polymers

Article
Polyetheretherketone Hybrid Composites with
Bioactive Nanohydroxyapatite and Multiwalled
Carbon Nanotube Fillers
Chen Liu 1 , Kai Wang Chan 1 , Jie Shen 2 , Cheng Zhu Liao 3 , Kelvin Wai Kwok Yeung 2
and Sie Chin Tjong 1, *
1 Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon,
Hong Kong, China; cliu266-c@[Link] (C.L.); kaiwchan8-c@[Link] (K.W.C.)
2 Department of Orthopedics and Traumatology, Li Ka Shing Faculty of Medicine, The University of Hong Kong,
Hong Kong, China; jayjayson909@[Link] (J.S.); wkkyeung@[Link] (K.W.K.Y.)
3 Department of Materials Science and Engineering, South University of Science and Technology of China,
Shenzhen 518055, China; liaocz@[Link]
* Correspondence: aptjong@[Link]; Tel.: +86-852-3442-7702

Academic Editor: Patrick van Rijn

Received: 25 October 2016; Accepted: 2 December 2016; Published: 8 December 2016

Abstract: Polyetheretherketone (PEEK) hybrid composites reinforced with inorganic nanohydroxyapatite

(nHA) and multiwalled carbon nanotube (MWNT) were prepared by melt-compounding and injection
molding processes. The additions of nHA and MWNT to PEEK were aimed to increase its elastic
modulus, tensile strength, and biocompatibility, rendering the hybrids suitable for load-bearing
implant applications. The structural behavior, mechanical property, wettability, osteoblastic cell
adhesion, proliferation, differentiation, and mineralization of the PEEK/nHA-MWNT hybrids were
studied. X-ray diffraction and SEM observation showed that both nHA and MWNT fillers are
incorporated into the polymer matrix of PEEK-based hybrids. Tensile tests indicated that the elastic
modulus of PEEK can be increased from 3.87 to 7.13 GPa by adding 15 vol % nHA and 1.88 vol %
MWNT fillers. The tensile strength and elongation at break of the PEEK/(15% nHA)-(1.88% MWNT)
hybrid were 64.48 MPa and 1.74%, respectively. Thus the tensile properties of this hybrid were
superior to those of human cortical bones. Water contact angle measurements revealed that the
PEEK/(15% nHA)-(1.88% MWNT) hybrid is hydrophilic due to the presence of nHA. Accordingly,
hydrophilic PEEK/(15% nHA)-(1.88% MWNT) hybrid promoted the adhesion, proliferation,
differentiation, and mineralization of murine MC3T3-E1 osteoblasts on its surface effectively on
the basis of cell culture, fluorescence microscopy, MTT assay, WST-1 assay, alkaline phosphatase
activity, and Alizarin red staining tests. Thus the PEEK/(15% nHA)-(1.88% MWNT) hybrid has the
potential to be used for fabricating load-bearing bone implants.

Keywords: polyetheretherketone; nanohydroxyapatite; multiwalled carbon nanotube; hybrid

nanocomposites; hydrophilic surface; osteoblasts; biocompatibility

1. Introduction
Nowadays, there is a substantial increase in the demand of bone implants due to an increase in
the numbers of patients suffering from traffic injury, aging, and bone disease [1]. The technological
development of biomaterials with excellent biocompatibility for bone replacements is considered of
critical importance. Metallic implants such as titanium alloy, CoCrMo alloy, and 316L stainless steel
have been widely used for fabricating load-bearing hip implants and bone fixation devices due to their
superior mechanical strength and ductility. However, metallic implants suffer from corrosion due to
the release of metal ions by interacting with body fluid containing 0.9% NaCl. Some ions (e.g., Ni2+ ,

Polymers 2016, 8, 425; doi:10.3390/polym8120425 [Link]/journal/polymers

Polymers 2016, 8, 425 2 of 19

Cr3+ and Co2+ ) can induce allergy, inflammation and cytotoxicity [2–4]. In addition, chloride anions can
attack passive films formed on metallic surfaces, leading to crevice and pitting corrosion [5,6]. Metallic
implants also experience a stress shielding effect due to a mismatch in stiffness between the implants
and host bone, resulting in a loss of bone density. In this respect, ceramics and polymers have been
investigated as possible bone substitute materials. In particular, hydroxyapatite [Ca10 (PO4 )6 (OH)2 ]
is of particular interest due to its resemblance to the mineral component of human bones. However,
hydroxyapatite (HA) cannot be used for load-bearing implants due to its brittle nature. Thus inorganic
HA in the form of particulates with sizes of several micrometers (mHA) are employed as reinforcing
fillers for high-density polyethylene (HDPE) [7]. The inclusion of bioactive mHA fillers (40 vol %)
renders HDPE with excellent bioactivity and biocompatibility.
Polymers with good processability and light weight exhibit beneficial effects for biomedical
applications [8,9]. Degradable polymers with low mechanical strength such as polylactic acid,
polyglycolic acid, and their blends are typically used for bone tissue engineering applications [10,11].
By contrast, non-degradable polymer such as polyetheretherketone (PEEK) with high temperature
durability, excellent radiation stability and high Young’s modulus has found clinical applications
as trauma fixation devices and spinal cages [12,13]. However, PEEK is bio-inert and hydrophobic,
and cannot osteo-integrate with adjacent bone tissues upon implantation [14]. The bioactivity of PEEK
can be greatly enhanced either by depositing HA coating on its surface [15], or by incorporating inorganic
HA whiskers [16] and mHA particulates into its matrix [17]. In the latter case, Abu Bakar et al. added
large mHA particulates up to 40 vol % into PEEK through melt compounding and injection-molding.
They found that the tensile modulus of PEEK/mHA composites rises with increasing mHA content.
However, the tensile strength of the composites decreases markedly as the mHA content increases.
This implies that the tensile strength of PEEK/mHA composites is lower than that of pure PEEK.
This is due to poor interfacial bonding between the fillers and PEEK. Further, large mHA particulates
often serve as stress concentrators, thereby fracturing into small fragments during tensile testing.
Recent progress in nanotechnology research has led to the development and synthesis of novel
nanomaterials with unique properties. Nanomaterials generally possess higher mechanical strength
and biocompatibility compared to their micron-sized counterparts. Nanohydroxyapatite (nHA) can be
synthesized by solid state reaction, co-precipitation, hydrothermal method, sol-gel process, etc. [18].
Synthetic nHA enhances protein adsorption, thereby promoting osteoblast adhesion, proliferation,
and differentiation [19]. Bone tissue is a biocomposite consisting of a collagen matrix and nHA. Human
cortical bone possesses an elastic modulus in the range of 7–30 GPa, tensile strength of 50–150 MPa
and fracture elongation of 1%–3% [20]. Therefore, polymer/nHA composites have been investigated
for making load-bearing hip prostheses and bone tissue scaffolds [21–26]. The additions of nHA to
polypropylene (PP) of low tensile stiffness and strength improve its tensile performance moderately.
The elastic modulus of PP/nHA nanocomposites is still much lower than that of cortical bone [21].
In this respect, PEEK with higher tensile stiffness and strength than PP is a potential matrix material of
biocomposites for forming load-bearing bone implants.
Ma et al. prepared PEEK/nHA nanocomposites through vibrating ball-mill mixing followed
by injection molding [27]. They found that that the elastic modulus of PEEK reaches a maximum
value of 4.6 GPa by adding 40 wt % (21.5 vol %) nHA. However, the modulus of this nanocomposite
is much smaller than the lower limit for the modulus of cortical bone, i.e., 7 GPa. Very recently,
we incorporated 4.4–21.5 vol % nHA rods to PEEK for enhancing its mechanical stiffness and
biocompatibility [23]. The results showed that the tensile modulus of PEEK-based composites
increases with increasing nHA content. It increases from 3.86 GPa (pure PEEK) to a maximum value
of 7.85 GPa by adding 21.5 vol % (40 wt %) nHA. The elastic modulus of PEEK/(21.5 vol % nHA)
nanocomposite reaches 7.85 GPa, being slightly higher than the lower bound in elastic modulus
of cortical bone. However, PEEK/(21.5 vol % nHA) nanocomposite is very brittle having a fracture
elongation of only 0.63%. These results reveal that the PEEK/(21.5 vol % nHA) nanocomposite is
unsuitable for making load-bearing implants due to its brittleness. To address this issue, carbon
Polymers 2016, 8, 425 3 of 19

nanofibers (CNFs) of 1.6–1.9 vol % are added to the PEEK/nHA composites for enhancing their
stiffness.
PolymersSo PEEK/(15
2016, 8, 425 vol % nHA)-(1.9 vol % CNF) biocomposite achieves a maximum elastic
3 of 18
modulus of 6.54 GPa, being smaller than that of cortical bone.
CNF)
As biocomposite
recognized, achieves
carbon a maximum
nanotubes elasticextraordinary
possess modulus of 6.54 GPa,tensile
high being smaller
modulusthan thatstrength.
and of
cortical bone.
Multiwalled carbon nanotubes (MWNTs) possess an elastic modulus of 0.9 TPa and tensile strength of
As recognized, carbon nanotubes possess extraordinary high tensile modulus and strength.
150 GPa [28]. Thus MWNTs are effective nanofillers for biocomposites as they promote the adhesion
Multiwalled carbon nanotubes (MWNTs) possess an elastic modulus of 0.9 TPa and tensile strength of
and proliferation of osteoblasts on their surfaces [29,30]. By incorporating into polymers, MWNTs
150 GPa [28]. Thus MWNTs are effective nanofillers for biocomposites as they promote the adhesion
enhance
and the mechanical
proliferation property on
of osteoblasts and biocompatibility
their surfaces [29,30]. of
Byresulting composites.
incorporating ThusMWNTs
into polymers, hybridizing
inorganic nHA with MWNTs can effectively generate PEEK-based nanocomposites with
enhance the mechanical property and biocompatibility of resulting composites. Thus hybridizing good tensile
stiffness and strength as well as excellent biocompatibility for biomedical load-bearing
inorganic nHA with MWNTs can effectively generate PEEK-based nanocomposites with good tensile applications.
stiffness and strength as well as excellent biocompatibility for biomedical load-bearing applications.
2. Experimental Section
2. Experimental Section
2.1. Materials
2.1. Materials
PEEK-Optima (molecular weight 20,800 g/mol) pellets and MWNTs were bought from Invibio
CompanyPEEK-Optima (molecular&weight
and Nanostructured 20,800 g/mol)
Amorphous pelletsInc.
Materials and(Los
MWNTs wereNM,
Alamos, bought fromrespectively.
USA), Invibio
Company and Nanostructured & Amorphous Materials Inc. (Los Alamos, NM, USA),
Nanohydroxyapatite (nHA) powders were purchased from Nanjing Emperor Nano Materials (Nanjing, respectively.
Nanohydroxyapatite (nHA) powders were purchased from Nanjing Emperor Nano Materials (Nanjing,
China). Transmission electron microscopy (TEM; Philips CM20, Philips, Amsterdam, The Netherlands)
China). Transmission electron microscopy (TEM; Philips CM20, Philips, Amsterdam, The
micrograph revealed nHA exhibiting a width of 20 nm and a length of 100 nm (Figure 1). PEEK pellets
Netherlands) micrograph revealed nHA exhibiting a width of 20 nm and a length of 100 nm (Figure
and nHA were
1). PEEK driedand
pellets in an
nHAoven 55 ◦ Cinovernight
wereatdried an oven at prior to melt-compounding.
55 °C overnight prior to melt-compounding.

