Preparation of polyetheretherketone composites with nanohydroxyapatite rods and carbon nanofibers having high strength, good biocompatibility and excellent thermal stability

Article abstract of DOI:10.1039/c5ra22134j

In recent years, research in the development of polymeric materials for orthopedic implants has become ever more important because the global demand of biocompatible implants has been steadily increasing. Bioinert polyetheretherketone (PEEK) is typically

Full text of DOI:10.1039/c5ra22134j

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Preparation of polyetheretherketone composites
with nanohydroxyapatite rods and carbon
nanofibers having high strength, good
Cite this: RSC Adv., 2016, 6, 19417
biocompatibility and excellent thermal stability
Kai Wang Chan,a Cheng Zhu Liao,b Hoi Man Wong,c Kelvin Wai Kwok Yeungc
a
*
and Sie Chin Tjong
In recent years, research in the development of polymeric materials for orthopedic implants has become
ever more important because the global demand of biocompatible implants has been steadily increasing.
Bioinert polyetheretherketone (PEEK) is typically reinforced with bioactive hydroxyapatite microparticles.
However, the tensile strength of conventional PEEK/hydroxyapatite microcomposites falls sharply with
increasing filler loading. To address low strength and high filler loading issues, nanohydroxyapatite rods
(nHA) and carbon nanofibers (CNF) were employed to reinforce PEEK. In this study, molded-grade PEEK
pellets, nHA and CNF fillers were melt-mixed and injection molded to form PEEK/nHA and hybrid PEEK/
nHA–CNF nanocomposites. The tensile and thermal properties, as well as the bioactivity and
biocompatibility of such nanocomposites, were investigated. Tensile test results showed that elastic
modulus of PEEK/nHA nanocomposites increases with increasing nHA content. The PEEK/9.3 vol% nHA
nanocomposite exhibited higher tensile strength than that of a conventional HAPEX microcomposite.
Thermogravimetric measurements indicated that the nHA addition improves the thermal stability of
PEEK. Thus, the PEEK/9.3 vol% nHA nanocomposite that had good mechanical, thermal and biological
performances was an attractive biomaterial for use in maxillofacial surgery. Furthermore, the tensile
property of the PEEK/15 vol% nHA–1.9 vol% nHA nanocomposite compared favorably with that of
human cortical bones. The results of biomineralization, alkaline phosphatase (ALP), 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 also showed that the PEEK/15 vol% nHA and PEEK/15
vol% nHA–1.9 vol% CNF nanocomposites exhibited excellent bioactivity and biocompatibility. The ALP
assay showed good activity of osteoblast cells on the composite specimens with high nHA content.
Moreover, CNF addition further increased the ALP activity of PEEK/15 vol% nHA nanocomposites. The
PEEK/15 vol% nHA–1.9 vol% CNF composite, with enhanced tensile strength and excellent
biocompatibility, shows large potential for load-bearing implant applications.
Received 22nd October 2015
Accepted 1st February 2016
DOI: 10.1039/c5ra22134j
www.rsc.org/advances
Autogras removed from the bones of patients and allogras
taken from cadavers are traditionally used for replacing bone
Introduction
tissue defects. Both autogras and allogras have many draw-
backs, including limited availability, immunological rejection
and possible disease transmission.3 Therefore, there is a high
demand for articial implants with excellent biocompatibility
for orthopedics. Metallic alloys are widely used for making bone
xation devices and implants. Metallic alloys offer benecial
advantages such as high mechanical exibility due to their
superior ductility and toughness.4
In recent years, there has been a substantial increase in the
number of accidents in the workplace, road traffic and from
sports activities. These result in unavoidable bone injuries.
Furthermore, the number of aging population has also
increased dramatically in developed and undeveloped coun-
tries.1,2 Elderly people have a higher risk of bone fractures that
imposes signicant social and economic burdens to our society.
Metallic materials, such as stainless steel, Co–Cr and Ti–6Al–
4V alloys, have been used extensively for fabricating load-
bearing hip implants. However, these implants undergo corro-
sion upon exposure to human body uids. Released ions from
the implants can cause allergic reactions, inammation and
aDepartment of Physics and Materials Science, City University of Hong Kong, Tat Chee
Avenue, Kowloon, Hong Kong. E-mail: aptjong@cityu.edu.hk
bDepartment of Materials Science and Engineering, South University of Science and
Technology of China, Shenzhen, China
cDepartment of Orthopedics and Traumatology, Li Ka Shing Faculty of Medicine, The
University of Hong Kong, Hong Kong
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cytotoxicity.5–9 The presence of 0.9% NaCl in body uid can also i.e. 50 wt% nHA (29.2 vol% nHA) to achieve an elastic modulus
induce pitting. The chloride ion attacks protective passivation of 6.73 GPa.59 Recently, Wang et al. fabricated PEEK/nHA
lms formed on metallic surfaces.10,11 The movement of an nanocomposites by mechanical mixing PEEK powders with
articial hip prostheses produces metallic wear debris that can nHA rods followed by injection molding.63 They reported that
lead to the secretion of cytokines by macrophages.12 These the fracture strength of all nanocomposites is poorer than that
cytokines then recruit more immune cells to the site of of pure PEEK. The poor performance of such PEEK/nHA
inammation and form giant cells.13 Metallic implants also composites resulted from the use of PEEK powders. In this
experience a stress shielding effect due to their elastic modulus study, we intend to employ nHA and molded-grade PEEK pellets
exceeds far more than that of cortical bones.
to fabricate PEEK/nHA biocomposites using injection molding.
