materials

Review
Thermomechanical Properties of Polylactic
Acid-Graphene Composites: A State-of-the-Art
Review for Biomedical Applications
Ilker S. Bayer
Smart Materials, Istituto Italiano di Tecnologia, 16163 Genoa, Italy; ilker.bayer@iit.it; Tel.: +39-380-387-6699

Received: 19 April 2017; Accepted: 30 June 2017; Published: 4 July 2017

Abstract: Due to its biodegradable and bioabsorbable characteristics polylactic acid (PLA) has
attracted considerable attention for numerous biomedical applications. Moreover, a number of tissue
engineering problems for function restoration of impaired tissues have been addressed by using
PLA and its copolymers due to their biocompatibility and distinctive mechanical properties. Recent
studies on various stereocomplex formation between enantiomeric PLA, poly(L-lactide) (PLLA) and
poly(D-lactide) (PDLA) indicated that stereocomplexation enhances the mechanical properties as
well as the thermal- and hydrolysis-resistance of PLA polymers. On the other hand, biomedical
application of graphene is a relatively new front with significant potential. Many recent reports have
indicated that understanding of graphene-cell (or tissue, organ) interactions; particularly the cellular
uptake mechanisms are still challenging. Therefore, use of graphene or graphene oxide properly
embedded in suitable PLA matrices can positively impact and accelerate the growth, differentiation,
and proliferation of stem cells, conceivably minimizing concerns over cytotoxicity of graphene.
As such, PLA-graphene composites hold great promise in tissue engineering, regenerative medicine,
and in other biomedical fields. However, since PLA is classified as a hard bio-polyester prone
to hydrolysis, understanding and engineering of thermo-mechanical properties of PLA-graphene
composites are very crucial for such cutting-edge applications. Hence, this review aims to present
an overview of current advances in the preparation and applications of PLA-graphene composites
and their properties with focus on various biomedical uses such as scaffolds, drug delivery, cancer
therapy, and biological imaging, together with a brief discussion on the challenges and perspectives
for future research in this field.

Keywords: polylactic acid; polylactide; graphene; PLA; polylactic acid-graphene composite

1. Introduction
Polylactic acid (PLA) polymers are synthesized and obtained from renewable agricultural
resources by polymerization of lactide, which is the cyclic di-ester of lactic acid [1]. For all practical
purposes, lactic acid can be considered as the monomer for PLA [2]. Recent progress in the capability
to manufacture the monomer in a cost effective way from renewable feedstocks has placed PLA
polymers at the forefront of emerging biodegradable soft materials science. Since lactic acid is a
chiral molecule existing in L and D isomers, “polylactic acid” refers to a family of polymers such as
poly-L-lactic acid (PLLA), poly-D-lactic acid (PDLA), and poly-D,L-lactic acid (PDLLA). The L-isomer
is a biological metabolite and constitutes the main fraction of PLA derived from renewable sources
since the majority of lactic acid from biological sources exists in this form [3]. Depending on the
composition of the optically active L- and D, L-enantiomers, PLA can crystallize in three forms (α, β,
and γ). Thorough understanding of the intrinsic structural, thermal, and physicochemical properties
of PLA polymers along with the knowledge of how such properties can be tuned and manipulated
with nanomaterials have fueled remarkable biotechnological interest in PLA nanocomposites [4–6].

Materials 2017, 10, 748; doi:10.3390/ma10070748 www.mdpi.com/journal/materials

Materials 2017, 10, 748 2 of 33
Materials 2017, 10, 748 2 of 33

The extensive
The extensivereview
reviewby by Södergårt
Södergårt and [7]
and Stold Stold [7] presents
presents differentdifferent polymerization
polymerization schemes
schemes associated
with lactic acid chemistry. A typical metal catalyst driven polymerization mechanism of lactic acid of
associated with lactic acid chemistry. A typical metal catalyst driven polymerization mechanism is
lactic acid is schematically illustrated in Figure 1. Dimerization of lactic acid needs a priori
schematically illustrated in Figure 1. Dimerization of lactic acid needs a priori removal of water in the removal
of water of
presence in acidic
the presence of The
catalysts. acidic catalysts.
formation of The
dimerformation
with high ofyield
dimer andwith high yield
selectivity and selectivity
requires the use of
requirescatalysts
special the use which
of special catalysts which
are primarily weaklyare primarily
basic. The useweakly basic.
of tin and The
zinc use of
oxides andtinorgano-stannates
and zinc oxides
and organo-stannates
and -titanates for this and -titanates
purpose haveforbeenthisreported
purpose[8].
have been reported
Although [8]. and
progress Although
recentprogress
advancesand in
recent advances in synthesis mechanisms, biocompatibility and biodegradation
synthesis mechanisms, biocompatibility and biodegradation dynamics of PLA polymers are beyond dynamics of PLA
polymers
the scope ofarethis
beyond theascope
review, number of this review, review
of excellent a number of excellent
articles review
are referred to articles are reading
for further referredonto
for further reading
these aspects [9,10]. on these aspects [9,10].