Figure 1. Transmission electron microscopy (TEM) image of nanohydroxyapatite (nHA).

Figure 1. Transmission electron microscopy (TEM) image of nanohydroxyapatite (nHA).
2.2. Preparation of Nanocomposites
2.2. Preparation of Nanocomposites
PEEK/(15 vol % nHA)-MWNT hybrids with chemical compositions as listed in Table 1 were
PEEK/(15
fabricated byvol % nHA)-MWNT
melt-mixing and injection hybrids
molding. Forwith chemicalpure
comparison, compositions
PEEK and binary as listed
PEEK/MWNTin Table 1
nanocomposites were also prepared. The compositions of binary
were fabricated by melt-mixing and injection molding. For comparison, pure PEEK and binary PEEK/MWNT and ternary
PEEK/(15 vol
PEEK/MWNT % nHA)-MWNTwere
nanocomposites nanocomposite
also [Link] thisThe
article were expressed
compositions in volume
of binary percentage. and
PEEK/MWNT
ternary PEEK/(15 vol % nHA)-MWNT nanocomposite in this article were expressed% in
From our previous work [23], elastic modulus and fracture strain of the PEEK/(15 vol nHA)volume
nanocomposite were 6.2 GPa and 2.71%, respectively. Thus the tensile performance of this composite can
percentage. From our previous work [23], elastic modulus and fracture strain of the PEEK/(15 vol % nHA)
be further improved by adding MWNTs. Accordingly, the nHA content of hybrid nanocomposites in
nanocomposite were 6.2 GPa and 2.71%, respectively. Thus the tensile performance of this composite
this study was fixed at 15 vol %.
can be further
In theimproved
melt-mixing by process,
adding MWNTs.
dried PEEK Accordingly, the nHA
pellets and nHA werecontent
initiallyofmixed
hybrid
in nanocomposites
an extruder
in this study
(C.W. was fixed
Brabender at 15 vol %.
Instruments, South Hackensack, NJ, USA) at a screw rotation speed of 30 rpm for 45
In the
min. Themelt-mixing process,ofdried
mixing temperatures PEEK
Brabender pellets
from hopper and nHA were
to extrusion initially
die were mixed at
maintained in360–380–
an extruder
(C.W.390–395–380–360
Brabender Instruments, South
°C, respectively. Hackensack,
The extrudates wereNJ, granulized
USA) at aby screw rotation
a pelletizer andspeed
loadedofinto30 rpm
for 45Brabender
min. Theagainmixingfor temperatures
second mixingofunder the same
Brabender fromconditions.
hopper toThe purposedie
extrusion waswereto achieve
maintained
homogeneous dispersion of ◦nanofillers
at 360–380–390–395–380–360 in the polymer
C, respectively. matrix. The
The extrudates extruded
were productsby
granulized were pelletized and
a pelletizer
Polymers 2016, 8, 425 4 of 19

loaded into Brabender again for second mixing under the same conditions. The purpose was to
achieve homogeneous dispersion of nanofillers in the polymer matrix. The extruded products were
pelletized again, dried overnight in an oven, and finally fed into an injection molder (Toyo TI-50H,
Toyo Machinery & Metal Co., Akashi, Japan) to make dog-bone tensile bars and circular disks. The disks
were mainly used for cell culture, cell viability, and alkaline phosphatase activity measurements.

Table 1. The compositions of binary PEEK/MWNT and ternary PEEK/(15 vol % nHA)-MWNT
nanocomposites.

MWNT nHA
Sample
vol % wt % vol % wt %
PEEK 0 0 0 0
PEEK/(0.93% MWNT) 0.93 1.5 0 0
PEEK/(1.88% MWNT) 1.88 3.0 0 0
PEEK/(15% nHA)-(0.93% MWNT) 0.93 1.5 15 30
PEEK/(15% nHA)-(1.88% MWNT) 1.88 3.0 15 30

2.3. Material Characterization

X-ray diffraction (XRD) patterns of the composite specimens were obtained using a Bruker D2
Phaser X-ray diffractometer (Karlsruhe, Germany) equipped with Cu-Kα radiation (λ = 0.154 nm) at
30 kV. The patterns were recorded from 10◦ to 60◦ . The morphologies of injection molded composite
specimens were examined in a field-emission SEM (Jeol JSM 7100F; Tokyo, Japan). The composites
were dipped in liquid nitrogen and then fractured by a hammer. The fractured surfaces were then
coated with a thin carbon film.
Differential scanning calorimetry (DSC) tests were determined with a TA Instruments model 2910
(TA Instruments, New Castle, DE, USA) under a protective nitrogen atmosphere. The previous thermal
history of the specimens was removed by an initial heating cycle of 20 ◦ C/min from ambient to
350 ◦ C, followed by holding for 3 min at this temperature. The specimens were then cooled at a rate of
20 ◦ C/min and the cooling traces were recorded accordingly.

2.4. Tensile Tests

Tensile tests were performed at room temperature using an Instron tester (model 5567,
Instron Corp., Norwood, MA, USA) at a crosshead speed of 10 mm/min. The elastic modulus of PEEK
and its nanocomposites was determined from the linear region of stress-strain curves. Five samples of
each composition were tested, and the average values were reported.

2.5. Water Contact Angle Tests

Water contact angles of nanocomposite specimens were measured using Rame Hart 500-F1
advanced goniometer equipped with DROP image software (Rame-Hart Instrument Co., Succasunna,
NJ, USA). Each test run was performed by producing a water droplet and its subsequent spreading
into a spherical cap on the specimen surface.

2.6. Cell Culture

Murine MC3T3-E1 pre-osteoblasts were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM,
Thermo Scientific, Pittsburgh, PA, USA) supplemented with 10% fetal bovine serum, penicillin,
and streptomycin. Injection molded disks were sliced into small round shapes for cell cultivation and
proliferation measurements. These samples were ground with SiC papers of different grades, followed
by rinsing with 70% ethanol and phosphate buffer saline (PBS) solutions. Rinsed samples were placed
in a 96-well plate. Afterwards, a cell suspension was pipetted at 104 cells per well. The 96-well plate
was incubated in a humidified atmosphere of 5% CO2 /95% air at 37 ◦ C for desired periods. The culture
Polymers 2016, 8, 425 5 of 19

medium was changed every two days. Following the incubation, the samples were washed with
phosphate-buffered saline (PBS) to remove unattached cells, fixed with 10% formaldehyde, dehydrated
in a graded series of ethanol. Dehydrated cells were critical point dried and sputter deposited with
gold in preparation for SEM examination. Moreover, micrographs of osteoblasts cultured on the
samples were taken at day 1 and day 3 using a fluorescence microscope. Cells were washed with PBS
and fixed with methanol-free formaldehyde for 30 min at each time point. After that, the samples were
washed with PBS and stained with Alexa Fluor 488 (A12379, ThermoFisher Scientific, Carlsbad, CA,
USA) at room temperature for 40 min to label F-actin filaments, and stained the nuclei with Hoechst
33342 (Sigma Aldrich, St. Louis, MO, USA) for 5 min. The samples were then washed three times and
stored in PBS before imaging.

2.7. Cell Metabolic Activity

The cell metabolic activity was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1)
assays. Rinsed samples (number of samples, n = 5) were placed in a 96-well plate. A suspension
of 104 cells per well was seeded into the 96-well plate. The plate was incubated in a humidified
atmosphere of 5% carbon dioxide in air at 37 ◦ C for 3, 7 and 10 days, respectively. The culture medium
was refreshed every 3 days. At days 3, 7 or 10, the medium was aspirated, then 10 µL MTT solution
(5 mg MTT:1 mL DMEM) was added to each well and incubated for 4 h at 37 ◦ C. Thus MTT reacted
with mitochondrial enzyme of viable osteoblasts, producing insoluble formazan crystals. The formazan
was dissolved in 10% sodium dodecyl sulfate (SDS)/0.01 M hydrochloric acid. The absorbance of
dissolved formazan was measured at a wavelength of 570 nm using a multimode detector (Beckman
Coulter DTX 880, Beckman Coulter Inc., Fullerton, CA, USA), with a reference wavelength of 640 nm.
Those wells with MTT and DMEM were used as a negative control.
For WST-1 assay, the samples (n ≥ 5) were first seeded with osteoblasts at 37 ◦ C for 3, 7 and
10 days, respectively. After cell culturing for every prescribed time period, tetrazolium salt was added
to each well followed by incubation for 4 h at 37 ◦ C to produce water soluble formazan. The amount
of water-soluble formazan was quantified by the absorbance at 450 nm. The cell metabolic activity
of both assays was calculated by comparing the absorbance of osteoblastic cells cultured on the
nanocomposite sample to that of the cells seeded on pure PEEK. The results were expressed in terms
of mean ± standard deviation (SD).