Polymers are attractive materials for fabricating bone To further enhance the mechanical performance of PEEK/nHA
implants because of their good processability and lightness in nanocomposites, carbon nanobers (CNFs) are simulta-
weight.14–19 Polyetheretherketone (PEEK) exhibits
a high neously added to the PEEK/nHA composites. Carbon nanobers
melting temperature (343 ꢀC), high temperature durability, generally exhibit good biocompatibility,64,65 and are very effec-
excellent radiation stability and high Young's modulus tive for reinforcing PEEK.66 Thus, may be feasible to produce
(3.8 GPa).20 Accordingly, PEEK has been increasingly used for biocompatible PEEK/nHA–CNF composites with good
making trauma, orthopedic and spinal xation devices.21–23 mechanical properties commercially in bulk quantities using
From a review study on recent biomedical applications of PEEK the injection molding process.
and its composites reinforced with carbon bers (CFs), PEEK is
currently used for fabricating cervical spine cages and lumbar
spinal fusions, whereas PEEK/CF composites are employed for
acetabular cup bearing components in hip and knee implants.24 Materials
Experimental
In addition, PEEK has also recently found application in
making custom implants for craniofacial surgery.25 PEEK is
Nano-hydroxyapatite rods were purchased from Nanjing
Emperor Nanomaterial (China).
A transmission electron
bioinert, thus it inhibits protein adsorption and cell adhe-
sion.26,27 Its biocompatibility can be improved either via surface
modications28,29 or by adding bioactive hydroxyapatite (HA)
llers in the form of whiskers and particulates.30–34 Abu Bakar
et al. incorporated large HA microparticulates (mHA) into
PEEK.32,33 They reported that the tensile modulus of the
composites increases, whereas the tensile strength decreases
with increasing ller content. The poor interfacial bonding
causes de-cohesion of mHA particles from the PEEK matrix.
In general, large HA particulates oen fracture into small
fragments during tensile loading. Large ller loading levels
oen lead to poor processability of polymer composites.34–38
Recent development in nanotechnology allows scientists to
synthesize various nanomaterials with enhanced chemical,
physical and mechanical properties.39–41 In particular, ceramic
nanomaterials with excellent biocompatibility show large
potential for use in the biomedical sector.42–50 Nanotechnology
opens new opportunities for developing novel biomaterials that
can mimic and reinforce bone tissues.51–53 Furthermore, only
low loading levels of nano-llers are needed for reinforcing
polymers.54–57
microscopy (TEM; Philips CM20) image of nHAs is shown in
Fig. 1. PEEK-Optima pellets and carbon nanobers were bought
from Invibio Company and Nanostructured & Amorphous
Materials Inc. (USA), respectively. Inorganic reagents, such as
CaCl2, NaCl, KCl, KH2PO4, NaHPO4, NaHCO3 and Na2SO4, were
supplied by Sigma-Aldrich Inc. (U.S.A.). They were used directly
without further purication. PEEK pellets and nHA were dried
ꢀ
in an oven at 55 C overnight prior to melt-compounding.
Preparation of nanocomposites
The chemical compositions of binary nHA/PEEK nano-
composites were listed in Table 1. Carbon nanobers were only
added to nHA/PEEK composites with high ller content to
create hybrid composites. The compositions of all composite
Bone tissue is a biocomposite consisting of a collagen matrix
and HA nanorods (nHA) in which cortical bone has an elastic
modulus of 7 GPa.58 Synthetic nHA promotes protein adsorp-
tion and osteoblast growth, leading to osseointegration.45
Therefore, nHA/polymer composites have recently been studied
for their clinical use in load-bearing hip prostheses and bone
tissue engineering.59–62 In a previous study, we have prepared
PEEK composites using nHA and PEEK powders via powder
processing and furnace sintering.59 In that process, both nHA
and PEEK powders are rst mechanically mixed followed by
furnace sintering. However, the powder processing technique is
inadequate to make PEEK nanocomposites with optimal
mechanical properties. It requires high ller loading, Fig. 1 TEM micrograph of HA nanorods.