Figure 1.1. Polymerization

Polymerization of of lactic
lactic acid
acid to
to produce
produce polylactic
polylactic acid
acid (PLA). Lactic acid cannot be
polymerized
polymerized directly
directlyasas
thethe
water produced
water prevents
produced the polymerization
prevents process.process.
the polymerization The acidThe
is therefore
acid is
first dimerized
therefore by heatingbyand
first dimerized afterwards
heating dimer undergoes
and afterwards ring opening
dimer undergoes ringpolymerization using tin
opening polymerization
octanoate as a catalyst.
using tin octanoate as a This method
catalyst. This avoids
methodthe formation
avoids of waterofduring
the formation water polymerization [7].
during polymerization [7].

PLA polymers
PLA polymershave havealready
alreadybeen been demonstrated
demonstrated as surgical
as surgical implant implant
materialsmaterials
[11–13],[11–13], drug
drug delivery
delivery systems [14,15], guided tissue and bone regeneration platforms,
systems [14,15], guided tissue and bone regeneration platforms, and also as porous scaffolds forand also as porous scaffolds
for the
the growth
growth of neo-tissue
of neo-tissue [16,17].
[16,17]. Use UseofofPLA
PLAininbiomedical
biomedicalapplications
applicationsisisnot not based
based only
only onon its
its
biodegradability but also on its thermo-mechanical properties including
biodegradability but also on its thermo-mechanical properties including its suitability for shape its suitability for shape
memory effects
memory effects [18].
[18]. Hence,
Hence, various
various medical
medical devices
devices have
have been
been prepared
prepared from different PLA
from different PLA types
types
including degradable
including degradablesutures,
sutures,drug drug releasing
releasing microscale
microscale particles,
particles, nanoparticles,
nanoparticles, and porous andscaffolds
porous
scaffolds for cellular applications [19]. Copolymers of PLA are also very
for cellular applications [19]. Copolymers of PLA are also very attractive biodegradable materials attractive biodegradable
materials
for medicine for [20,21].
medicine For[20,21].
instance,Forbiodegradation
instance, biodegradation of poly(lactic-co-glycolic
of poly(lactic-co-glycolic acid) (PLGA) acid) (PLGA)
occurs by
occurs by hydrolysis leading to metabolite monomers, lactic acid, and glycolic
hydrolysis leading to metabolite monomers, lactic acid, and glycolic acid. Because these two monomers acid. Because these
twoendogenous
are monomers are andendogenous and easily
easily metabolized metabolized
by the body via the byKrebs
the body
cycle,via the Krebs
a minimal cycle, atoxicity
systemic minimal is
systemic toxicity is associated with the use of PLGA as biomedical
associated with the use of PLGA as biomedical material and drug releasing vehicle [22]. material and drug releasing
vehicle [22]. to Rasal et al. [23], PLA polymers have a number of limitations. These can be summarized
According
According to Rasal et limitations,
in three groups: mechanical al. [23], PLA polymers have
biodegradation ratesaand number of limitations.
byproducts, chemical These
reactivitycanand
be
summarized
hydrolytic in three groups:
degradation. PLA ismechanical
very brittlelimitations, biodegradation
polymer. Although rates
its tensile and byproducts,
strength and elastic chemical
modulus
are comparable to poly(ethylene terephthalate) its poor toughness limits its use intensile
reactivity and hydrolytic degradation. PLA is very brittle polymer. Although its strength
applications and
where
elastic modulus are comparable to poly(ethylene terephthalate) its poor toughness
plastic deformation at higher stress levels is required such as implant screws and fracture fixation plates. limits its use in
applications
For instance,where plastic
when poly- deformation
L -lactic at higher stress
acid interference screws levels
wereisused
required suchfixation
for graft as implant
duringscrews and
anterior
fracture fixation plates. For instance, when poly- L-lactic acid interference screws
cruciate ligament (ACL) reconstruction, the devices were reported to be mechanically weaker than their were used for graft
fixation counterparts
metallic during anterior and cruciate ligament
often fractured (ACL)
during reconstruction,
implantation the devices
[23]. Secondly, were reported rate
its biodegradation to be
is
mechanically weaker than their metallic counterparts and often fractured during
considered to be quite slow (3 years or more), which depends on its crystallinity and molecular weight. implantation [23].
Secondly,
This causesitsconcerns
biodegradation rate is considered
over secondary to be quitefor
surgical procedures slow (3 years
implants or more),
made with PLAwhich depends
[24]. However, on
its crystallinity
recent and molecular
studies indicate that laserweight. This causes
treatment of PLA concerns
polymers overcansecondary
accelerate surgical procedures for
their biodegradation
implants made with PLA [24]. However, recent studies indicate that
rates [25]. Moreover, acidic byproducts during its degradation in vivo have been causing laser treatment of PLA polymers
concerns
can accelerate their biodegradation rates [25]. Moreover, acidic byproducts during its degradation in
vivo have been causing concerns in surgical applications [26,27]. Thirdly, an important limitation is
Materials 2017, 10, 748 3 of 33