2.8. Cellular Differentiation

The alkaline phosphatase (ALP) activity of osteoblasts on PEEK and its nanocomposites (number
of samples, n = 5) was measured with a colorimetric assay kit (No. 2900-500, Stanbio Laboratory,
Boerne, TX, USA). The kit used colorless 4-nitrophenyl phosphate as a substrate. The enzyme ALP of
the cells hydrolyzed the substrate to color 4-nitrophenol and an inorganic phosphate. In the process,
test samples were placed in each well of a 48-well plate. Osteoblasts were seeded on the samples
for 3, 7 and 14 days. The culture medium was changed every 3 days. At each time period, the cells
were rinsed with PBS, and lysed with 0.1% triton X-100 at 4 ◦ C for 30 min. The cell lysates were
then centrifuged at 4 ◦ C followed by pipetting 10 µL supernatant liquid of each sample in a 96-well
plate. Finally, p-nitrophenyl phosphate was introduced into the plate. The absorbance of p-nitrophenol
formed was measured using a spectrophotometer (Beckman Coulter DTX 880) at 405 nm. The ALP
activity was normalized to the total protein level of the samples determined by the Bio-Rad protein
Assay (Bio-Rad, Hercules, CA, USA).
Calcium mineralization of differentiating osteoblast on PEEK and its nanocomposites was
measured with Alizarin red-S (Sigma Aldrich, Darmstadt, Germany) after cultivation with MC3T3-E1
pre-osteoblasts for 28 days. Disk samples of 10 mm diameter (number of samples, n = 5) were placed
in a 24-well plate. At the end of the experiment, cell cultures were fixed in 4% paraformaldehyde,
washed twice with PBS and then stained with Alizarin red-S. To quantify calcium mineralization,
Polymers 2016, 8, 425 6 of 19

the alizarin red stained cultures were incubated with 10% cetyl-pyridinium chloride for 30 min in each
well to release calcium-bound alizarin red into solution. Then extracted supernatant was transferred
to a 96-well plate. The optical absorbance of the released alizarin red was measured at 562 nm using
a spectrophotometer.
Polymers 2016, 8, 425 6 of 18
2.9. Statistical Analysis for Cellular Tests
2.9.
AllStatistical Analysis
cell viability andfor Cellular Tests assays were performed in triplicate for each treatment and the
differentiation
results were expressed
All cell viabilityasand
mean ± standardassays
differentiation deviation
were(SD). A two-way
performed ANOVA
in triplicate for was
each used to determine
treatment and
the significance level of difference and p < 0.05 was considered to be statistically significant. to
the results were expressed as mean ± standard deviation (SD). A two-way ANOVA was used
determine the significance level of difference and p < 0.05 was considered to be statistically significant.
3. Results and Discussion
3. Results and Discussion
3.1. Structure and Morphology
3.1. Structure and Morphology
Figure 2a shows the XRD patterns of PEEK/(15% nHA)-(0.93% MWNT) and PEEK/(15%
FigureMWNT)
nHA)-(1.88% 2a shows hybrids.
the XRD patterns
For theof purposes
PEEK/(15%of nHA)-(0.93%
comparison, MWNT)
XRD and PEEK/(15%
patterns nHA)-
of pure PEEK,
(1.88% MWNT) hybrids. For the purposes of comparison, XRD patterns of
PEEK/(0.93% MWNT) and PEEK/(1.83% MWNT) composites are depicted in Figure 2b. Pure PEEKpure PEEK, PEEK/(0.93%
MWNT) and PEEK/(1.83% MWNT) composites are depicted in Figure 2b. Pure PEEK exhibits
exhibits characteristic peaks at around 2θ = 18.8◦ , 20.7◦ , 22.9◦ and 28.9◦ , corresponding to
characteristic peaks at around 2θ = 18.8°, 20.7°, 22.9° and 28.9°, corresponding to (110), (111), (200)
(110), (111), (200) and (211) crystalline planes, respectively [31]. The patterns of PEEK/MWNT
and (211) crystalline planes, respectively [31]. The patterns of PEEK/MWNT nanocomposites show
nanocomposites show additional peak at 26.6◦ due to the (002) reflection of nanotubes. For both
additional peak at 26.6° due to the (002) reflection of nanotubes. For both PEEK/(15% nHA)-(0.93%
PEEK/(15%
MWNT) and nHA)-(0.93%
PEEK/(15%MWNT) and PEEK/(15%
nHA)-(1.88% MWNT) hybrids, nHA)-(1.88% MWNT)
the peaks arising hybrids,
from the peaks
the crystalline arising
planes
fromofthe
nHAcrystalline
fillers can planes of observed
be clearly nHA fillers can be clearly
in addition observed
to the PEEK in addition
reflections. to the
Moreover, thePEEK reflections.
(002) peak of
Moreover, the (002) peakoverlaps
of nHA with
located ◦
nHA located at ~26.4° the at ~26.4
(002) peakoverlaps
of [Link] the (002) peak of MWNTs.

Figure 2. X-ray diffraction (XRD) patterns of (a) PEEK/(15% nHA)-(0.93% MWNT) and
Figure 2. X-ray diffraction (XRD) patterns of (a) PEEK/(15% nHA)-(0.93% MWNT) and PEEK/(15% nHA)-
PEEK/(15% nHA)-(1.88% MWNT) hybrids and (b) pure PEEK and PEEK/MWNT nanocomposites.
(1.88% MWNT) hybrids and (b) pure PEEK and PEEK/MWNT nanocomposites.

Figure 3a,b are the SEM images of PEEK/MWNT nanocomposites showing the homogeneous
dispersion of MWNT fillers in the PEEK matrix. It can be seen that the PEEK matrix is quite rough,
indicating the ductile nature of the nanocomposites. Figure 3c shows the SEM image of
PEEK/(15% nHA)-(1.88% MWNT) hybrid. Some nHA agglomerates can be readily seen due to higher
nHA filler loading in the hybrid.
Polymers 2016, 8, 425 7 of 19

Figure 3a,b are the SEM images of PEEK/MWNT nanocomposites showing the homogeneous
dispersion of MWNT fillers in the PEEK matrix. It can be seen that the PEEK matrix is quite
rough, indicating the ductile nature of the nanocomposites. Figure 3c shows the SEM image of
PEEK/(15% nHA)-(1.88% MWNT) hybrid. Some nHA agglomerates can be readily seen due to higher
nHA filler2016,
Polymers loading
8, 425 in the hybrid. 7 of 18

Figure 3. Scanning electron microscopy (SEM) images of (a) PEEK/(0.93 vol % MWNT);
Figure 3. Scanning electron microscopy (SEM) images of (a) PEEK/(0.93 vol % MWNT); (b) PEEK/
(b) PEEK/(1.88 vol % MWNT) and (c) PEEK/(15% nHA)-(1.88% MWNT) nanocomposites. Black arrows
(1.88 vol % MWNT) and (c) PEEK/(15% nHA)-(1.88% MWNT) nanocomposites. Black arrows indicate
indicate MWNT fillers.
MWNT fillers.

3.2. Crystallization Behavior

3.2. Crystallization Behavior
Figure 4 shows typical DSC cooling scans of neat PEEK and its nanocomposites. The crystallization
Figure 4 shows
temperature typical
(Tc) and the DSC cooling scans
crystallization of neat(ΔH
enthalpy PEEK and
c) can beits nanocomposites.
determined The crystallization
from these curves, and
temperature
tabulated in(TTable
c ) and the degree
2. The crystallization enthalpy
of crystallinity (Χc) (∆H c ) can
of PEEK andbeitsdetermined fromisthese
nanocomposites curves,
calculated
and tabulated
from in Tableequation:
the following 2. The degree of crystallinity (Xc ) of PEEK and its nanocomposites is calculated
from the following equation: ∆
% 100% (1)
1 Φ ∆
where ΔHm is the melting enthalpy of the 100% crystalline PEEK, i.e., 130 J/g [31], and Φ is the weight
fraction of the filler of the nanocomposites. The Χc values of the specimens studied are also listed in
Table 2. DSC results indicate that the additions of low MWNT levels to PEEK increase its crystallization
temperature. The Tc value of pure PEEK is 281.14 °C, and increases to 285.91 and 286.58 °C by adding
Polymers 2016, 8, 425 8 of 19

∆Hc
Xc ( % ) = × 100% (1)
(1 − Φ) × ∆Hm

where ∆Hm is the melting enthalpy of the 100% crystalline PEEK, i.e., 130 J/g [31], and Φ is the weight
fraction of the filler of the nanocomposites. The Xc values of the specimens studied are also listed in
Table 2.2016,
Polymers DSC8,results
425 indicate that the additions of low MWNT levels to PEEK increase its crystallization 8 of 18
temperature. The Tc value of pure PEEK is 281.14 ◦ C, and increases to 285.91 and 286.58 ◦ C by
0.93%
addingand 1.88%
0.93% MWNTs,
and respectively.
1.88% MWNTs, These demonstrate
respectively. that MWNTs
These demonstrate thatserve as effective
MWNTs serve asnucleation
effective
sites for PEEK
nucleation sitescrystallites on coolingon
for PEEK crystallites from melting
cooling fromtemperature, leading to
melting temperature, an increase
leading of the degree
to an increase of the
of crystallinity.
degree By adding
of crystallinity. 15% nHA
By adding 15%tonHAthe to
PEEK/(0.93% MWNT)
the PEEK/(0.93% and PEEK/(1.83%
MWNT) MWNT),
and PEEK/(1.83% the Tc
MWNT),
values
the Tc appear to decrease.
values appear Moreover,
to decrease. Xc valuesXof
Moreover, PEEK/(15%
c values nHA)-(0.93%MWNT)
of PEEK/(15% nHA)-(0.93% and PEEK/(15%
MWNT) and
nHA)-(0.93% MWNT) hybrids
PEEK/(15% nHA)-(0.93% MWNT)also hybrids
decreasealso
duedecrease
to the agglomeration of nHA fillers
due to the agglomeration as shown
of nHA fillers asin
Figure 4.
shown in Figure 4.

Figure 4.
Figure Differential scanning
4. Differential scanning calorimetry
calorimetry (DSC)
(DSC) cooling
cooling curves
curves of
of PEEK
PEEK and
and its
its nanocomposites.
nanocomposites.

Table 2.
Table Crystallization parameters
2. Crystallization parameters of
of PEEK
PEEK and
and its
its nanocomposites.
nanocomposites.