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Table
nanocomposites
1 The compositions of PEEK/nHA and PEEK/nHA–CNF
seeding and growth 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;
a cell suspension was pipetted at 104 cells per well. The plate
was placed inside an incubator under a humidied atmo-
sphere of 5% CO2/95% air at 37 ꢀC for 1 and 3 days. The
culture medium was changed every two days. Following the
incubation, the samples were washed with phosphate-
buffered saline (PBS), xed with 10% formaldehyde and
dehydrated in a graded series of ethanol baths. Dehydrated
cells were critical point dried and sputter-deposited with gold
in preparation for SEM examination.
nHA
wt%
CNF
wt%
Specimens
vol%
vol%
PEEK
PEEK/4.4 vol% nHA
PEEK/9.3 vol% nHA
PEEK/15 vol% nHA
PEEK/21.5 vol% nHA
PEEK/9.3 vol% nHA–1.6 vol% CNF
PEEK/15 vol% nHA–1.9 vol% CNF
0
10
20
30
40
20
30
0
0
0
0
0
0
2
2
0
0
0
0
0
1.6
1.9
4.4
9.3
15
21.5
9.3
15
Cell viability
samples in this article were expressed in volume percentage.
Dried PEEK pellets and nHA were initially compounded in
a Brabender compounder at a screw rotation speed of 30 rpm
for 45 min. The mixing temperatures of Brabender from hopper
to extrusion die were maintained at 360–380–390–395–380–
360 ꢀC. The extrudates were cut into small pellets by a pelletizer
and fed into the Brabender again for a second mixing under the
same conditions to achieve a homogeneous dispersion of
nanollers in the polymer matrix. The extruded products were
pelletized again, dried overnight in an oven and nally fed into
an injection molder (Toyo TI-50H) to produce dog-bone tensile
bars and circular disks. The disks were mainly used for cell
culture, cell viability and alkaline phosphatase activity
measurements.
Osteoblastic cell viability was assessed using 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. A suspension with 104 cells was
seeded in each well (96-well plate) containing test samples
(number of specimens, n ¼ 5). The plate was incubated in
ꢀ
a humidied atmosphere of 5% carbon dioxide in air at 37 C
for 3, 7 and 10 days. The culture medium was refreshed every
three days. At days 3, 7 or 10, the medium was aspirated. Then,
10 mL of MTT solution (5 mg MTT : 1 mL DMEM) was added to
each well and incubated for 4 h at 37 ꢀC. In the test process, the
tetrazolium ring of MTT salt was cleaved by mitochondrial
enzymes, i.e. succinic dehydrogenase from viable osteoblasts,
forming insoluble formazan crystals. MTT formazan is insol-
uble in water, thus, an organic solvent was needed to solubilize
the crystals. The formazan was dissolved in 10% sodium
dodecyl sulfate (SDS)/0.01 M hydrochloric acid (100 mL). The
absorbance of dissolved formazan was measured at a wave-
length of 570 nm using a multimode detector (Beckman Coulter
DTX 880), with a reference wavelength of 640 nm. The results
were expressed in terms of mean ꢂ standard deviation (SD).
MTT tests were repeated at least twice.
For the WST-1 assay, the samples (n $ 3) were rst cultured
with osteoblasts at 37 ꢀC for 3, 7 and 10 days, respectively. Aer
cell culturing for every prescribed time period, tetrazolium salt
was added to each well followed by incubation for 4 h at 37 ꢀC.
The cleaved tetrazolium product was water soluble, thus elim-
inating the solubilization step as required for the MTT assay.
The amount of formazan formed was directly proportional to
the number of metabolically active cells in the culture. The
amount of water-soluble formazan was quantied by the
absorbance at 450 nm. WST-1 tests were repeated at least twice.
Statistical signicance was assessed by the Student's t-test, with
a signicance level of p < 0.05 as compared to pure PEEK.
Material characterization and tensile tests
The morphological features of the as-fabricated composite
specimens, and adhered osteoblasts on specimen surfaces,
were examined in a SEM (Jeol JSM 820). The composites were
dipped in liquid nitrogen and then fractured by a hammer. The
fractured surfaces were then coated with a thin carbon lm.
Tensile tests were performed at room temperature using an
Instron tester (model 5567) at a crosshead speed of 1 mm
minꢁ1. The elastic modulus of PEEK-based composites was
determined from the linear region of stress–strain curves. Five
samples of each composition were tested, and the average
values were reported.
Thermogravimetric analysis
Thermogravimetric experiments were carried out using the
TGA1 STARe system (Mettler Toledo AG, Switzerland) in
a nitrogen atmosphere from 50 to 800 C at 10 C minꢁ1. The
temperatures at 10% weight loss (T10%) and 30% weight loss
(T30%) were determined from the weight loss vs. temperature
curves.