in surgical applications [26,27]. Thirdly, an important limitation is its water sensitivity, which is
reflected through interaction with moisture, resulting in hydrolytic destruction or hydrolysis [28,29].
Nonetheless, aforementioned limitations can be addressed by using PLA copolymers, adding bio-based
plasticizers, designing PLA nanocomposites, grafting other polymers [30] and blending PLA polymers
with faster biodegrading polymers or enzymes to name but a few [31]. Stereo-complexes of PLLA
and PDLA deserve special attention due to the fact that PLA stereo-complexes have higher melting
temperature (or heat resistance), mechanical performance, and hydrolysis-resistance compared to
those of neat PLLA and PDLA [32].
Graphene, on the other hand, has been at the forefront in biotechnological applications [33–36].
Graphene is a free-standing 2D crystal with one-atom thickness. It is in fact an allotrope of carbon
comprising layers of six-atom rings in a honeycombed network and can be theoretically viewed as a
true planar aromatic macromolecule [37,38]. Graphene exhibits remarkable properties [39], such as
unusual electronic flexibility as well as high planar surface area (~2630 m2 /g), outstanding mechanical
strength (Young’s modulus, ~1100 GPa) and unparalleled thermal conductivity (~5000 W/m/K).
As such, it is a highly popular nanoscale additive for reinforced biopolymers [40]. Biosafety of
graphene and graphene derivatives is attracting more and more attention as we need to learn the
fate of graphene and its derivatives in vivo once it invasively enters into a biological system [39–41].
It has now been acknowledged that the biological effect of graphene is complex and largely affected by
the exposure dose and methods as well as the chemical functionalization, lateral size, thickness, and
surface properties [42,43].
Among graphene derivatives, graphene oxide (GO) has been widely explored for in vitro and
in vivo drug delivery and imaging, taking advantage of its high solubility and stability in physiological
solutions, low cost and scalable production, and facile biological/chemical functionalization [41–44].
Several studies reported good biocompatibility [45,46]. Nevertheless, granuloma formation,
inflammation, and thrombus formation in mice have also been observed after GO exposure [47,48].
Both graphene and graphene oxide can be adapted to function as smart biomedical devices as long
as their surfaces are properly functionalized. Figure 2, for instance, schematically illustrates several
potential functionalization approaches to render graphene and graphene oxide cytocompatible both
in vitro and in vivo. Among them proteins, small biological molecules, DNA and peptides are very
popular and promising. Note that ideally graphene is a single atomic layer planar material but almost
all the polymer nanocomposites made with graphene utilize flakes that are made up of many layers
of “real grapheme” bundled stacked up form. They are generally referred to as graphene platelets,
graphene nanoplatelets, graphene nanosheets, etc. and are denoted by GPs, GnPs, xGnPs, etc.
As such, thermo-mechanical properties of PLA polymers are directly related to their biomedical
performance when interfaced with biological systems, since these properties can be used to optimize
important design criteria such as modulus, strength, morphology, crystallinity, biocompatibility, and, in
turn, these properties can affect cell response, tissue regeneration, and in vivo degradation. Moreover,
recent research indicates that thermal effects on the crystallinity of not only PLA polymers but also other
bio-polyesters is very critical for cell biocompatibility and drug release dynamics [49]. Since, nowadays
graphene and its derivatives have been shown to influence thermo-mechanical properties of many
semi-crystalline plastics including biopolymers, carefully designed and engineered nanocomposites
of PLAs with graphene and its derivatives can be used to remediate certain structural, thermal,
and degradation related drawbacks of PLA polymers making them more attractive for biomedical
applications. Moreover, research efforts on biopolymer-graphene/GO-based scaffold materials for cell
culture is an ever expanding field that deserves special attention since graphene and GO are able to
accelerate growth, differentiation, and proliferation of stem cells, and therefore hold great promise in
tissue engineering, regenerative medicine, and other biomedical fields.
Materials 2017, 10, 748 4 of 33
Materials 2017, 10, 748 4 of 33

Figure 2. Although graphene oxide has been demonstrated to be cytocompatible in vitro, its
Figure 2. Although graphene oxide has been demonstrated to be cytocompatible in vitro, its compatibility
compatibility in vivo in tissue sites relevant for biomedical device application is yet to be fully
in vivo in tissue sites relevant for biomedical device application is yet to be fully understood. Promising
understood. Promising results may be achieved by proper functionalization. Graphene and its
results may be achieved by proper functionalization. Graphene and its derivatives have been reported
derivatives have been reported to be functionalized with avidin–biotin, peptides, nucleic acid,
to be functionalized with avidin–biotin, peptides, nucleic acid, proteins, aptamers, small molecules,
proteins, aptamers, small molecules, bacteria, and cells through physical adsorption or chemical
bacteria, and cells through physical adsorption or chemical conjugation. Reproduced from [37,45].
conjugation. Reproduced from [37,42].

