of M-HAP exhibited the diffraction peaks (2h)
at 28.8°, 29.8°, 30.4°, 31.1°, 34.5°, 41.0° and 53.4° respectively
which could be assigned to the M-HAP (ICDD card no. 09-
0432) (Uskoković et al., 2017). The XRD peaks confirmed
the formation of M-HAP and comparable with the JCPDS
card, 09-0432. Subsequently, the XRD spectrum obtained for
M-HAP/GO composite coated Ti alloy (Fig. 2b) showed peaks
at 2h values of 14.9°, 28.1°, 29.5°, 30.3°, 31.3°, 34.7°, 40.9° and
53.2°. The peaks range at 28.1°, 29.5°, 30.3°, 31.3°, 34.7°, 40.9°
and 53.2 were corresponding to crystalline nature of M-HAP.
A peak observed at 14.9° can be assigned to the diffraction of
(0 0 2) planes of graphite (ICDD card no.: 13-0148), which
confirms the incorporation of GO into M-HAP. The XRD
pattern of M-HAP/PCL/GO composites was further investi-
gated. In Fig. 2c, the peaks were identified at 2h values of
14.8°, 20.1°, 23.3° 28.3°, 29.6, 30.1, 31.2°, 34.6°, 41.1° and
53.6°. The defined peak signals at 2h values of 28.3°, 29.6,
30.1, 31.2°, 34.6°, 41.1° and 53.6°, correspond to M-HAP.
The peaks at 14.8°, 20.1° and 23.3° represented GO/PCL
(PCL (10 wt%) in GO (1 wt%). Since, PCL is a semi-
crystalline polymer: the XRD peaks for PCL can be recognized
at 2h = 20.1° and 23.3° which can be represented to (1 1 0) and
(2 0 0), respectively (ICDD card no. 74-566). It is noteworthy
964 N. Murugan et al.

that the peak range becomes slightly broader than the peak sig- 3.2. Mechanical characterization of the coatings
nals observed for HAP, which indicates that the M-HAP/PCL/
GO composite had a low crystalline nature. The XRD data 3.2.1. Adhesion strength
confirmed the incorporation of PCL with M-HAP/GO coat- The mechanical strength of the composite coating on the
ing. In addition, the peaks point out the formation of the M- implants is one of the most significant properties for orthope-
HAP/PCL/GO composite on the Ti alloy. dic applications. Here, the adhesion strength of the M-HAP/
PCL and different concentrations of PCL in GO reinforced
3.1.2. Surface morphology analysis M-HAP/PCL composite on Ti alloy was evaluated. Fig. 4
FE-SEM images show the surface morphologies of M-HAP/ shows the adhesion strength for M-HAP/PCL (PCL 10 wt
GO and M-HAP/PCL/GO composite coating obtained with %), M-HAP/PCL/GO (PCL 5 wt%, GO 1 wt%), M-HAP/
various concentration of PCL (5 wt%, 10 wt% and 15 wt%). PCL/GO (PCL 10 wt%, GO 1 wt%) and M-HAP/PCL/GO
The morphology of M-HAP/GO (GO 1 wt%) appeared flake (PCL 15 wt%, GO 1 wt%) coatings were (21.3 ± 0.4 MPa),
like structure with uneven pores in between them (Fig. 3a).
As shown in Fig. 3b, the morphological feature of M-HAP/
PCL/GO (PCL 5 wt%) composite coated Ti alloy exhibits
the well-defined flake like layered film around micro-
structure arrangement with few irregular pores in between
them. Similarly, the morphology of the M-HAP/PCL/GO
composite coated Ti alloy surface (Fig. 3c) showed obvious
evidence that PCL (10 wt%) uniformly covered on GO and
mineralized HAP with porous-like structure. The GO allows
the surface to have the adequate hydrophilicity and numerous
functional groups as nucleation sites. With the higher concen-
tration of PCL up to 15 wt%, this porous-like structure exter-
nally forming on the surface paves the way for the detachment
of the composite coating from the Ti alloy surface (Fig. 3d).