ꢀ
ꢀ
Alkaline phosphatase activity
Cell culture
Alkaline phosphatase (ALP) is an enzyme secreted by osteo-
Human osteoblasts (Saos-2) were cultured in Dulbecco's blasts during osteogenesis and acts as the marker for their
Modied Eagle's Medium (DMEM) supplemented with 10% differentiation. The ALP activity of each sample (number of
fetal bovine serum, penicillin and streptomycin. Injection specimens, n ¼ 5) was assessed by a colorimetric assay kit (no.
molded disks were sliced into small rectangles for cell 2900-500, Stanbio Laboratory, Boerne, Texas) according to
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the manufacturer's instructions. The kit employed colorless
4-nitrophenyl phosphate as a substrate. The enzyme ALP of
the cells hydrolyzed the substrate to colored 4-nitrophenol
and an inorganic phosphate. In the measurements, test
samples were placed in each well of a 24-well plate. Osteo-
blasts were cultured on the samples for 3, 7 and 14 days. The
culture medium was changed every three days. At selected
days 3, 7 or 14, 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 placing 10 mL of the
supernatant of each sample in a 96-well plate. Finally, p-
nitrophenyl phosphate was added to the plate. The absor-
bance of p-nitrophenol formed was measured using a spec-
trophotometer (Beckman Coulter DTX 880) at 405 nm. The
rate at which p-nitrophenol formed was directly proportional
to ALP activity. The ALP activity was normalized to the DNA
content of the samples. DNA standard (calf thymus DNA,
Ultrapure D-4764; Sigma Aldrich) and Hoechst 33258 dye
(Sigma Aldrich) were used in the tests. Hoechst 33258 nucleic
acid dye emitted blue uorescence at 465 nm when bound to
double-stranded DNA.
Biomineralization test
The PEEK-based nanocomposites were immersed in a simu-
lated body uid (SBF) solution at 37 ꢀC for 21 days for assessing
their bioactivity. This solution was prepared according to the
corrected Kokubo protocol by dissolving the required amounts
of reagent grade chemicals, including NaCl, NaHCO3, KCl, K2-
HPO4$3H2O, MgCl2$6H2O, CaCl2$2H2O and Na2SO4, into
distilled water to obtain desired ion concentrations: Na+ (142
mM), ꢁK+ (5 mM), Ca2+ (2.5 mM), Mg2+ (1.5 mM), Clꢁ (147.8 mM),
ꢁ
2ꢁ
HCO3 (4.2 mM), HPO4 (1 mM) and SO4 (0.5 mM). The pH
value of the solution was adjusted to 7.4 using tris-
(hydroxymethyl)-aminomethane and 1 M HCl at 37 ꢀC.67,68 Aer
immersion, the specimens were removed from the solution,
rinsed with distilled water, dried, and then examined by SEM as
well as by X-ray diffraction (XRD; Bruker, USA) under CuKa
radiation at 30 kV.
Results and discussion
Morphology
Fig. 2a shows the SEM image of the PEEK/4.4 vol% nHA nano-
composite. It can be observed that the nHA llers are dispersed
uniformly in the polymer matrix. The polymer matrix is quite
ductile as characterized by the presence of a rough surface. By
increasing the nHA content to 15 vol%, most nHA llers are still
dispersed homogeneously, but a few of the nHA llers tended to
aggregate to form small agglomerates. For the PEEK/15 vol%
nHA–1.9 vol% CNF composite, CNF llers can be readily
observed in Fig. 2b. As is widely recognized, polymer
Fig. 2 SEM micrographs showing fracture surfaces of (a) PEEK/4.4%
nHA and (b) PEEK/15% nHA–1.9% CNF nanocomposites. Black arrow: Fig. 3 Stress–strain curves of pure PEEK, PEEK/nHA and PEEK/nHA–
CNF; white arrow: nHA.
CNF nanocomposites.
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Fig.
5
Load–displacement curves of pure PEEK and PEEK/nHA
Fig. 4 Tensile strength versus nHA volume content for PEEK/nHA
nanocomposites.
nanocomposites prepared by mechanical mixing PEEK powders with
nHA followed by injection molding.63
nanocomposites are generally reinforced with very low loading
levels of llers (e.g. 0.2–2 vol%) to achieve the desired chemical,
physical or mechanical properties. However, polymer nano-
composites for bone implant applications require the additions
of higher nHA loadings because the polymeric matrix is bio-
inert. Such nHA llers can anchor osteoblasts and promote
their growth on the surfaces of polymer nanocomposites. It
should be noted that the nHA ller content of PEEK/nHA
nanocomposites are much lower than the mHA loadings in
conventional PEEK/mHA micro-composites.
gauge length of the tensile specimen. Therefore, Young's
modulus is determined as the change in stress divided by
change in strain in the linear portion of the stress–strain
curve.70 Therefore, the elastic modulus of their PEEK and PEEK/
nHA composites cannot be determined from the load–
displacement curves, as shown in Fig. 5.
Fig. 6a shows the variation of elastic modulus with nHA
content for PEEK/nHA nanocomposites. The elongation vs. nHA
volume content plot of these nanocomposites is depicted in
Fig. 6b. It can be observed that the stiffness of PEEK/nHA
nanocomposites increases continuously with increasing nHA
content. At 21.5 vol% nHA loading, the stiffness of the PEEK/
nHA composite reaches 7.85 GPa and exceeds the lower limit
of cortical bone with a value of 7 GPa.58 Despite the high stiff-
ness of the PEEK/21.5% nHA nanocomposite, its tensile
strength (44.51 MPa) and elongation at break (0.69%) are lower
than those of human cortical bone. The tensile stress and
fracture strain of cortical bone are 50 MPa and 1%, respectively.