2. Thermal Effects on the Crystallinity of PLA Polymers

2. Thermal Effects on the Crystallinity of PLA Polymers
One of the most important performance comparison criteria between biodegradable polymers and
One of the most important performance comparison criteria between biodegradable polymers
conventional oil-based polymers is thermal stability along with moisture retention issues. Although
and conventional oil-based polymers is thermal stability along with moisture retention issues.
mechanical properties of PLA polymers match those of poly(ethylene terephthalate) (PET) polyesters,
Although mechanical properties of PLA polymers match those of poly(ethylene terephthalate) (PET)
for instance, their thermal stability is not as high as PET polymers [50]. Thermal stabilization of PLA
polyesters, for instance, their thermal stability is not as high as PET polymers [50]. Thermal
polymers are closely related to their crystallization behavior. Moreover, PLA’s wider application
stabilization of PLA polymers are closely related to their crystallization behavior. Moreover, PLA’s
has been limited by its relatively slow crystallization rate from the practical application viewpoint.
wider application has been limited by its relatively slow crystallization rate from the practical
However, new recent research shows that crystallization of PLA polymers can be tuned by preparing
application viewpoint. However, new recent research shows that crystallization of PLA polymers can
homo-composites (based purely on PLA polymers with different structures) [51].
be tuned by preparing homo-composites (based purely on PLA polymers with different structures) [51].
A comprehensive review by Tan et al. [52] illustrates that diverse isomeric forms of PLA could
A comprehensive review by Tan et al. [52] illustrates that diverse isomeric forms of PLA could
provide great opportunities for thermal and mechanical enhancement through stereo-complexation.
provide great opportunities for thermal and mechanical enhancement through stereo-complexation.
In their review, recent developments in thermal and mechanical enhancement of PLA via
In their review, recent developments in thermal and mechanical enhancement of PLA via stereo-
stereo-complexation in different polymeric systems were presented. They include enantiomeric
complexation in different polymeric systems were presented. They include enantiomeric PLA
PLA homopolymers, PLA-based block and graft copolymers, as well as enantiomeric PLA materials
homopolymers, PLA-based block and graft copolymers, as well as enantiomeric PLA materials
having unique architectures such as cyclic, star, dendritic and comb-shaped morphologies. Insightful
having unique architectures such as cyclic, star, dendritic and comb-shaped morphologies. Insightful
discussions on the influence of crystal structure and intermolecular interactions between PLLA and
discussions on the influence of crystal structure and intermolecular interactions between PLLA and
PDLA in different polymeric systems on performance enhancement of the resultant materials were
PDLA in different polymeric systems on performance enhancement of the resultant materials were
discussed. The “enhanced” PLA polymers with diverse functions oriented toward engineering
discussed. The “enhanced” PLA polymers with diverse functions oriented toward engineering
materials and their biomedical significance in various areas were also covered in their review. Figure 3a
materials and their biomedical significance in various areas were also covered in their review. Figure
schematically shows that almost all mechanical, thermal, and hydrolytic properties of PLA polymer
3a schematically shows that almost all mechanical, thermal, and hydrolytic properties of PLA
can be improved by stereo-complexation. As an example, incorporating stereo-complex PLA in PDLLA
polymer can be improved by stereo-complexation. As an example, incorporating stereo-complex PLA
polymer, thus forming homocomposites, grants unprecedented shape recovery ability to the neat
in PDLLA polymer, thus forming homocomposites, grants unprecedented shape recovery ability to
PDLLA resin (see Figure 3b).
the neat PDLLA resin (see Figure 3b).
Materials 2017, 10, 748 5 of 33
Materials 2017, 10, 748 5 of 33