Sample
Sample TTc c(◦(°C)
C) ∆HΔH c (J/g) X (%)
c (J/g) c Xc (%)FWHM
FWHM
(◦ C) (°C)
PEEK
PEEK 281.14
281.14 59.38
59.38 45.6845.68 7.57 7.57
PEEK/(0.93% MWNT)
PEEK/(0.93% MWNT) 285.91
285.91 59.26
59.26 46.2846.28 8.81 8.81
PEEK/(1.88%
PEEK/(1.88% MWNT)
MWNT) 286.58
286.58 59.85
59.85 47.4647.46 9.62 9.62
PEEK/(15% nHA)-(0.93% MWNT)
PEEK/(15% nHA)-(0.93% MWNT) 284.90
284.90 36.87
36.87 41.4041.40 9.21 9.21
PEEK/(15% nHA)-(1.88% MWNT) 284.73 35.97 41.30 9.07
PEEK/(15% nHA)-(1.88% MWNT) 284.73 35.97 41.30 9.07

Furthermore, the
Furthermore, the full
full width
width at
at half-maximum
half-maximum (FWHM)
(FWHM) of of crystallization
crystallization peak
peak of
of PEEK
PEEK increases
increases
with increasing MWNT content. The FWHM values of PEEK and its nanocomposites
with increasing MWNT content. The FWHM values of PEEK and its nanocomposites are also listed are also listed
in Table
in Table 2.
2. This
Thisincrease
increaseininthe
thewidth
widthofof
thethe crystallization
crystallization peak
peak of PEEK/MWNT
of PEEK/MWNT nanocomposites
nanocomposites due
to MWNT additions could be attributed to a wider crystal size distribution [32]. However,[32].
due to MWNT additions could be attributed to a wider crystal size distribution However,
the addition of
the addition
15% nHA to theof 15% nHA to thenanocomposites
PEEK/MWNT PEEK/MWNT nanocomposites leads to aofslight
leads to a slight decrease decrease
the FWHM of the FWHM
of crystallization
of crystallization
peak and a marked peak and a marked
decrease decreaseThus
in crystallinity. in crystallinity. Thusof
a large amount a large
nHA amount of nHA
fillers that fillers that
agglomerate in
agglomerate in the PEEK matrix cannot act as effective sites for
the PEEK matrix cannot act as effective sites for polymeric chain [Link] chain nucleation.

3.3. Tensile Behavior

3.3. Tensile Behavior
Figure 5 shows the stress-strain curves of PEEK and its nanocomposites. The tensile parameters
Figure 5 shows the stress-strain curves of PEEK and its nanocomposites. The tensile parameters of
of these samples and human cortical bone are summarized in Table 3. It is obvious that the
these samples and human cortical bone are summarized in Table 3. It is obvious that the elastic modulus
elastic modulus of PEEK can be increased from 3.87 to 4.25 GPa by adding 1.88 vol % MWNTs.
of PEEK can be increased from 3.87 to 4.25 GPa by adding 1.88 vol % MWNTs. The elongation at
The elongation at break of PEEK is reduced from 67.1% to 56.48% by adding 1.88 vol % MWNTs.
break of PEEK is reduced from 67.1% to 56.48% by adding 1.88 vol % MWNTs. However, the
However, the PEEK/(15% nHA)-(1.88% MWNT) hybrid still exhibits ductile feature as evidenced
PEEK/(15% nHA)-(1.88% MWNT) hybrid still exhibits ductile feature as evidenced by the SEM
by the SEM micrograph as shown in Figure 3b. MWNTs with exceptionally high stiffness also
micrograph as shown in Figure 3b. MWNTs with exceptionally high stiffness also exhibit high
mechanical flexibility upon the application of large deformation strains [33]. Thus the additions of
low MWNT contents to PEEK enhance its tensile stiffness and strength, with a small reduction in tensile
ductility. On the other hand, the addition of 15 vol % nHA to PEEK increases it stiffness to 6.2 GPa, but
with a drastic reduction in fracture elongation to 2.71% due to the brittle nature of nHA [23]. From
Polymers 2016, 8, 425 9 of 19

exhibit high mechanical flexibility upon the application of large deformation strains [33]. Thus the
additions of low MWNT contents to PEEK enhance its tensile stiffness and strength, with a small
reduction in tensile ductility. On the other hand, the addition of 15 vol % nHA to PEEK increases
it stiffness to 6.2 GPa, but with a drastic reduction in fracture elongation to 2.71% due to the
brittle nature of nHA [23]. From Table 3, the addition of 1.88 vol % MWNT to PEEK/(15% nHA)
leads to a further increase in elastic modulus from 6.20 to 7.13 GPa. The tensile strength and
elongation at break of the PEEK/(15% nHA)-(1.88% MWNT) hybrid are 64.48 MPa and 1.74%,
respectively.
Polymers 2016, 8, Therefore,
425 all the tensile properties of PEEK/(15% nHA)-(1.88% MWNT) hybrid are
9 of 18
superior to those of human cortical bone. This demonstrates that the PEEK/(15% nHA)-(1.88% MWNT)
demonstrates
hybrid can bethat the for
used PEEK/(15% nHA)-(1.88%
fabricating MWNT)
load-bearing bone hybrid can be
implants inused
terms forof
fabricating
mechanicalload-bearing
property
bone implants From
consideration. in terms of 2,
Table mechanical
PEEK/(15% property consideration.
nHA)-(0.93% MWNT)From Table 2, PEEK/(15%
and PEEK/(15% nHA)-(0.93%
nHA)-(1.88% MWNT)
MWNT) exhibit
hybrids and PEEK/(15%
lower XnHA)-(1.88% MWNT)with
c values comparing hybrids exhibit
binary lower Xc values
PEEK/MWNT comparingAs
composites. with
a binary
result,
PEEK/MWNTnHA)-(0.93%
PEEK/(15% composites. MWNT)
As a result,
and PEEK/(15%
PEEK/(15%nHA)-(0.93%
nHA)-(1.88%MWNT)
MWNT)and PEEK/(15%
hybrids exhibit nHA)-
lower
(1.88% strength
tensile MWNT)than hybrids
binaryexhibit lower tensile
PEEK/MWNT strength
composites than binary
as shown PEEK/MWNT
in Table composites
3. As recognized, polymers as
shown
and in Table
their 3. As recognized,
composites with higherpolymers
degree ofand their composites
crystallinity exhibit with
betterhigher
tensiledegree of crystallinity
strength due to the
exhibit better
presence of moretensile strength
ordered due to
polymer the presence of more ordered polymer crystallites.
crystallites.

Figure
Figure 5.
5. Tensile stress-strain curves
Tensile stress-strain curves of
of PEEK
PEEK and
and its
its nanocomposites.
nanocomposites.

Table 3. Tensile
Table 3. properties of
Tensile properties of PEEK
PEEK and
and its
its nanocomposites.
nanocomposites.

Sample Elastic modulus (GPa) Tensile stress (Mpa) Elongation (%)

Sample Elastic modulus (GPa) Tensile stress (Mpa) Elongation (%)
PEEK 3.87 ± 0.10 80.06 ± 0.49 67.10 ± 13.40
PEEK MWNT)
PEEK/(0.93% 4.21±± 0.10
3.87 0.11 83.38±
80.06 0.49
± 0.78 67.10 ±±13.20
57.25 13.40
PEEK/(0.93%
PEEK/(1.88%MWNT)
MWNT) 4.25±± 0.11
4.21 0.85 82.08±
83.38 0.78
± 3.68 57.25 ±±24.90
56.48 13.20
PEEK/(1.88%
PEEK/(15% MWNT)
nHA)-(0.93% MWNT) 6.83±± 0.85
4.25 0.20 70.99±
82.08 3.68
± 7.51 2.32 ±±1.15
56.48 24.90
PEEK/(15% nHA)-(0.93% MWNT) 6.83 ± 0.20 70.99 ± 7.51 2.32 ± 1.15
PEEK/(15% nHA)-(1.88% MWNT) 7.13 ± 0.12 64.48 ± 8.51 1.74 ± 0.58
PEEK/(15% nHA)-(1.88% MWNT) 7.13 ± 0.12 64.48 ± 8.51 1.74 ± 0.58
PEEK/(15% nHA) [23] 6.20 ± 0.13 70.56 ± 3.22 2.71 ± 0.34
PEEK/(15% nHA) [23] 6.20 ± 0.13 70.56 ± 3.22 2.71 ± 0.34
Human cortical bone [20]
Human cortical bone [20] 7–30
7–30 50–150
50–150 1–3
1–3

3.4. Wettability
3.4. Wettability
Contact angle is a measure of the wettability of a solid when a liquid comes in contact with it. A
Contact angle is a measure of the wettability of a solid when a liquid comes in contact with
hydrophobic solid surface forms when the water contact angle is larger than 90°. In this case, the water
it. A hydrophobic solid surface forms when the water contact angle is larger than 90◦ . In this case,
droplet does not spread but rather forms a spherical cap resting on the solid surface with a contact angle.
the water droplet does not spread but rather forms a spherical cap resting on the solid surface with
By contrast, the hydrophilic solid surface shows a water contact angle smaller than 90°. Figure 6 shows
a contact angle. By contrast, the hydrophilic solid surface shows a water contact angle smaller than 90◦ .
the appearances of water droplets and contact angles on the surfaces of all samples studied. Pure
Figure 6 shows the appearances of water droplets and contact angles on the surfaces of all samples
PEEK displays a contact angle of 106.58° ± 1.13°, revealing its hydrophobic nature. Adding 0.93 vol %
studied. Pure PEEK displays a contact angle of 106.58◦ ± 1.13◦ , revealing its hydrophobic nature.
and 1.88 vol % MWNTs to PEEK decreases its water contact angles. At 1.88 vol % MWNT loading, the
Adding 0.93 vol % and 1.88 vol % MWNTs to PEEK decreases its water contact angles. At 1.88 vol %
contact angle reduces to 99.07° ± 0.50°. However, contact angle drops markedly to 84.08° ± 0.65° and 76.99°
MWNT loading, the contact angle reduces to 99.07◦ ± 0.50◦ . However, contact angle drops markedly
± 0.78° by hybridizing 15% nHA with 0.93% MWNTs and 1.88% MWNTs, respectively. This is due to
the presence of nHA, which is hydrophilic in nature. Thus hydrophilic PEEK/(15% nHA)-(0.93%
MWNT) and PEEK/(15% nHA)-(1.88% MWNT) hybrids favor adhesion of osteoblasts on their
surfaces.
Polymers 2016, 8, 425 10 of 19

to 84.08◦ ± 0.65◦ and 76.99◦ ± 0.78◦ by hybridizing 15% nHA with 0.93% MWNTs and 1.88% MWNTs,
respectively. This is due to the presence of nHA, which is hydrophilic in nature. Thus hydrophilic
PEEK/(15% nHA)-(0.93% MWNT) and PEEK/(15% nHA)-(1.88% MWNT) hybrids favor adhesion of
Polymers 2016, on
osteoblasts 8, 425
their surfaces. 10 of 18

Figure
Figure 6.
6. Contact
Contact angles
angles formed
formed by
by spreading
spreading of
of water
water droplets on PEEK
droplets on PEEK and
and its
its nanocomposites.
nanocomposites.