The PEEK/21.5% nHA nanocomposite with a tensile strain of
0.69% is brittle, as expected, and unlikely to be used as the
material for making bone implants. In this respect, particular
attention is paid to the tensile behavior of the PEEK/15% nHA
nanocomposite with tensile strain of 2.71% and tensile stress of
70.56 MPa because both tensile values are higher than those of
cortical bone. The elastic modulus of the PEEK/15% nHA
nanocomposite is 6.2 GPa (Fig. 6). By adding 1.9 vol% CNF to
the PEEK/15% nHA nanocomposite, the modulus increases to
6.54 GPa, whereas the tensile stress and fracture strain also
increase to 71.67 MPa and 2.83%, respectively.
Mechanical properties
Fig. 3 shows the stress–strain curves of pure PEEK, PEEK/nHA
and PEEK/nHA–CNF nanocomposites. The nHA additions
exhibit a benecial effect in enhancing the elastic modulus of
PEEK at the expense of tensile ductility. Furthermore, the
tensile strength of PEEK/nHA nanocomposites increases
slightly with nHA content up to 9.3 vol%; therefore, it decreases
with increasing nHA content (Fig. 4). Thus, 4.4–9.3 vol% nHA
llers can provide mechanical interlocking with the PEEK
matrix, thereby reinforcing the polymer matrix. On the contrary,
the tensile strength of PEEK/nHA nanocomposites as reported
by Wang et al. (Fig. 5) is lower than that of pure PEEK, and falls
rapidly with increasing ller content.63 Such tensile results
indicate the poor load-bearing capacity of the nanocomposites.
The poor performance of their PEEK/nHA composites is
attributed to the use of PEEK powders rather than injection
molded PEEK pellets. PEEK powders are unsuitable for injec-
tion molding purposes for fabricating nanocomposites.
Furthermore, Wang et al. did not employ an extensometer to
measure tensile strain of the specimens during tensile testing.
As known, Young's modulus of a material is determined from
the linear slope of stress–strain curves and not from the linear
slope of the load–displacement curves. From Hooke's law, stress
varies linearly with elastic modulus and strain in the elastic
region.69 To measure strain, an extensometer is attached to the
As aforementioned, metallic implants have many drawbacks
for orthopedic applications, including corrosion problem,
cytotoxicity of metallic ions and stress shielding effect. In the
latter case, the extremely large elastic modulus of metallic
implants (e.g. Ti–6Al-4V alloy (110 GPa) and Co–Cr alloy (240
GPa)) can lead to a stress shielding effect, causing bone loss and
eventual implant loosening. Stress shielding in the femur
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Fig. 7 TGA curves of pure PEEK, PEEK/9.3 vol% nHA and PEEK/15 vol%
nHA–1.9 vol% CNF nanocomposites.
Medical devices must be sterilized to prevent the introduction
of pathogens into the body. On the contrary, polyethylene is
unable to withstand high temperature and pressure conditions
during the sterilization process. Irradiation of polyethylene
results in the scission of molecular chains and the formation of
degradation products.73 In this study, the PEEK/15 vol% nHA–
1.9 vol% CNF bio-nanocomposite with good mechanical
performance shows potential for use as a material for load-
bearing implants in orthopedics.
Thermal stability
Fig. 6 (a) Elastic modulus as a function of filler content, and (b) TGA measurements have been used to provide the substantiate
elongation at break vs. nHA content for PEEK/nHA nanocomposites.
evidence for high thermal stability of PEEK and its nano-
composites (Fig. 7). Highly stable PEEK can prevent thermal
degradation during sterilization. TGA results of representative
occurs when some of the loads are taken by the prosthesis and
shielded from going to the bone. As a consequence, polymeric
materials with low elastic modulus appear to be attractive for
use in orthopedics. High-density polyethylene (HDPE) rein-
forced with 40 vol% mHA particles (HAPEX) has been developed
commercially for biomedical applications. The elastic modulus,
tensile stress and fracture strain of the HAPEX composite are
4.29 GPa, 20.67 MPa and 2.6%, respectively.71 However, the
tensile strength of HAPEX (20.67 MPa) is far below the strength
of cortical bone (50 MPa). Thus HAPEX can only be used as non-
load bearing biocomposites for maxillofacial surgery, orbital
oor prosthesis and middle ear implants.72 In the present study,
the PEEK/9.3 vol% nHA nanocomposite with a favorable
mechanical performance, i.e. elastic modulus of 4.92 GPa,
tensile stress of 81.23 MPa and fracture strain of 7.62%, can
replace HAPEX for use in maxillofacial surgery. Moreover, its
PEEK matrix with the high melting temperature of 343 ꢀC,
excellent radiation stability and low moisture absorption,
provides high levels of resistance for sterilization.20 Therefore,
the molecular chains of PEEK do not degrade either during
autoclave sterilization or gamma-ray radiation treatment.