Figure 3. (a) Schematic representation of thermal and mechanical enhancement possibilities of PLA
Figure 3. (a) Schematic representation of thermal and mechanical enhancement possibilities of PLA via
via stereo-complexation of PLA in different polymeric systems, including enantiomeric PLA
stereo-complexation of PLA in different polymeric systems, including enantiomeric PLA homopolymers
homopolymers and PLA-based block and graft copolymers, as well as enantiomeric PLA materials;
and PLA-based block and graft copolymers, as well as enantiomeric PLA materials; (b) Photographs of
(b) Photographs of the shape recovery of neat PDLLA and the blend samples of PDLLA with
the shape recovery of neat PDLLA and the blend samples of PDLLA with increasing stereo-complex
increasing stereo-complex PLA loadings [52].
PLA loadings [52].
Hirata and Kimura [53] conducted a careful experimental study on thermomechanical properties
ofHirata
stereoblock PLAs with
and Kimura [53] different
conducted PLLA/PDLA block compositions.
a careful experimental study on They synthesized stereoblock
thermomechanical properties
PLAs (sb-PLAs) having abundant enantiomeric compositions of PLLA
of stereoblock PLAs with different PLLA/PDLA block compositions. They synthesized stereoblock by solid-state polycondensation
PLAsof (sb-PLAs)
the melt blends
havingof mediumenantiomeric
abundant molecular weight prepolymers.
compositions of PLLA Theirby stereoblock PLA chemical
solid-state polycondensation
synthesis
of the method
melt blends is shownmolecular
of medium in Figure 4a. Resulting
weight sb-PLAs were
prepolymers. Theircast into polymer
stereoblock PLAfilms by solution
chemical synthesis
deposition. Dynamic mechanical analysis measurements signified the retention
method is shown in Figure 4a. Resulting sb-PLAs were cast into polymer films by solution deposition. of storage modulus
above melting point Tm of homo-chiral crystals (160 °C). They argued that PLLA-rich sb-PLAs can be
Dynamic mechanical analysis measurements signified the retention of storage modulus above melting
used as more robust and high-performance resins compared to PLA; minimizing the use of D-lactic
point Tm of homo-chiral crystals (160 ◦ C). They argued that PLLA-rich sb-PLAs can be used as more
acid which is more expensive than L-lactic acid.
robust and high-performance resins compared to PLA; minimizing the use of D-lactic acid which is
Yuryev et al. [54] studied surface crystallinity on films of poly(L-lactide), poly(L/D-lactide) and
more expensive
their thanpoly(
blends with L -lactic acid.
D-lactide). The isothermal spherulitic crystal growth rate and its dependence
Yuryev et al. [54] studied
on temperature were measured surface
usingcrystallinity
tapping mode onatomic
films of poly(
force L -lactide),
microscopy poly(
and L / Disothermal
ex-situ -lactide) and
theircrystallization.
blends with poly( Further, they also investigated the crystallization kinetics of blends of poly(dependence
D -lactide). The isothermal spherulitic crystal growth rate and its L-lactide)
on temperature were
and poly(L/D-lactide) measured using
with poly( tapping The
D-lactide). mode atomiccrystalline
typical force microscopy
structureand ex-situ polymer
of PDLA isothermal
crystallization.
annealed at 100 Further,
°C cantheybe seenalso
ininvestigated
the atomic force themicroscope
crystallization
imagekinetics
in Figure of4b.
blends of poly(
Resultant L -lactide)
crystalline
anddomain
poly(L/growth
D-lactide)ratewith
as a poly( D-lactide).
function of PDLAThe typical
in PLLA is crystalline structure
shown in Figure of any
4c. At PDLAgivenpolymer annealed
temperature
below◦
at 100 C can be seen in the atomic force microscope image in Figure 4b. Resultant crystallineofdomain
150 °C, adding more PDLA (as percentage in stereoblock polymer) results in lowering the
spherulite growth rate compared to pure PLLA. These polymers are still
growth rate as a function of PDLA in PLLA is shown in Figure 4c. At any given temperature below rich in PLLA (not more than
150 20% PDLA; more
◦ C, adding see Figure
PDLA 4c)(as
butpercentage
with muchin better crystalline
stereoblock structure.
polymer) results in lowering of the spherulite
Perego et al. [55] analyzed several samples
growth rate compared to pure PLLA. These polymers are still rich in of PLA polymers with
PLLA different molecular
(not more than 20%weights
PDLA;
and tacticity to investigate the effect of molecular weight and crystallinity on mechanical properties.
see Figure 4c) but with much better crystalline structure.
Injection molded PLA specimens were annealed to promote their crystallization. From the
Perego et al. [55] analyzed several samples of PLA polymers with different molecular weights and
characterization data, PLLA showed more interesting mechanical properties than PDLA, and its
tacticity to investigate the effect of molecular weight and crystallinity on mechanical properties.
behavior significantly improved with crystallization. This is attributed to the stereo-regular nature of
Injection
the poly(molded PLA specimens were annealed to promote their crystallization. From the
L-lactic acid) chain. Thermally annealed specimens retained higher values of tensional and
characterization
flexural modulus data,of PLLA showed
elasticity, impactmore interesting
strength, and heat mechanical
resistance. properties than PDLA,
They also showed and its
that while
behavior significantly improved with crystallization. This is attributed
PDLLA and amorphous PLLA presented a heat resistance very near to their glass transition to the stereo-regular nature
of the poly( L -lactic acid) chain. Thermally annealed specimens retained
temperature (Tg), annealed PLLA demonstrated a better behavior in the sense that it was more stable higher values of tensional
andatflexural modulus
temperatures of 50of °Celasticity, impact
or higher, due to itsstrength,
crystallinityandinduced
heat resistance.
stability. They also showed that
while PDLLA and amorphous PLLA presented a heat resistance very near to their glass transition
temperature (Tg ), annealed PLLA demonstrated a better behavior in the sense that it was more stable
at temperatures of 50 ◦ C or higher, due to its crystallinity induced stability.
Materials 2017, 10, 748 6 of 33
Materials 2017, 10, 748 6 of 33

Figure 4. (a) Synthesis of sb-PLAs with high-molecular weight by solid-state polycondensation (SSP)
Figure 4. (a) Synthesis of sb-PLAs with high-molecular weight by solid-state polycondensation (SSP)
of the melt blend (PLLA/PDLA); (b) Typical crystalline structure of PDLA annealed at ◦110 °C.
of the melt blend (PLLA/PDLA); (b) Typical crystalline structure of PDLA annealed at 110 C. The
The square image size is 100 μm; (c) Isothermal spherulite radius growth rates for PLLA and its blends
square image size is 100 µm; (c) Isothermal spherulite radius growth rates for PLLA and its blends
with PDLA.
with PDLA. Reproduced
Reproduced from
from [53,54].
[53,54].