From the literature, enhancing the hydrophilicity of the polymer surface leads to increased cell
From the literature, enhancing the hydrophilicity of the polymer surface leads to increased cell
spreading and adhesion. Upon implantation into the body, water molecules interact with the material
spreading and adhesion. Upon implantation into the body, water molecules interact with the material
surfaces followed by protein adsorption. These surface proteins play a key role in cell adhesion,
surfaces followed by protein adsorption. These surface proteins play a key role in cell adhesion,
proliferation, and subsequent differentiation [34,35]. The bound proteins on the material surface
proliferation, and subsequent differentiation [34,35]. The bound proteins on the material surface
provide recognition sites for cell adhesion via specific cell receptors such as integrins [35,36].
provide recognition sites for cell adhesion via specific cell receptors such as integrins [35,36].
3.5. Cellular Adhesion
3.5. Cellular Adhesion
The initial cell-biomaterial interactions mimic to a certain extent the natural communication of
The initial cell-biomaterial interactions mimic to a certain extent the natural communication of cells
cells with extracellular matrix (ECM). Cell adhesion is mediated by the integrins of cell surface receptor
with extracellular matrix (ECM). Cell adhesion is mediated by the integrins of cell surface receptor and
and the ECM proteins (e.g., fibronectin, collagen), which in turn are sensitive to the chemical
the ECM proteins (e.g., fibronectin, collagen), which in turn are sensitive to the chemical composition
composition and wettability of biomaterials [37]. Integrins are transmembrane proteins that bind ECM
and wettability of biomaterials [37]. Integrins are transmembrane proteins that bind ECM and
and interact with the cytoskeleton at focal adhesion complexes. Once integrins adhere to such
interact with the cytoskeleton at focal adhesion complexes. Once integrins adhere to such molecules,
molecules, they are activated and clustered into nascent adhesions [37,38]. Therefore, osteoblast adhesion
they are activated and clustered into nascent adhesions [37,38]. Therefore, osteoblast adhesion on the
on the biomaterial surface is evidenced by markedly expedited cell spreading and dense actin fiber
biomaterial surface is evidenced by markedly expedited cell spreading and dense actin fiber formation.
formation. PEEK exhibits a relatively hydrophobic surface, thereby limiting cell attachment. By
PEEK exhibits a relatively hydrophobic surface, thereby limiting cell attachment. By adding bioactive
adding bioactive nHA and MWNTs, the resulting PEEK hybrids become hydrophilic as discussed
nHA and MWNTs, the resulting PEEK hybrids become hydrophilic as discussed above. Figure 7a–c
above. Figure 7a–c shows the fluorescence micrographs of osteoblasts cultured on the PEEK/(15%
shows the fluorescence micrographs of osteoblasts cultured on the PEEK/(15% nHA)-(0.93% MWNT)
nHA)-(0.93% MWNT) hybrid for 1 and 3 days, respectively. These micrographs show extensive
hybrid for 1 and 3 days, respectively. These micrographs show extensive spreading of osteoblasts and
spreading of osteoblasts and distinct actin stress fibers, especially for hybrids cultivated with
distinct actin stress fibers, especially for hybrids cultivated with osteoblasts for 3 days. Furthermore,
osteoblasts for 3 days. Furthermore, more distinct actin stress fibers are observed on the PEEK/(15%
more distinct actin stress fibers are observed on the PEEK/(15% nHA)-(1.88% MWNT) hybrid as
nHA)-(1.88% MWNT) hybrid as expected (Figure 8a,b). The higher expression level of actin and focal
expected (Figure 8a,b). The higher expression level of actin and focal adhesion contacts demonstrate
adhesion contacts demonstrate enhanced cellular activity and stable cytoskeleton, thereby facilitating
enhanced cellular activity and stable cytoskeleton, thereby facilitating progressive development of
progressive development of cellular processes [39]. For comparison, fluorescence micrographs of
cellular processes [39]. For comparison, fluorescence micrographs of osteoblasts cultured on the
osteoblasts cultured on the PEEK/(0.93% MWNT) and PEEK/(1.88% MWNT) nanocomposites for 3
PEEK/(0.93% MWNT) and PEEK/(1.88% MWNT) nanocomposites for 3 days are shown in Figure 9a,b,
days are shown in Figure 9a,b, respectively. These images reveal that osteoblast can attach and spread
respectively. These images reveal that osteoblast can attach and spread on the PEEK/MWNT composite
on the PEEK/MWNT composite surfaces in the absence of nHA, but with lower amounts of the cell
surfaces in the absence of nHA, but with lower amounts of the cell adhesion. It is noted that these
adhesion. It is noted that these fluorescence figures provide qualitative observations only.
fluorescence figures provide qualitative observations only.
Polymers 2016, 8, 425 11 of 19
Polymers 2016, 8, 425 11 of 18
Polymers 2016, 8, 425 11 of 18
Polymers 2016, 8, 425 11 of 18

Figure 7. Fluorescence micrographs of osteoblasts cultured on PEEK/(15% nHA)-(0.93% MWNT) hybrid

Figure 7. Fluorescence micrographs of osteoblasts cultured on PEEK/(15% nHA)-(0.93% MWNT)
and stained
Figure for F-actinmicrographs
7. Fluorescence cytoskeletonof(green) and nucleus
osteoblasts cultured(blue) for (a) 1 nHA)-(0.93%
on PEEK/(15% day and (b) 3MWNT) days; (c)hybrid
High
hybrid [Link]
Figure
for F-actin
Fluorescence
cytoskeleton
micrographs of(green)
(green)
osteoblasts
and on
cultured
nucleus
PEEK/(15%
(blue) for (a) 1 day
nHA)-(0.93% MWNT)
and hybrid
(b) 3 days;
magnification
and stained forimage showing
F-actin the morphology
cytoskeleton andofnucleus
well-organized
(blue) foractin
(a) 1filaments
day andof (b)a 3cell at day
days; (c) 3.
High
(c) High
andmagnification
stained forimage
magnification image
F-actin showing
cytoskeleton
showing the morphology
(green)
the morphology of well-organized
andofnucleus (blue) for
well-organized actinand
(a) 1filaments
actin day filaments
(b)a cell
of of
3 days; a(c)
cell
at day at day 3.
High
3.
magnification image showing the morphology of well-organized actin filaments of a cell at day 3.

Figure 8. Fluorescence micrographs of osteoblasts cultured on PEEK/(15% nHA)-(1.88% MWNT)

hybrid and
Figure stained for F-actin
8. Fluorescence cytoskeleton
micrographs (green) and
of osteoblasts nucleus
cultured on(blue) at (a) day
PEEK/(15% 1 and (b) day
nHA)-(1.88% 3.
MWNT)
Figure 8. Fluorescence micrographs of osteoblasts cultured on PEEK/(15% nHA)-(1.88% MWNT)
Figure and
hybrid 8. Fluorescence micrographs
stained for F-actin of osteoblasts
cytoskeleton cultured
(green) and on(blue)
nucleus PEEK/(15%
at (a) daynHA)-(1.88% MWNT)
1 and (b) day 3.
hybrid and and
hybrid stained for for
stained F-actin cytoskeleton
F-actin cytoskeleton(green)
(green) and nucleus(blue)
and nucleus (blue)atat
(a)(a)
dayday 1 and
1 and (b) day
(b) day 3. 3.

Figure 9. Fluorescence micrographs of osteoblasts cultured on (a) PEEK/(0.93% MWNT) and (b)
PEEK/(1.88%
Figure MWNT) nanocomposites
9. Fluorescence micrographs ofand stained for
osteoblasts F-actin cytoskeleton
cultured (green) and
on (a) PEEK/(0.93% nucleus
MWNT) (blue)
and (b)
Figure
for 9. Fluorescence
3 days.
PEEK/(1.88% micrographs ofand
MWNT) nanocomposites osteoblasts
stained forcultured on (a) PEEK/(0.93%
F-actin cytoskeleton MWNT)
(green) and and
nucleus (b)
(blue)
Figure 9. Fluorescence micrographs of osteoblasts cultured on (a) PEEK/(0.93% MWNT) and
PEEK/(1.88%
for 3 days. MWNT) nanocomposites and stained for F-actin cytoskeleton (green) and nucleus (blue)
(b) PEEK/(1.88%
The
for 3 adherence
days.
MWNT) nanocomposites
and spreading and stained
of osteoblasts for F-actin
on a material cytoskeleton
surface can also be(green) andthrough
examined nucleus
(blue)
SEM Thefor adherence
3 days.
imaging. and
Figure spreading
10a,b of osteoblasts
show the on a material
SEM micrographs surface can also
of PEEK/(1.88% MWNT)be examined through
nanocomposites
SEM The
seeded adherence
imaging.
with and
Figure
osteoblasts spreading
10a,b
for 2show of
and 4the osteoblasts
SEM
days, on a material
micrographs
respectively. surface
of PEEK/(1.88%
Apparently, can also
MWNT)
osteoblasts be examined
attach through
nanocomposites
firmly on these
SEMadherence
seeded
The imaging. Figure 10a,b
with osteoblasts for 2show
and spreading 4 the
and of SEM
days, micrographs
respectively.
osteoblasts of PEEK/(1.88%
Apparently,
on a material MWNT)
osteoblasts
surface can benanocomposites
attach
also firmly on these
examined through
SEM seeded
imaging. withFigure
osteoblasts
10a,bforshow
2 andthe
4 days,
SEMrespectively.
micrographs Apparently, osteoblastsMWNT)
of PEEK/(1.88% attach firmly on these
nanocomposites
seeded with osteoblasts for 2 and 4 days, respectively. Apparently, osteoblasts attach firmly on these
Polymers 2016, 8, 425 12 of 19