specimens are listed in Table 2. Apparently, PEEK exhibits very
high T10% and T30% decomposition temperatures, demon-
strating its high thermal stability. The increase of thermal
decomposition temperature is oen regarded as an indicator
for an improvement in thermal stability. The decomposition
tempꢀerature of PEEK is considerably higher than that of HDPE
(335 C).74 Furthermore, the 9.3% nHA addition increases the
ꢀ
T30% of PEEK from 588 to 598 C. Hybridizing CNF with nHA
can further increase the T30% value of PEEK to 645 ꢀC. The
excellent thermal properties of PEEK are attributed to the
stability of its aromatic backbone. PEEK consists of bulky
molecules that cannot volatilize easily. Therefore, weight loss is
not observed until the thermal scission products of PEEK are
Table 2 TGA results of PEEK and representative composites
Specimen
T10% (ꢀC)
T30% (ꢀC)
PEEK
572
577
577
588
598
645
PEEK/9.3% nHA
PEEK/15% nHA–1.9% CNF
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volatilized. Thermal degradation of PEEK involves chain scis- mass when compared with pure PEEK. The mass loss can be
sions of the ether and ketone bonds, giving rise to low molec- further decreased to 30% by incorporating 15% nHA and 1.9%
ular volatiles such as diphenyl ether and phenol at high CNF into PEEK. This result implies that very low CNF loading
temperatures.75,76
markedly improves the thermal stability of PEEK. Carbon
From Fig. 7, PEEK decomposes more intensely above 600 ꢀC, nanobers can generally be classied as multi-walled carbon
resulting in the volatilization of around 45% of the polymer nanotubes with larger diameters (less than 500 nm). The
mass. In the presence of 9.3% nHA, large mass loss also occurs benecial effect of carbonaceous nanomaterials such as carbon
above 600 ꢀC, causing volatilization of about 35% of the polymer nanotubes in enhancing thermal stability of polymers has been
Fig. 8 SEM micrographs of PEEK/9.3% nHA nanocomposite cultured Fig. 9 SEM micrographs of PEEK/15% nHA nanocomposite cultured
with osteoblasts for (a) 1 and (b) 3 days.
with osteoblasts for (a) 1 and (b) 3 days.
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reported in the literature. The stabilization effect of carbon that carbon nanobers promote osteoblast adhesion.65 As
nanotubes on the polymer is explained by a barrier effect of the aforementioned, the MTT assay requires an additional solu-
nanotubes that hinder the diffusion of the degradation prod- bilization step to dissolve colored formazan crystals resulting
ucts from the bulk of the polymer onto the gas phase.77
from the metabolic activity of mitochondria. Such formazan
crystals do not dissolve completely in an organic solvent,
particularly in the presence of carbonaceous nanomaterials
such as carbon nanotubes.78 Those undissolved crystals in
a solvent can lead to low colorimetric results in the spectro-
photometric measurement, resulting in low cell viability. In
contrast, the WST-1 assay obtains water soluble formazan and
would not form insoluble clusters such as MTT. Therefore, the
WST-1 assay yields more reliable results for cell viability.
Fig. 11 shows the cell viability measured by the WST-1 assay for
PEEK-based nanocomposites. It is evident that the WST-1
assay obtains considerably higher cell viability than the MTT
assay. From Fig. 10 and 11, the MTT and WST-1 tests cover 3, 7
and 10 days. At day 3, the results show low cell viability.
Additional 7 and 10 days are needed for testing cell viability to
achieve higher cell viability. In this respect, the difference in
cell viability of each sample becomes more apparent.
It is worth noting that nanomaterials may induce a cytotoxic
effect on biological cells. Grabinski et al. indicated that cyto-
toxicity of carbon materials is dependent on their dimensions.64
Carbon bers (10 mm diameter) and CNF (100 nm diameter) did
not signicantly affect the viability of mouse keratinocytes,
whereas multi-walled carbon nanotubes (10 nm diameter)
reduced cell viability greatly. The cytotoxic action of carbon
nanotubes in general is caused by suspending them indepen-
dently in the cell culture medium. As such, independent carbon
nanoparticles can penetrate cell membranes and reside inside
the cytoplasm.79,80 Our previous study showed that the polymer
matrix of nanocomposites is an effective material for encapsu-
lating CNFs.81 Therefore, CNFs embedded rmly in the polymer
matrix of nanocomposites act as excellent substrates for the
adhesion, growth and viability of osteoblasts.
Cellular adhesion and viability
The adherence of cells to the material surface is evaluated
through SEM imaging. Fig. 8a and b and 9a and b are the SEM
micrographs of PEEK/9.3% nHA and PEEK/15% HA nano-
composites cultured with osteoblasts for 1 and 3 days, respec-
tively. The cells anchor tightly on the surfaces of these
composite specimens aer cultivation for 1 day. The cells then
grow and spread atly on the surfaces such that many neighbor
cells join with each other through cytoplasmic extension aer 3
days of cultivation. Consequently, entire surface of the PEEK/
15% HA nanocomposite is covered with osteoblasts (Fig. 9b).