The surface morphological evaluation of the M-HAP/PCL/
GO (10 wt% of PCL) composite coating displayed an entire
surface coverage and uniform arrangement. It was evident that
the porous M-HAP/PCL/GO structure can offer a consider-
able effectiveness in the mechanical strength of the coated sub-
strate. The minerals substituted HAP and GO could be
deposited evenly with the PCL molecules along the surface.
Moreover, the interconnected network like porous structure Fig. 4 Adhesion strength of M-HAP/PCL and M-HAP/PCL/
seemed to be sufficient for orthopedic applications. GO (with different wt.% of PCL) composite coatings on Ti alloys.

Fig. 3 FE-SEM images of (a) M-HAP/GO composite coatings on Ti alloy and M-HAP/PCL/GO composite at different concentrations
of PCL (b) 5 wt%, (c) 10 wt% and (d) 15 wt% composite coatings on Ti alloys.
In vitro and in vivo characterization of mineralized hydroxyapatite/polycaprolactone-graphene oxide
Fig. 5 Micro-hardness of M-HAP, M-HAP/PCL and M-HAP/
PCL/GO composite coatings on Ti alloys.
(27.5 ± 0.5 MPa), (29.6 ± 0.4 MPa) and (26.8 ± 0.6 MPa),
respectively. These results indicated that GO could effectively
increase the binding strength of the coatings. The high adhe-
sion strength of coated M-HAP/PCL/GO (PCL 10 wt%, GO
1 wt%) (29.6 ± 0.4 MPa) composite can be considered a suit-
able biomaterial for high load bearing applications. These
results conclusively proved that even low concentrations of
GO may improve the mechanical property of M-HAP/PCL.
3.2.2. Vickers micro-hardness
The mechanical properties of the composite coated samples
were examined by micro-hardness tester. The micro-hardness
(Hv) values for the M-HAP, M-HAP/PCL (PCL 10 wt%)
and M-HAP/PCL/GO (PCL 10 wt%) composite coatings are
shown in Fig. 5. The micro-hardness value of M-HAP, M-
HAP/PCL and M-HAP/PCL/GO material was found to be
(315 ± 4 Hv), (370 ± 3 Hv) and (455 ± 5 Hv), respectively.
The M-HAP/PCL/GO composite had higher micro-hardness
(Hv) value compared to M-HAP and M-HAP/PCL composite.
This may be due to the presence GO which comprises epoxide
and hydroxyl groups on the basal plane as well as carboxyl and
carbonyl moieties on its edges. These functional groups
favored the interfacial bonding between HAP and polymeric
matrices. It proved that GO could improve the mechanical
property of M-HAP/PCL composite coating (Peng et al.,
2017). The M-HAP/PCL/GO composite may be employed
for biomedical applications. The overall synthetic process of
M-HAP/PCL/GO composite is shown in Fig. 6.
3.3. Biological studies
3.3.1. Antimicrobial test
To investigate the antibacterial activity of composite, varying
doses (25 mL, 50 mL, 75 mL, 100 mL and 125 mL) of control,
M-HAP, M-HAP/GO (1 wt% GO) and M-HAP/PCL/GO
(10 wt% PCL) composite were tested against both Escherichia
965
Fig. 6 The schematic illustration for the formation mechanism
of M-HAP/PCL/GO composite.
coli (Gram-negative) and Staphylococcus aureus (Gram-
positive) bacteria the dominant bacterial causative agents for
postsurgical infections associated with the implants. In Fig. 7
(a and b), the graphs show the inhibition zone of different
composite treatment. Among these, M-HAP/PCL/GO com-
posite treated sample induced higher antibacterial activity.