In a highly cited work, Carrasco et al. [56] processed PLA resin with injection and
In a highly cited work, Carrasco et al. [56] processed PLA resin with injection and extrusion/injection
extrusion/injection as well as annealing. These thermally processed PLAs were studied in order to
as well asthe
analyze annealing.
variationsThese thermally
in chemical processed
structure, PLAs were
thermal studied inand
degradation, order to analyzeproperties
mechanical the variations
(see
Figure 5). Processing of PLA demonstrated evidence for a decrease in molecular weightProcessing
in chemical structure, thermal degradation, and mechanical properties (see Figure 5). due to chainof
PLA demonstrated evidence for a decrease in molecular weight due to
scission. They found that mechanical processing led to quasi disappearance of crystal structurechain scission. They found
that mechanical
whereas it was processing
recovered led afterto post-annealing.
quasi disappearance Afterofcareful
crystal analysis
structureofwhereas
Fourierit transform
was recovered and
after post-annealing. After careful analysis of Fourier transform
Nuclear Magnetic Resonance spectroscopy chemical shifts and peak areas, they affirmed and Nuclear Magnetic Resonance
that the
spectroscopy
chemical chemicalofshifts
composition PLA and peakchange
did not areas, after
they processing,
affirmed that butthethechemical
proportion composition
of methyl of PLA
groups
did not change after processing, but the proportion of methyl groups increased,
increased, thus indicating the presence of a different molecular environment. In fact, they showed thus indicating the
presence of a different molecular environment. In fact, they showed that annealed
that annealed specimens possessed higher values of tensional and flexural modulus of elasticity, Izod specimens possessed
higher values
impact strength,of tensional and flexural
and heat resistance. modulus
After of elasticity,
annealing, samples Izod impact
showed anstrength,
increase and heat resistance.
in Young modulus
After annealing, samples showed an increase in Young modulus (5–11%) and
(5–11%) and in yield strength (15–18%), which was explained by the higher degree of crystallinity in yield strength (15–18%),of
which was explained by the higher degree of crystallinity of annealed
annealed materials, with a subsequent decrease in chain mobility. Extruded/injected polymers materials, with a subsequent
decreaseainsignificant
showed chain mobility.
increase Extruded/injected
in elongation at breakpolymers showed
(32–35% more),a significant
comparedincrease
to injectedin elongation
materials.
This was attributed to a higher number of chains due to chain scissions in reprocessednumber
at break (32–35% more), compared to injected materials. This was attributed to a higher materialsof
chains
(see due to
Figure 5).chain scissions in reprocessed materials (see Figure 5).
As can be
As can be seen
seen from
from this
this section,
section, crystallization
crystallization of of PLA
PLA polymers
polymers is is aa very
very complex
complex thermal
thermal
process. Many factors such as post annealing, aging, stereocomplexation,
process. Many factors such as post annealing, aging, stereocomplexation, cooling or heating rates, cooling or heating rates,
plasticizers not only
plasticizers not only play
play aa significant
significant role
role in
in the
the crystallization temperature but
crystallization temperature but also
also in in the
the shape,
shape,
size, type, and density of the crystals that would nucleate and form, eventually
size, type, and density of the crystals that would nucleate and form, eventually determining the final determining the final
thermomechanical properties of the
thermomechanical properties of the polymer. polymer.
Materials 2017, 10, 748 7 of 33
Materials 2017, 10, 748 7 of 33

Figure 5. (a) Representative stress vs. strain curves from tensile testing for processed PLA polymers.
Figure 5. (a) Representative stress vs. strain curves from tensile testing for processed PLA polymers.
Nomenclature; PLA-I: injection molded; PLA-EI: extruded and injection molded; PLA-IA: injection
Nomenclature; PLA-I: injection molded; PLA-EI: extruded and injection molded; PLA-IA: injection
molded and annealed PLA-EIA: extruded, injection molded and annealed; (b) Physical appearance of
molded and annealed PLA-EIA: extruded, injection molded and annealed; (b) Physical appearance of
the thermo-processed specimens [56].
the thermo-processed specimens [56].
3. Effect of PLA Polymers Crystallinity on Cells
3. Effect of PLA Polymers Crystallinity on Cells
The unique crystalline properties of PLA polymers render them attractive platforms for
The unique
controlling crystalline
cellular properties
behavior. In fact,of PLA polymers render
homo-composites of PLAthem attractive
polymers withplatforms
different for controlling
crystallinity
cellular behavior. PLA
or engineering In fact, homo-composites
polymers of PLAgradients
with crystallinity polymershavewithbeen
different crystallinity
inspired or engineering
by the occurrence of
PLA polymers
natural tissueswith crystallinity
presenting gradientsvariations
continuous have beenofinspired
propertiesby the
alongoccurrence of natural
one direction, suchtissues
as
ligament-to-bone,
presenting continuous tendon-to-bone,
variations of andproperties
dentin-to-enamel
along interfaces [57,58].such
one direction, The pioneering work by
as ligament-to-bone,
Simon Jr. et al.and
tendon-to-bone, [58] dentin-to-enamel
demonstrated a rapid method
interfaces for screening
[57,58]. cell proliferation
The pioneering work byon PLLA/PDLLA
Simon Jr. et al. [58]
blends. Strip-shaped
demonstrated films containing
a rapid method for screening a gradient in polymer
cell proliferation composition were
on PLLA/PDLLA blends.prepared and
Strip-shaped
characterized. Cells were cultured on the films and adhesion and proliferation
films containing a gradient in polymer composition were prepared and characterized. Cells were were assessed using
high-throughput,
cultured on the filmsautomated
and adhesion microscopy. More specifically,
and proliferation they combined
were assessed automated fluorescence
using high-throughput, automated
microscopy with a combinatorial approach for creating polymer
microscopy. More specifically, they combined automated fluorescence microscopy with blend gradients to yield a rapid
a combinatorial
screening
approach formethod
creating forpolymer
characterizing
blend cell proliferation
gradients to yieldonapolymer blends. method for characterizing
rapid screening
Strip-shaped blend films were annealed to allow PLLA to crystallize as shown in Figure 6a.
cell proliferation on polymer blends.
Fourier transform infrared (FTIR) micro-spectroscopy was used to determine the composition in the
Strip-shaped blend films were annealed to allow PLLA to crystallize as shown in Figure 6a.
gradients and atomic force microscopy was used to characterize surface topography (also shown in
Fourier transform infrared (FTIR) micro-spectroscopy was used to determine the composition in the
Figure 6a). Osteoblasts were cultured on the gradients and proliferation was assessed by automated
gradients and atomic force microscopy was used to characterize surface topography (also shown in
counting of cells using fluorescence microscopy as shown in Figure 6c; whereas Figure 6b
Figure 6a). Osteoblasts were cultured on the gradients and proliferation was assessed by automated
demonstrates their control experiments on glass slides. Surface roughness varied with composition,
counting of cellsregions
PDLLA-rich using fluorescence
were smooth microscopy as shown inregions
whereas PLLA-rich Figure 6c;
werewhereas
rough.Figure 6b demonstrates
The enhanced cell
their control experiments on glass slides. Surface roughness varied with
proliferation on PDLLA-rich regions of the gradients correlated with this smooth topography composition, PDLLA-rich
but the
regions
authorswerecouldsmooth whereas
not exactly PLLA-rich
conclude regions
whether thiswere
was rough.
a result The enhanced
of blend cell proliferation
composition or surfaceon
PDLLA-rich regions of the gradients correlated with this smooth topography
roughness since both parameters had been varied across the gradients. They argued that for the case but the authors could
notofexactly concludeblends,
PLLA-PDLLA whether this was
smooth a result of blends
PDLLA-rich blend composition
could be best or surface roughness
suited for supporting sincecell
both
parameters hadAdhesion
proliferation. been varied was across
similarthe gradients.
in all regions ofThey argued that
the gradients afterfor
onethe case
day of PLLA-PDLLA
of observation but
blends, smooth
after five days PDLLA-rich
proliferation wasblends could be
enhanced onbest suited for supporting
the PDLLA-rich ends of the cell proliferation. Adhesion
gradients.
was similar in all regions of the gradients after one day of observation but after five weight
Sladago et al. [59] reported a study to evaluate how crystallinity and molecular of PLLA
days proliferation
wasinfluenced
enhancedthe patterns
on the of cell ends
PDLLA-rich adhesion,
of theproliferation,
gradients. and cell morphology parameters. Four
conditions
Sladago were tested:
et al. [59] low molecular
reported a study to weight amorphous
evaluate and semi-crystalline
how crystallinity and molecular hot-pressed
weight of PLLA
PLLA
disks (PLLA1-A and PLLA1-SC), and high molecular weight amorphous and semi-crystalline
influenced the patterns of cell adhesion, proliferation, and cell morphology parameters. Four conditions hot-
pressed
were tested:PLLA disks (PLLA2-A
low molecular weightand PLLA2-SC).
amorphous and For the cell culture
semi-crystalline studies they
hot-pressed PLLA chose
disksa (PLLA1-A
human
osteosarcoma cell line (SaOS-2). Disks were immersed in a cell suspension
and PLLA1-SC), and high molecular weight amorphous and semi-crystalline hot-pressed PLLA disks containing 5 × 10 4 cells/mL