Bioinert PEEK does not promote osteoblastic adhesion and
proliferation. The nHA llers serve as effective sites for the
adhesion and growth of osteoblasts. SEM images of cell-
cultured nanocomposites at day 1 and 3 are presented herein
to display the difference of cell coverage on the specimen
surfaces. Lower cell attachment is observed at day 1, whereas
nearly or full cell coverage is observed at day 3. Above day 3,
entire surfaces are covered with osteoblasts.
Fig. 10 shows the MTT results for PEEK and its nano-
composites. A dramatic increase in cell viability can be
observed on PEEK/nHA nanocomposites at every test time
period compared to the PEEK control. Furthermore, the
addition of CNF to PEEK/9.3% nHA and PEEK/15% nHA
composites does not impair the viability of osteoblasts. In
contrast, a slight increase in cell viability is found for the
PEEK/9.3% nHA–1.6% CNF and PEEK/15% nHA–1.9% CNF
composites. Comparing with single-walled and multi-
walled carbon nanotubes, carbon nanobers have been re-
ported to have higher cell viability.64 Price et al. also reported
Fig. 10 MTT assay results showing cell viability of human osteoblasts Fig. 11 WST-1 assay results showing cell viability of human osteoblasts
(Saos-2) grown on PEEK and its nanocomposites for 3, 7 and 10 days. * (Saos-2) grown on PEEK and its nanocomposites for 3, 7 and 10 days. *
represents p < 0.05.
represents p < 0.05.
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on carbon nanobers increases with decreasing ber diameter
in the range of 60–200 nm.84
Alkaline phosphatase activity
Alkaline phosphatase is an important component in hard tissue
formation and is highly expressed in mineralized tissue cells.
Proliferating osteoblasts show alkaline phosphatase activity,
and this is greatly enhanced during in vitro bone formation.
Alkaline phosphatase catalyzes hydrolysis of phosphate esters
in alkaline buffer and produces phenol and inorganic phos-
phate. Fig. 12 shows the ALP activity of PEEK and its nano-
composites at days 3, 7 and 14. The ALP enzymatic assay results
reveal good ALP activity level of osteoblasts on the PEEK/nHA
composites at day 14. In particular, ALP activity increases with
increasing nHA content of the composites. This is because nHA
llers promote osteoblastic cell adhesion and differentiation. In
addition, synthetic nHA exhibits excellent osteoconductivity
and biocompatibility. As recognized, osteoblastic cell differen-
tiation plays an important role in osteogenesis, or bone
formation, at early stages.82 ALP is an enzyme produced by cells
that participate in bone tissue mineralization through the
deposition of minerals on the extracellular matrix (ECM)
molecules.
Bioactivity
SBF is a solution that mimics the inorganic ion composition of
human plasma. Apatite-forming ability is one of the major
mechanisms of chemical bioactivity.86 By performing SBF
immersion, the apatite layer forming ability, or bioactivity, of
our PEEK/nHA and PEEK/nHA–CNF nanocomposites can be
assessed. The SBF test is widely used by researchers to evaluate
the capability of test samples to form an apatite layer on their
surfaces.68,86–89 The main difference between SBF and the inor-
ganic component of plasma is the carbonate concentration:
4.2 mM in SBF and 27 mM in plasma. The carbonate deciency
in SBF is compensated by a greater concentration of chloride
From the osteoblastic differentiation model proposed by
Stein and Lian,83 bone cells proliferate greatly up to 7–14 days
and then begin to secrete ECM proteins and produce ALP
differentiation markers for matrix mineralization. Upon
completion of adhesion and proliferation, osteoblastic differ-
entiation is then initiated to secrete proteins, minerals and
collagens for bone tissue mineralization. It is interesting to
observe that the ALP activity of the PEEK/15% nHA nano-
composite can be increased by adding CNF. In a previous study,
the hybridization of nHA ller with CNF promotes osteoblastic
adhesion and proliferation.81 CNFs and multi-walled carbon
nanotubes have been reported to enhance osteoblastic adhe-
sion and differentiation by promoting protein–material inter-
actions.65,84,85 Elias et al. demonstrated that osteoblast adhesion,
proliferation, alkaline phosphatase activity, and ECM secretion
Fig. 13 (a) Plan-view and (b) cross-sectional SEM micrographs of the
Fig. 12 ALP activity normalized to DNA content of human osteoblasts PEEK/9.3% nHA nanocomposite after soaking in SBF solution for 21
(Saos-2) grown on PEEK and its nanocomposites for 3, 7, and 14 days. days.