Polymers 2016, 8, 425 12 of 18

Polymers 2016, 8,
nanocomposite 425
surfaces after cultivation for 2 days. At day 4, they then proliferate and spread 12 of 18flatly

on thenanocomposite
surfaces in which surfacesmany
after cultivation
neighbor cellsfor 2 days. At day
link with 4, they
each thenthrough
other proliferate and spread extension.
cytoplasmic flatly
nanocomposite
on the surfaces surfaces
in which after
manycultivation
neighbor for 2 days.
cells link At day
with 4, they
each otherthen proliferate
through and spread
cytoplasmic flatly
extension.
The PEEK/(1.83% MWNT) nanocomposite surface is almost covered with osteoblasts after culturing
on the surfaces in which many neighbor cells link with each other through cytoplasmic
The PEEK/(1.83% MWNT) nanocomposite surface is almost covered with osteoblasts after culturing extension.
for 4 The
days. It is evident that nanocomposite
MWNTs act assurface effective sites covered
for osteoblastic adhesion. culturing
Interestingly,
for 4PEEK/(1.83% MWNT)
days. It is evident that MWNTs act as is almost
effective sites for with osteoblasts
osteoblastic [Link]
Interestingly,
osteoblasts attach
for 4 days. It and spread
is evident entirely
that MWNTs on the PEEK/(15% nHA)-(0.93% MWNT) hybrid surface cultured
osteoblasts attach and spread entirelyactonasthe
effective sites for
PEEK/(15% osteoblasticMWNT)
nHA)-(0.93% [Link]
Interestingly,
surface
for 2 cultured
days (Figure
osteoblasts 11a).and
for attach
2 days At day
(Figure 4, the
spread
11a). At osteoblastic
entirely
day 4,onthethe feature remains
PEEK/(15%
osteoblastic unchanged
nHA)-(0.93%
feature remains MWNT)(Figure
unchanged 11b).surface
hybrid
(Figure 11b).
cultured for 2 days (Figure 11a). At day 4, the osteoblastic feature remains unchanged (Figure 11b).

FigureFigure 10. images

10. SEM SEM images of PEEK/(1.88%
of PEEK/(1.88% MWNT)
MWNT) nanocomposite
nanocomposite cultured
cultured withwith osteoblasts
osteoblasts for2 and
for (a)
Figure
(a) 2 and10.
(b)SEM images of PEEK/(1.88% MWNT) nanocomposite cultured with osteoblasts for
4 days.
(b) 4 days.
(a) 2 and (b) 4 days.

Figure 11. SEM images of PEEK/(15% nHA)-(0.93% MWNT) hybrid cultured with osteoblasts for
FigureFigure
(a) 2 and
11. 11.(b)SEM
SEM images
4images
days. of PEEK/(15%nHA)-(0.93%
of PEEK/(15% nHA)-(0.93% MWNT)MWNT)hybrid hybridcultured
cultured with osteoblasts
with for for
osteoblasts
(a) 2 and (b) 4 days.
(a) 2 and (b) 4 days.
3.6. Cellular Metabolic Activity
3.6. Cellular Metabolic Activity
3.6. Cellular
TheMetabolic
MTT andActivityWST-1 assays can measure viable cell number [40], while Mwenifumbo et al.
The MTT that
demonstrated and MTT
WST-1 assays
assay can measure
is related viable cellactivity
to cell metabolic number[41]. [40],Awhile Mwenifumbo
live/dead stain would et be
al.
The
used to measure cell viability. Figure 12 shows the results of MTT measurements for PEEK and its et al.
MTT
demonstrated and WST-1
that MTT assays
assay is can measure
related to cell viable
metabolic cell number
activity [41]. [40],
A while
live/dead Mwenifumbo
stain would be
demonstrated
used to measure
nanocomposites. that MTT cell assay is Figure
viability.
PEEK/(0.93% related
MWNT) 12to cellPEEK/(1.88%
shows
and metabolic
the results activity
of [41].
MTT measurements
MWNT) A live/dead
composites stain
for PEEK
exhibit and
higher would
its be
cell
nanocomposites.
used metabolic
to measure PEEK/(0.93% MWNT) and PEEK/(1.88% MWNT) composites
activity comparing to pure PEEK specimen, especially after cultivation for 7 days. Thus and
cell viability. Figure 12 shows the results of MTT measurements exhibit higher
for PEEKcell
metabolic
its nanocomposites.
MWNT fillers activity comparing
PEEK/(0.93%
promote the growthto pure
MWNT) PEEK andspecimen,
of osteoblasts especially MWNT)
PEEK/(1.88%
on the nanocompositeafter surfaces.
cultivation
compositesforbeen
It has 7 days.
exhibitThus
reported higher
MWNT fillers
in the literature
cell metabolic promote
activity the
that comparing growth
carbon nanotubes of osteoblasts
to pure mayPEEK on the nanocomposite
exhibitspecimen,
cytotoxic effects surfaces.
towards
especially after It has
biological been
cultivation reported
cells [42,43].
for 7 days.
in thecytotoxicity
literature that carbon nanotubes by may exhibit carbon
cytotoxic effects towards biological
the cells
cell [42,43].
ThusTheMWNT fillers promote is mainly caused
the growth individual
of osteoblasts onnanotubes dispersed
the nanocomposite insurfaces. culture
It has been
The
mediumcytotoxicity
or organic is mainly
[Link] As by
such, individual
individual carbon
carbon nanotubes
nanotubes dispersed
can penetratein the
cell cell culture
membrane
reported in the literature that carbon nanotubes may exhibit cytotoxic effects towards biological
medium
and reside or inorganic suspensions.
the cytoplasm As such,
[44,45]. On the individual
other hand, carbon nanotubeseffects
no cytotoxic can penetrate cell membrane
are observed in carbon
cells [42,43].
and resideThe in cytotoxicity
the cytoplasm is[44,45].
mainlyOn caused
the by individual
other hand, no carboneffects
cytotoxic nanotubesare dispersed
observed in the cell
in carbon
nanotube fillers of polymer nanocomposites [21–23]. This is because carbon nanotube fillers are
culture medium
nanotube or organic
fillers suspensions. As such, individual carboncarbonnanotubes can penetrate are cell
embedded firmlyofwithin
polymer nanocomposites
the matrix of polymer[21–23]. This is because
nanocomposites. In this case, they nanotube
serve as fillers
the active
membrane
sites for the attachment of osteoblasts. From Figure 12, a marked increase in cell metabolic activity in
embedded and reside
firmly in the
within cytoplasm
the matrix of[44,45].
polymer On the other
nanocomposites. hand,In no
this cytotoxic
case, they effects
serve are
as theobserved
active
carboncan nanotube
sitesbeforseen
the in fillers
attachment of polymer nanocomposites
of osteoblasts.
both PEEK/(15% From MWNT)
nHA)-(0.93% Figure 12, [21–23].
and Thisincrease
a marked
PEEK/(15% is nHA)-(1.88%
because carbon
in cell nanotube
metabolic
MWNT) activityfillers
hybrids.
can be
are embedded seen
Thus the presence in both
firmly withinPEEK/(15% nHA)-(0.93%
the matrix
of inorganic nHA inofsuch MWNT)
polymer
hybrids and PEEK/(15%
nanocomposites. nHA)-(1.88%
offers a beneficialIneffectthis case, MWNT) hybrids.
they serve
in enhancing theiras the
Thus
sitestheforpresence
activebiocompatibility. of inorganic
the attachment
From of nHA
the water in such
osteoblasts.
contact hybrids
From Figure
measurements, offers a beneficial
12,
these a marked
hybrids effect
are in enhancing
increase
hydrophilic in due
cell to their
metabolic
the
biocompatibility.
presence
activity can beofseen nHA. From
On the
in both the water contact
contrary, PEEK/(0.93%
PEEK/(15% measurements,
nHA)-(0.93%MWNT) MWNT)these
and hybrids
PEEK/(1.83%
and are
PEEK/(15% hydrophilic
MWNT) due to
composites
nHA)-(1.88% the
MWNT)
presence
exhibit of nHA.
hydrophobic On the
behavior contrary, PEEK/(0.93%
because ofnHA the absence MWNT) and PEEK/(1.83% MWNT) composites
hybrids. Thus the presence of inorganic in suchofhybrids
nHA. Asoffers
discussed above, increased
a beneficial effect insurface
enhancing
exhibit hydrophobic behavior because of the absence of nHA. As discussed above, increased surface
their biocompatibility. From the water contact measurements, these hybrids are hydrophilic due to
the presence of nHA. On the contrary, PEEK/(0.93% MWNT) and PEEK/(1.83% MWNT) composites
Polymers 2016, 8, 425 13 of 19

Polymers 2016, 8, 425 13 of 18

exhibit hydrophobic behavior because of the absence of nHA. As discussed above, increased surface
hydrophilicity
hydrophilicity enhances
enhancesprotein
proteinadsorption
adsorptionand
andcell
cell spreading.
spreading. Thus
Thus hydrolytic
hydrolytic materials
materials favor
favor the
adhesion and proliferation of osteoblasts on their surfaces [34–46].

Figure
Figure 12.
12.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
bromide(MTT) assay
(MTT) results
assay showing
results cell
showing
metabolic activity
cell metabolic of murine
activity MC3T3-E1
of murine MC3T3-E1pre-osteoblasts grown
pre-osteoblasts on PEEK
grown and and
on PEEK its nanocomposites
its nanocompositesfor 1,for
3
and 7 days.
1, 3 and * represents
7 days. p < 0.05.
* represents Significance
p < 0.05. is assessed
Significance for each
is assessed for time-point assayed.
each time-point assayed.