and kept in culture for periods up to two weeks. They concluded that cell viability was not affected
(PLLA2-A and PLLA2-SC). For the cell culture studies they chose a human osteosarcoma cell line
(SaOS-2). Disks were immersed in a cell suspension containing 5 × 104 cells/mL and kept in culture
Materials 2017, 10, 748 8 of 33

Materials 2017, 10, 748 8 of 33

for periods up to
Materials 2017, 10,two
748 weeks. They concluded that cell viability was not affected by the different 8 of tested
33
conditions. However, cell proliferation was increased in the high molecular weight amorphous
by the different tested conditions. However, cell proliferation was increased in the high molecular samples
by
andweighttheseemed
cells different to tested
have conditions.
higher However,
adhesion cell proliferation
patterns was increased in the
on semi-crystalline high molecular
amorphous samples and cells seemed to have higher adhesion samples.
patterns onThey attributed
semi-crystalline these
weight
observations amorphous
to different samples and
ratesthese cells
of integrin seemed to
interactionhave higher adhesion
with therates
substrate patterns on semi-crystalline
leadinginteraction
to different patterns
samples. They attributed observations to different of integrin with the of
samples. They attributed these observations to different rates of integrin interaction with the
focalsubstrate
adhesion point to
leading formation. Figure 7ofshows
different patterns a summary
focal adhesion of formation.
point their results including
Figure 7 shows scanning electron
a summary
substrate leading to different patterns of focal adhesion point formation. Figure 7 shows a summary
of their results
micrographs of theincluding
cells onscanning
the PLLAelectron micrographs
surfaces of the cells on the PLLA surfaces (Figure 7a,b).
(Figure 7a,b).
of their results including scanning electron micrographs of the cells on the PLLA surfaces (Figure 7a,b).