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ions, therefore the electroneutrality of solution is maintained.68 to negatively charged surfaces. Thus, Ca2+ ions in the SBF are
Fig. 13a is a SEM micrograph showing the morphology of an attracted towards the nHA of the nHA/PEEK nanocomposites.
apatite layer deposited on the surface of the PEEK/9.3% nHA Furthermore, nHA llers also contain Ca2+ ions on their
3ꢁ
nanocomposite aer soaking in SBF solution for 21 days. Many surfaces. As more positive charge is built-up, PO4 ions from
globular apatite particles can be observed on the composite the SBF are also attracted to this site, forming calcium phos-
surface. X-ray energy dispersive spectrum (EDS) of the globular phate deposits that eventually crystallize into an apatite layer.91
apatite reveals the presence of calcium and phosphorus. The A cross-sectional SEM image of the apatite layer formed on the
nHA llers act as nucleation sites for apatite deposition. The surface of this nanocomposite is shown in Fig. 13b. Apparently,
formation mechanism of apatite on the surface of nHA can be a dense apatite layer is deposited on the composite surface aer
attributed to the ion exchange between nHA and the SBF solu- soaking for 21 days. Fig. 14a and b are the SEM micrographs
tion.90,91 The OHꢁ and PO43ꢁ groups of the nHA llers give rise showing plan-view and cross-sectional images of the PEEK/15%
nHA–1.9% CNF nanocomposite. The plan-view micrograph
reveals the presence of more apatite nodules on the surface of
this composite with higher nHA content. The nodules then
form a compact and continuous layer over the surface. A simi-
larly dense apatite layer can be observed on the surface of this
nanocomposite (Fig. 14b), demonstrating its good bioactivity.
Comparing with HA llers of micrometer dimension, the
nHA llers with a large surface area favor formation of more
3ꢁ
apatite nodules as higher Ca2+ ions and PO4
groups are
concentrated on their surfaces. Moreover, nHAs with a large
surface area provides more nucleation sites for apatite nodules.
The nucleating effect of apatite nodules increases with nHA
content in the nanocomposites. Recently, Yang et al. reported
that the incorporation of nHA into polycaprolactone (PCL)
facilitates the precipitation of apatite nodules on their surface
due to the enhanced dissolution of nHA and the subsequent
release of Ca2+ ions, which favors apatite deposition.92
A bone-like hydroxyapatite layer generally obtains a Ca/P
ratio close to 1.65, which is the value reported for bone. The
Ca/P ratio values determined from the EDS proles as shown in
Fig. 13a and 14a are 1.62 and 1.65, respectively. Fig. 15 shows
the XRD pattern of the mineralized layer on the surface of
a representative PEEK/15% nHA nanocomposite aer immer-
sion in SBF for 21 days. The diffraction peaks of PEEK in the
Fig. 14 (a) Plan-view and (b) cross-sectional SEM images of the PEEK/
15% nHA–1.9% CNF nanocomposite after soaking in SBF solution for Fig. 15 XRD pattern of mineralized layer deposited on the PEEK/15%
21 days.
nHA nanocomposite after immersion in SBF for 21 days.
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pattern are indexed.93,94 The small thickness of the deposited
layer induces PEEK peaks. In addition, nHA peaks can be
readily observed in the pattern. All the nHA planes in the
pattern have been indexed in accordance with the Joint
Committee on Powder Diffraction Standards (JCPDS no. 09-
0432) for hydroxyapatite, Ca10(PO4)6(OH)2. From these results,
the mineralized layer formed on the composite surface is
hydroxyapatite.
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Conclusions
In this study, we presented the preparation, biochemical,
mechanical and thermal characterizations of PEEK/nHA and
PEEK/nHA–CNF nanocomposites for bone replacements in
orthopedics. Our results showed that the PEEK/9.3% nHA
nanocomposite with good mechanical, thermal and biological
performances can be considered as a biomaterial for use in
maxillofacial surgery. This nanocomposite exhibited higher
tensile strength than a conventional HAPEX composite rein-
forced with 40 vol% HA micro-particles. Furthermore, tensile
test results revealed that the PEEK/15% nHA–1.9% CNF nano-
composite exhibits comparable tensile properties with human
cortical bones. TGA measurements showed that the incorpora-
tion of nHA ller or CNF and nHA hybrid llers into PEEK
increases the thermal stability. Hybridization of CNF with nHA
llers was found to increase the T30% value of PEEK from 588 ꢀC
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ꢀ
to 645 C, and to effectively reduce mass loss at high tempera-
tures. As such, thermal degradation of the PEEK hybrid nano-
composite can be avoided during sterilization treatment.
Accordingly, PEEK/15% nHA–1.9% CNF nanocomposite shows
high potential for use as a biomaterial for load-bearing
implants.
MTT and WST-1 results demonstrated that cell viability of
osteoblasts increases with increasing nHA content in the PEEK/
nHA nanocomposites. In addition, a PEEK/nHA nanocomposite
with high nHA content (i.e. 15%) exhibited higher ALP activity
compared to pure PEEK. The presence of the nHA mineral
phase enhanced ALP activity, an early marker of bone forma-
tion. Moreover, CNF addition further increased the ALP activity
of the PEEK/15% nHA nanocomposite.
´
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This study is supported by a Strategic Research Grant (Project No.
7004384), City University of Hong Kong, and Shenzhen Science
and Technology Project Grant (JCYJ20150630145302228), China.
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