It is worth-noting that MTT measurements tend to give lower cellular metabolic activity for carbon
It is worth-noting that MTT measurements tend to give lower cellular metabolic activity for carbon
nanotubes due to the formation of insoluble formazan products [47]. MTT assay is a colorimetric test
nanotubes due to the formation of insoluble formazan products [47]. MTT assay is a colorimetric test
based on the reduction of the yellowish, water-soluble tetrazolium salt into a water-insoluble, purple
based on the reduction of the yellowish, water-soluble tetrazolium salt into a water-insoluble, purple
formazan product by mitochondrial enzyme succinate dehydrogenase of living cells in the culture.
formazan product by mitochondrial enzyme succinate dehydrogenase of living cells in the culture.
The reduction occurs only when reductase enzymes are active. Dead cells are incapable of converting
The reduction occurs only when reductase enzymes are active. Dead cells are incapable of converting
MTT into formazan. MTT assay needs an additional solubilization step to dissolve color formazan
MTT into formazan. MTT assay needs an additional solubilization step to dissolve color formazan
crystals. The intensity of the color gives a relative count of viable cells. Such formazan crystals do not
crystals. The intensity of the color gives a relative count of viable cells. Such formazan crystals do not
dissolve completely in an organic solvent, particularly in the presence of carbon nanotubes [47]. The
dissolve completely in an organic solvent, particularly in the presence of carbon nanotubes [47].
undissolved crystals in a solvent give low colorimetric readings in the spectrophotometric
The undissolved crystals in a solvent give low colorimetric readings in the spectrophotometric
measurements, causing low cell metabolic activity. By contrast, WST-1 assay produces water soluble
measurements, causing low cell metabolic activity. By contrast, WST-1 assay produces water
formazan and does not form insoluble clusters like MTT. Therefore, WST-1 assay generates reliable
soluble formazan and does not form insoluble clusters like MTT. Therefore, WST-1 assay generates
results for assessing cell metabolic activity. Figure 13 shows the cell metabolic activity measured by
reliable results for assessing cell metabolic activity. Figure 13 shows the cell metabolic activity
the WST-1 assay for the PEEK-based nanocomposites. Apparently, WST-1 assay gives considerably
measured by the WST-1 assay for the PEEK-based nanocomposites. Apparently, WST-1 assay gives
higher cell metabolic activity than MTT assay. At day 7, the amount of viable cells for PEEK/(15%
considerably higher cell metabolic activity than MTT assay. At day 7, the amount of viable cells for
nHA)-(0.93% MWNT) and PEEK/(15% nHA)-(1.88% MWNT) hybrids is relatively high as compared
PEEK/(15% nHA)-(0.93% MWNT) and PEEK/(15% nHA)-(1.88% MWNT) hybrids is relatively high as
to PEEK/(0.93% MWNT) and PEEK/(1.83% MWNT) composites.
compared to PEEK/(0.93% MWNT) and PEEK/(1.83% MWNT) composites.
For the MTT and WST assays, the amount of formazan crystals produced is directly proportional
to the number of metabolically active cells in the culture. Both assays reveal that, after 7 days,
cells cultured on the PEEK/(15% nHA)-(0.93% MWNT) and PEEK/(15% nHA)-(1.88% MWNT)
hybrids exhibit a significantly higher metabolic activity than on the PEEK control specimen.
As aforementioned, nHA enhances protein adsorption, thereby promoting osteoblast adhesion,
proliferation, and differentiation [19]. In other words, proteins that can induce osteoblast adhesion,
including fibronectin and vitronectin, have been reported to have higher adsorption rates on
nHA [19,48]. Moreover, osteoblastic cells also attach and survive on the MWNT materials, producing
higher metabolic activity on the basis of MTT results [41]. Therefore, PEEK/(15% nHA)-(0.93% MWNT)
and PEEK/(15% nHA)-(1.88% MWNT) hybrid nanocomposites exhibit enhanced cell activity compared
to the control PEEK specimen.

Figure 13. 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) assay results

showing cell metabolic activity of murine MC3T3-E1 pre-osteoblasts grown on PEEK and its
nanocomposites for 1, 3 and 7 days. * represents p < 0.05. Significance is assessed for each time-point
assayed.
formazan and does not form insoluble clusters like MTT. Therefore, WST-1 assay generates reliable
results for assessing cell metabolic activity. Figure 13 shows the cell metabolic activity measured by
the WST-1 assay for the PEEK-based nanocomposites. Apparently, WST-1 assay gives considerably
higher cell metabolic activity than MTT assay. At day 7, the amount of viable cells for PEEK/(15%
nHA)-(0.93%
Polymers MWNT) and PEEK/(15% nHA)-(1.88% MWNT) hybrids is relatively high as compared
2016, 8, 425 14 of 19
to PEEK/(0.93% MWNT) and PEEK/(1.83% MWNT) composites.

Polymers 2016, 8, 425 14 of 18

For the MTT and WST assays, the amount of formazan crystals produced is directly proportional
to the number of metabolically active cells in the culture. Both assays reveal that, after 7 days, cells
cultured on the PEEK/(15% nHA)-(0.93% MWNT) and PEEK/(15% nHA)-(1.88% MWNT) hybrids
exhibit a significantly higher metabolic activity than on the PEEK control specimen. As
aforementioned, nHA enhances protein adsorption, thereby promoting osteoblast adhesion,
proliferation, and differentiation [19]. In other words, proteins that can induce osteoblast adhesion,
including fibronectin and vitronectin, have been reported to have higher adsorption rates on nHA [19,48].
Moreover, osteoblastic cells also attach and survive on the MWNT materials, producing higher metabolic
activity on the
Figure 13. basis of MTT results [41]. Therefore, PEEK/(15% nHA)-(0.93% MWNT) and PEEK/(15%
Figure 13. 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium
2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) assay
(WST-1) results
assay
nHA)-(1.88%
showing MWNT)
cell hybrid
metabolic nanocomposites
activity of murine exhibit
MC3T3-E1 enhanced cell
pre-osteoblastsactivity
grown compared
results showing cell metabolic activity of murine MC3T3-E1 pre-osteoblasts grown on PEEK and itson PEEK to the
and control
its
PEEKnanocomposites
specimen.
nanocompositesfor for1,1, 3 and
3 and 7 days.
7 days. * represents
* represents p < 0.05.
p < 0.05. Significance
Significance is assessed
is assessed for eachfor each time-point
time-point assayed.
assayed.
3.7. Cellular Differentiation and Mineralization
3.7. Cellular Differentiation and Mineralization
Alkaline phosphatase (ALP) activity and calcium mineralization are mostly used as biochemical
Alkaline phosphatase (ALP) activity and calcium mineralization are mostly used as biochemical
markers for early and late differentiation of osteoblast cells, respectively [49,50]. Bone formation
markers for early and late differentiation of osteoblast cells, respectively [49,50]. Bone formation
involves a series of events that occur in the initial proliferation and differentiation stages, followed by
involves a series of events that occur in the initial proliferation and differentiation stages, followed
mineralization. Proliferating osteoblasts show alkaline phosphatase activity in vitro; this is greatly
by mineralization. Proliferating osteoblasts show alkaline phosphatase activity in vitro; this is greatly
enhanced during early differentiation of osteoblasts. Alkaline phosphatase catalyzes hydrolysis of
enhanced during early differentiation of osteoblasts. Alkaline phosphatase catalyzes hydrolysis
p-nitrophenyl phosphate in alkaline buffer and produces phenol and inorganic phosphate. An enhanced
of p-nitrophenyl phosphate in alkaline buffer and produces phenol and inorganic phosphate.
level of this enzyme is required just before the onset of bone mineralization, providing localized
An enhanced level of this enzyme is required just before the onset of bone mineralization, providing
enrichment of inorganic phosphate for nHA nucleation and proliferation. Figure 14 shows the ALP
localized enrichment of inorganic phosphate for nHA nucleation and proliferation. Figure 14 shows
activity for the osteoblastic cells exposed to PEEK and its nanocomposites. Apparently, very low ALP
the ALP activity for the osteoblastic cells exposed to PEEK and its nanocomposites. Apparently,
activity is detected after 3 days for all samples investigated. At day 14, alkaline phosphatase activity
very low ALP activity is detected after 3 days for all samples investigated. At day 14, alkaline
of osteoblasts grown on the hybrids, especially PEEK/(15% nHA)-(1.88% MWNT) increases markedly
phosphatase activity of osteoblasts grown on the hybrids, especially PEEK/(15% nHA)-(1.88% MWNT)
in comparison to those observed in pure PEEK and PEEK-MWNT nanocomposites. This
increases markedly in comparison to those observed in pure PEEK and PEEK-MWNT nanocomposites.
demonstrates the ability of this hybrid nanocomposite to enhance early osteoblast differentiation.
This demonstrates the ability of this hybrid nanocomposite to enhance early osteoblast differentiation.

14. Alkaline
Figure 14.
Figure phosphatase (ALP)
Alkaline phosphatase (ALP) activity
activity normalized
normalized to
to protein
protein content
content of
of murine
murine MC3T3-E1
MC3T3-E1
pre-osteoblasts cultured on PEEK and its nanocomposites for 1, 3 and 7 days. * represents p
pre-osteoblasts cultured on PEEK and its nanocomposites for 1, 3 and 7 days. * represents p << 0.05.
0.05.

Finally, Alizarin
Alizarin Red staining
staining after
after 28
28 days
days of cell culture is used to determine
determine the presence of
calcium minerals on the PEEK-based nanocomposites. Bone forming cells containing calcium deposits
are stained dark red by the Alizarin red solution. Prior to staining, white nodules can be seen on the
PEEK/MWNT and PEEK/(15% nHA)-MWNT nanocomposites. However, large calcified nodules are
more abundant in the hybrids (Figure 15). White mineralized nodules were stained a dark red color
by alizarin red. Mineralized nodule formation indicates the final stages of osteoblastic differentiation at
28 days. To clarify that the nHA fillers of PEEK-based hybrids do not contribute to dark red staining,
Polymers 2016, 8, 425 15 of 19