The antibacterial activity with the agar disc diffusion method
(at 37 °C for 24 h) can be seen in Fig. 8, which shows the inhi-
bition zones for the M-HAP/PCL/GO composite coated sam-
ple with both E. coli (lower panel) and S. aureus (upper panel)
at different doses (25 lL, 50 lL, 75 lL, and 100 lL). It was
noted from the images that for M-HAP/PCL/GO composite
coatings, the inhibitory zone on E. coli strain was 10.5, 12,
14.5, 16.5 and 20.5 mm and S. aureus strain was 10, 11, 14
and 16 mm respectively. At the final concentration (100 lL),
the M-HAP/PCL/GO composite coated sample revealed the
superior rate of antimicrobial activity when compared with
other doses. In addition, the antibacterial activity of the M-
HAP/PCL/GO composite coating against E. coli was slightly
higher than S. aureus bacteria. This is likely due to variation
in the structure of the bacteria’s’ cell wall. The gram positive
S. aureus cell wall had thickness ranges from 20 to 80 nm
owing to the presence of double layer of peptidoglycan and
the cell wall of gram negative E. coli strain had a reduced
thickness ranges from 1.5 to 10 nm because of single layer pep-
tidoglycan (Kaviya et al., 2011). The earlier studies suggests
that Mg2+ and Zn2+ may have contributed in targeted killing
of S. aureus including strains that are antibiotic-resistant
966
Fig. 7 Antibacterial activity of control, M-HAP, M-HAP/GO
and 10 wt% PCL/M-HAP/GO composite coatings on Ti alloy
samples with different concentrations of 25 mL, 50 mL, 75 mL, 100
mL and 125 mL against S. aureus (a) and E. coli (b).
Fig. 8 Photographic images for the zone of inhibition of 10 wt%
PCL/M-HAP/GO composite coating against S. aureus and E. coli.
(Xie and Yang 2016). The results of this test confirmed that
these ions improve the antibacterial effect and furthermore,
that the M-HAP/PCL/GO composite has a greater level of
antibacterial activity against both E. coli and S. aureus at
37 °C for 24 h.
N. Murugan et al.
3.3.2. Cell proliferation
In order to assess the cell proliferation or cell viability of
human MG63 osteoblast cells in conjunction with M-HAP,
M-HAP/PCL (10 wt% PCL) and M-HAP/PCL/GO (10 wt%
PCL) composites, samples were quantitatively evaluated by
MTT assay. As shown in Fig. 9, the nontoxic features of all
M-HAP, M-HAP/PCL and M-HAP/PCL/GO composite
coatings were revealed. The cell proliferative effects of M-
HAP, M-HAP/PCL and M-HAP/PCL/GO composites were
tested for 24 hr. at the intervals of 1 day, 3 days and 5 days.
The MG63 osteoblast cells cultured on the M-HAP/PCL/GO
composite (10 mg/mL) coated sample displayed outstanding
viability after 3 and 5 days of incubation at pH 7.4, which indi-
cates the dramatic increase in cell viability. These comparative
results confirmed that M-HAP/PCL/GO composite coated Ti
alloy would be a good candidate material for additional
research in orthopedic applications such as bone implant scaf-
folding. With the success of the MG63 cell proliferation at the
5 days of incubation. The M-HAP/PCL/GO was deemed a
suitable material for the in vitro biocompatibility study.
3.3.3. In vivo biocompatibility study of the composite coating
To evaluate the in vivo biocompatibility and new bone forma-
tion nature of the M-HAP/PCL (PCL 10 wt%) and M-HAP/
PCL/GO (PCL 10 wt%) composite coated Ti alloy implant
was made and used on male Wistar rats using the hematoxylin
and eosin (H&E) staining histological study. The M-HAP/
PCL and M-HAP/PCL/GO composite coated Ti alloy was
implanted into the surgical site for 14 and 28 days. After the
insertion, the surgical places were healed in both M-HAP/
PCL and M-HAP/PCL/GO composites. After the period of
14 & 28 days, the implants were successfully removed from
the bone of experimental Wistar rats using forceps. To study
the interaction between the implant and surrounding tissue after
14 and 28 days, both the M-HAP/PCL composite and the M-
HAP/PCL/GO composite coated Ti alloy were assessed. From
Fig. 10(a and b), the formation of connective fibrous tissue
Fig. 9 Bar diagrams showing the% cell viability of HOS MG63
cells with M-HAP, M-HAP/PCL and 10 wt% PCL/M-HAP/GO
composite coatings on Ti alloy for 1, 3 and 5 days of incubation.