are stained dark red by the Alizarin red solution. Prior to staining, white nodules can be seen on the
PEEK/MWNT and PEEK/(15% nHA)-MWNT nanocomposites. However, large calcified nodules are
more abundant in the hybrids (Figure 15). White mineralized nodules were stained a dark red color by
Polymers 2016,
alizarin red.8,Mineralized
425 nodule formation indicates the final stages of osteoblastic differentiation 15 of at
18
Polymers 2016, 8, 425 15 of 18
28 days. To clarify that the nHA fillers of PEEK-based hybrids do not contribute to dark red staining,
PEEK/(0.93% MWNT)
PEEK/(0.93% composite
MWNT) composite
composite andand PEEK/(15%
and PEEK/(15% nHA)-MWNT
PEEK/(15%nHA)-MWNT
nHA)-MWNThybridshybrids without
hybrids without cells
without cells are
cells are also
are also stained
also stained
stained
PEEK/(0.93% MWNT)
with Alizarin
with Alizarin RedRed (Figure 16).
(Figure 16). Apparently,
16). Apparently, these specimens
Apparently, these specimens do do not
not display dark
display dark red
dark red color,
red color, indicating
color, indicating
indicating
with Alizarin Red (Figure these specimens do not display
that the
that the nanohydroxyapatite
the nanohydroxyapatite
nanohydroxyapatiteininthe in the hybrids
thehybrids does
hybridsdoes not
doesnot contribute
notcontribute
contribute to the staining. Figure 17 shows the
that to to
thethe staining.
staining. Figure
Figure 17 shows
17 shows the
optical density
the optical density(OD) of calcium mineralization of MC3T3-E1 pre-osteoblasts cultured on PEEK and
optical density (OD)(OD) of calcium
of calcium mineralization
mineralization of MC3T3-E1
of MC3T3-E1 pre-osteoblasts
pre-osteoblasts cultured
cultured on PEEK
on PEEK and
its nanocomposites
and its nanocomposites for 28
for days.
28 Low
days. Lowmineral
mineraldeposition
deposition level occurs
level occurs ininpure
pure PEEK
PEEK as
as expected.
expected.
its nanocomposites for 28 days. Low mineral deposition level occurs in pure PEEK as expected.
Increased mineral
Increased mineral deposition is is found inin the PEEK/(0.93%
PEEK/(0.93% MWNT) and and PEEK/(1.83% MWNT) MWNT)
Increased mineral deposition
deposition is found
found in thethe PEEK/(0.93% MWNT) MWNT) and PEEK/(1.83%
PEEK/(1.83% MWNT)
nanocomposites comparing
nanocomposites comparing withwith neat
with neat PEEK.
neat PEEK. However,
PEEK. However,
However, highhigh mineralization level
high mineralization
mineralization can
level can
can bebe observed in
be observed in
nanocomposites comparing level observed in
both hybrids,
both hybrids, especially
especially in
in the
the PEEK/(15%
PEEK/(15% nHA)-(1.88%
nHA)-(1.88% MWNT)
MWNT) sample.
sample. Increased
Increased mineralization in
mineralization in
both hybrids, especially in the PEEK/(15% nHA)-(1.88% MWNT) sample. Increased mineralization in
cultured osteoblasts
cultured osteoblasts implies
osteoblasts implies increased
implies increased calcium
increased calcium deposition.
calcium deposition.
deposition. AsAs previously
As previously mentioned,
previously mentioned,
mentioned, the the presence
the presence
presence of of
of
cultured
nHA in
nHA in PEEK-based
PEEK-based hybrids
hybrids converts
converts hydrophobic
hydrophobic PEEKPEEK to to hydrophilic,
hydrophilic, thereby
thereby promoting
promoting protein
protein
nHA in PEEK-based hybrids converts hydrophobic PEEK to hydrophilic, thereby promoting protein
adhesion, and
adhesion, subsequent
and subsequent osteoblastic
subsequent osteoblastic cell
osteoblastic cell attachment,
cell attachment, proliferation,
attachment, proliferation, differentiation,
proliferation, differentiation,
differentiation, andand eventual
and eventual
adhesion, and eventual
calcium mineralization.
calcium mineralization.
mineralization.
calcium

Figure 15.
Figure 15. Representative photographs
Representative photographs showing
photographs showing calcium
showing calcium mineralization
calcium mineralization (before
mineralization (before staining)
(before staining) on
staining) on
on
Figure 15. Representative
PEEK/(0.93% MWNT),
PEEK/(0.93% MWNT), PEEK/(15%
PEEK/(15% nHA)-(0.93%
nHA)-(0.93% MWNT)
MWNT) and
and PEEK/(15% nHA)-(1.88%
PEEK/(15% nHA)-(1.88% MWNT)
PEEK/(0.93% MWNT), PEEK/(15% nHA)-(0.93% MWNT) and PEEK/(15% nHA)-(1.88% MWNT)
nanocomposites cultured
nanocomposites with murine MC3T3-E1 pre-osteoblasts for 28 days (top); Calcified nodules
cultured with
nanocomposites cultured murine MC3T3-E1 pre-osteoblasts for 28 days (top); Calcified nodules
appeared as color on these
these samples after
after staining with
with Alizarin red-S
red-S (bottom).
appeared as aa dark
dark red
red color
color on
on these samples
samples after staining
staining with Alizarin
Alizarin red-S (bottom).
(bottom).

Figure 16. Photographs of Alizarin Red stained PEEK/(0.93% MWNT), PEEK/(15% nHA)-(0.93%
Figure
Figure 16.
16. Photographs of of
Photographs Alizarin RedRed
Alizarin stained PEEK/(0.93%
stained MWNT),
PEEK/(0.93% PEEK/(15%
MWNT), nHA)-(0.93%
PEEK/(15% nHA)-
MWNT) and PEEK/(15% nHA)-(1.88% MWNT) nanocomposites without cell exposure.
MWNT) and PEEK/(15%
(0.93% MWNT) nHA)-(1.88%
and PEEK/(15% MWNT)MWNT)
nHA)-(1.88% nanocomposites withoutwithout
nanocomposites cell exposure.
cell exposure.
Polymers 2016, 8, 425 16 of 19
Polymers 2016, 8, 425 16 of 18

Figure 17.
Figure 17. Optical
Optical absorbance
absorbance of
of MC3T3-E1
MC3T3-E1 pre-osteoblasts cultured on
pre-osteoblasts cultured on PEEK
PEEK and
and its
its nanocomposites
nanocomposites
for 28
for 28 days
days and
and stained
stained with
with Alizarin
Alizarin red.
red. ** represents
represents pp << 0.05.
0.05.

4. Conclusions
4. Conclusions
Melt-mixing and
Melt-mixing and injection
injection molding
molding techniques
techniques have
have been
been successfully
successfully adapted
adapted forfor fabricating
fabricating
PEEK/(15% nHA)-MWNT hybrid nanocomposites. Combining the
PEEK/(15% nHA)-MWNT hybrid nanocomposites. Combining the advantages of inorganic advantages of inorganic nHAnHAand
MWNTs,
and MWNTs,the resulting hybrid nanocomposites
the resulting show good show
hybrid nanocomposites tensile good
property and excellent
tensile propertybiocompatibility.
and excellent
biocompatibility. In particular, the elastic modulus of PEEK can be increasedbyfrom
In particular, the elastic modulus of PEEK can be increased from 3.87 to 7.13 GPa adding3.8715tovol % nHA
7.13 GPa
and 1.88 vol % MWNT. The tensile strength and elongation at break of the
by adding 15 vol % nHA and 1.88 vol % MWNT. The tensile strength and elongation at break of PEEK/(15% nHA)-(1.88%
MWNT)
the hybrid are
PEEK/(15% 64.48 MPa and
nHA)-(1.88% MWNT)1.74%,hybrid
respectively. Thus
are 64.48 MPatheand
tensile properties
1.74%, of this hybrid
respectively. are
Thus the
superior to those of human cortical bone. Water contact angle measurements reveal
tensile properties of this hybrid are superior to those of human cortical bone. Water contact angle that pure PEEK is
hydrophobic with
measurements a water
reveal that contact angleisofhydrophobic
pure PEEK 106°. Addingwith1.88% MWNTs
a water to PEEK
contact angledecreases
of 106◦ . its water
Adding
contact angles to 98°. The contact angle drops markedly to 77° by ◦adding 1.88%
1.88% MWNTs to PEEK decreases its water contact angles to 98 . The contact angle drops markedly MWNTs and 15% nHA
hybrid fillers to PEEK. Accordingly, hydrophilic PEEK/(15% nHA)-(1.88%
to 77◦ by adding 1.88% MWNTs and 15% nHA hybrid fillers to PEEK. Accordingly, hydrophilic MWNT) hybrid favors the
adhesion, proliferation
PEEK/(15% nHA)-(1.88% andMWNT)
differentiation
hybrid of osteoblasts
favors on its surface
the adhesion, based and
proliferation on the results of cell
differentiation of
culture, fluorescence microscopy, MTT assay, WST-1 assay, ALP, and Alizarin
osteoblasts on its surface based on the results of cell culture, fluorescence microscopy, MTT red staining [Link],
Thus
the PEEK/(15%
WST-1 assay, ALP, nHA)-(1.88%
and Alizarin MWNT) hybridtests.
red staining has the
Thuspotential to be used
the PEEK/(15% for fabricating
nHA)-(1.88% load-bearing
MWNT) hybrid
orthopedic implants.
has the potential to be used for fabricating load-bearing orthopedic implants.
Acknowledgments: This work was supported by the Applied Research Grant (No. 9667126), City University of
Acknowledgments:
Hong Kong, ShenzhenThis work was
Science and supported
Technologyby the Applied
Project Research
Grant (Grant [Link] (No. 9667126), City University
JCYJ20150630145302228), China and
of Hong Kong, Shenzhen Science and Technology Project Grant (Grant No. JCYJ20150630145302228), China
Shenzhen Knowledge Innovation Program of Basic Research Items of Guangdong Province
and Shenzhen Knowledge Innovation Program of Basic Research Items of Guangdong Province (Grant (Grant No.
JCYJ20140414090541811),
No. China.
JCYJ20140414090541811), China.
Author Contributions:Chen
Author Contributions: Chen LiuLiu
carried out the
carried drop
out thewater
dropcontact
waterangle test, SEM
contact examination
angle test, SEMofexamination
nanocompositeof
features, fluorescence
nanocomposite microscopic
features, fluorescenceimaging, MTT assay,
microscopic WST-1
imaging, MTTassay,
assay,ALP
WST-1assay, andALP
assay, Alizarin red-S
assay, and staining.
Alizarin
Kai Wang
red-S ChanKai
staining. fabricated pure PEEK
Wang Chan and composite
fabricated pure PEEK materials using melt-compounding
and composite and injection molding
materials using melt-compounding and
processes as well as performed tensile measurements. Jie Shen performed cell culture for all assays and SEM
injection molding processes as well as performed tensile measurements. Jie Shen performed
examination of osteoblastic cells. Cheng Zhu Liao carried out XRD and DSC tests. Kelvin Wai Kwok Yeung cell culture for all
assays and SEM examination of osteoblastic cells. Cheng Zhu Liao carried out XRD and DSC tests.
supervised cell cultivation, MTT, WST-1, ALP, and Alizarin red-S staining tests. Sie Chin Tjong designed the Kelvin Wai
project and wrote
Kwok Yeung the manuscript.
supervised cell cultivation, MTT, WST-1, ALP, and Alizarin red-S staining tests. Sie Chin Tjong
designed the project and wrote
Conflicts of Interest: The authors thedeclare
manuscript.
no conflict of interest.
Conflicts of Interest: The authors declare no conflict of interest.
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