In vitro and in vivo characterization of mineralized hydroxyapatite/polycaprolactone-graphene oxide
Fig. 10 Histological analysis of in vivo bone formation ability for M-HAP/PCL (10 wt% PCL) composite coated Ti alloy group (I) (a, b)
and M-HAP/PCL/GO (PCL 10 wt%) composite coated Ti alloy group (II) (c, d) at 14 and 28 days of implantation.
encompassing fibroblastic cells along with the rim around the
implants was found significantly induce newly formed spongy
bone (trabecular bone) at 14 days and 28 days in M-HAP/
PCL. In addition, the Wistar rats implant with M-HAP/PCL/
GO composite coated (Group II) showed no obvious clinical
infection at 14 days and 28 days (Fig. 10c and d). Hence, these
results implied that the Histological observation showed no
occurrence of infection in the M-HAP/PCL/GO composite
coated Ti alloy implant samples. The group II implants exhib-
ited the most constructive healing response by the presence of
newly formed spongy bone and no presence of inflammation
was observed. It was found that there was new bone formation
after 28 days post-operation, which suggests that the area of
newly formed bone gradually increased between 14 and 28 days.
Besides, the ingrowths of abundant osteoblast like cells detected
around the implants which suggest that M-HAP/PCL/GO com-
posite coated materials offer the scaffolding or the development
of new bone and tissue response. The findings also implied that
GO not only possess in vitro osteogenesis enhancing ability, but
also excellent in vivo bone-forming ability (Peng et al., 2017).
The results displayed no unfavorable local effects such as
marked hematoma or edema with the composites up to 28 days
postoperatively. These results further support that the M-HAP/
PCL/GO composite coated Ti alloy has positive outcomes in the
orthopedic application as a better bone regenerator in bone
replacement material.
4. Conclusions
In summary, we reported a novel porous implant material for
orthopedic applications. The biomaterial composite coating
967
was made by 1 wt% of GO and mineral substituted HAP with
different concentrations (5, 10 and 15 wt%) of PCL. These
composite coatings showed considerable improvement in the
morphological, mechanical and biological properties of the
metallic implant. The FT-IR, XRD and FE-SEM confirmed
that the successful coating of M-HAP/PCL/GO composite
material on Ti alloy. The addition of GO in the PCL/M-
HAP matrix also had its benefits. With the addition of GO,
the adhesion strength and the hardness for the composite
increased. The cell viability and proliferation activity of M-
HAP/PCL/GO on MG63 osteoblast cells was also evaluated
by MTT assay. The obtained findings revealed M-HAP/
PCL/GO had a superior cell viability rate in contrast to all
the other coated materials. Furthermore, the in vitro and
in vivo studies confirmed the developed M-HAP/PCL/GO
composite could establish the direct bonds with bone tissue
after implantation. Therefore, these results support that the
M-HAP/PCL/GO composite had good biocompatible,
mechanical and bactericidal properties and hence, is a favor-
able implant material for future orthopedic and dental
applications.
Acknowledgements
Dr. AKS acknowledges the major financial support from the
DST-SERB, New Delhi, India (Ref. No.: ECR/2016/001446).
Dr. NM acknowledges the postdoctoral fellowship from
SRM Research Institute, SRM Institute of Science and Tech-
nology. Additional thanks for technical writing support to
Kari Jordan, Department of Biological Systems Engineering,
University of Wisconsin-Madison, Madison, Wisconsin, USA.
968
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