Figure
Figure 6. (a)
6. (a) Top:Top:
AnAn image
image ofofthe
thebirefringence
birefringence from
from aa PLLA-PDLLA
PLLA-PDLLAgradient gradientis shown. TheThe
is shown. higher
higher
Figure 6. (a) Top: An image of the birefringence from a PLLA-PDLLA gradient is shown. The higher
crystallinity
crystallinity of the
of the PLLA-rich
PLLA-rich end endof of
thethe gradient
gradient causesitittotobebemore
causes morebirefringent
birefringent than
than the
the PDLLA-rich
PDLLA-
crystallinity of the PLLA-rich end of the gradient causes it to be more birefringent than the PDLLA-
end.rich
The
rich
end.
end.
The gradient
gradient shown
The gradient
shown
is 52isis
shown mm52 mm
long.
52 mm
long. Bottom:FTIR-reflectance-transmission
Bottom:
long.
FTIR-reflectance-transmission microspectroscopy
microspectroscopy
Bottom: FTIR-reflectance-transmission microspectroscopy
(RTM)
(RTM) map
mapmap of a PLLA-PDLLA
of aofPLLA-PDLLA composition
composition gradient. The strip film has been outlined withwith
a black
(RTM) a PLLA-PDLLA compositiongradient. Thestrip
gradient. The stripfilm
filmhas
hasbeen
been outlined
outlined a black
with a black
line. Blue corresponds to PDLLA rich regions and orange to PLLA-rich regions (see color bar below
line.line.
BlueBlue
corresponds
corresponds to to
PDLLA
PDLLArich richregions
regions and
and orange
orange to toPLLA-rich
PLLA-richregions
regions (see
(see color
color barbar
belowbelow
map); (b) Cell adhesion and proliferation on the PLLA-PDLLA gradients and on control glass slides.
map); (b) Cell
map); adhesion
(b) Cell adhesion and
andproliferation
proliferationon on the
the PLLA-PDLLA
PLLA-PDLLA gradients gradientsandandonon control
control glass
glass slides.
slides.
Adhesion data for 1 day and for proliferation data for 4 days are plotted; (c) Same data as b on the
Adhesion
Adhesiondatadataforfor
1 day
1 dayandandforforproliferation
proliferation data for 44 days
data for daysare areplotted;
plotted;(c)(c) Same
Same data
data as basonb the
on the
PLLA-PDLLA gradient substrate [58].
PLLA-PDLLA
PLLA-PDLLA gradient
gradientsubstrate
substrate [58].
[58].

Figure 7. SEM micrographs of human osteoblasts like cells after two weeks in culture (a) amorphous
Figure 7. SEM micrographs of human osteoblasts like cells after two weeks in culture (a) amorphous
PLLA1;
Figure (b) micrographs
7. SEM semi-crystalline
of PLLA1;
human (c) Cell viability
osteoblasts measured
like cells fromweeks
after two the optical density
in culture (a)(O.D.) at
amorphous
PLLA1; (b) semi-crystalline PLLA1; (c) Cell viability measured from the optical density (O.D.) at
490 (b)
PLLA1; nm;semi-crystalline
and (d) Cell proliferation
PLLA1; (c) assays on SaOS-2
Cell viability cells grown
measured from theonoptical
PLLA density
disks, (PLLA1—low
(O.D.) at 490 nm;
490 nm; and (d) Cell proliferation assays on SaOS-2 cells grown on PLLA disks, (PLLA1—low
and molecular weight; PLLA2—high
(d) Cell proliferation assays on molecular weight;
SaOS-2 cells grown A—amorphous;
on PLLA disks, SC—Semi-crystalline)
(PLLA1—low molecular
molecular weight; PLLA2—high molecular weight; A—amorphous; SC—Semi-crystalline) [59].
[59]. weight;
PLLA2—high molecular weight; A—amorphous; SC—Semi-crystalline) [59].
Materials 2017, 10, 748 9 of 33
Materials 2017, 10, 748 9 of 33

Li et
etal.al.[60]
[60]
usedused
PLLAPLLA
filmsfilms
havinghaving
normalnormal spherulites
spherulites (Figure
(Figure 8a), banded8a),spherulites
banded spherulites
(Figure 8b),
(Figure 8b), and amorphous
and amorphous surfaces
surfaces (Figure 8c)(Figure 8c) as
as model model substrates
substrates to conductto conduct a systematic
a systematic assessment
assessment of the
of the
role forrole for polymer
polymer crystallization
crystallization inducedinduced surface morphologies
surface morphologies on celland
on cell growth growth
contactand contact
guidance.
guidance. Figure
Figure 8 shows 8 shows
that that the orientation
the orientation of cells wasofalong
cells the
wascrystal
along the crystal radial
spherulite spherulite radialon
direction direction
normal
on normal and banded spherulite surfaces. However, on amorphous surfaces,
and banded spherulite surfaces. However, on amorphous surfaces, cells spread out in a random cells spread out
in a random
fashion because fashion becausesamples
amorphous amorphous samples
do not contain doany
notspecial
containpatterned
any special patterned
structure structure
(Figure 8d,f).
(Figure
The cell8d,f).
shape,The cell shape,
spreading, andspreading,
orientation and orientation
were wereby
investigated investigated
fluorescentby fluorescent
staining staining
techniques in
techniques
order to find in the
order to findcorrelation
possible the possible correlation
between between
the cells the cells
behavior andbehavior and the crystallization
the crystallization morphology
morphology
of PLLA. of PLLA.

Figure 8.
Figure 8. Polarized
Polarized optical
optical images
images of
of PLLA
PLLA films
films crystallized
crystallized atat various
various crystallization
crystallization temperature
temperature
(Tc ) values. (a) Tc = 150 C; normal spherulite surface (b) Tc = 145 C; ring-banded spherulite (c)
(T c) values. (a) Tc = 150 °C; ◦ normal spherulite surface (b) T c = 145 °C;
◦ ring-banded spherulite (c)
melt-quenchedsample;
melt-quenched sample;amorphous
amorphous surface.
surface. TheThe scale
scale bar represents
bar represents 100Stereo
100 µm. μm. Stereo microscope
microscope images
images
of of MC3T3-E1
MC3T3-E1 cells cultured
cells cultured on PLLAon PLLA surfaces;
surfaces; (d) normal (d) normal spherulite
spherulite surface; (e)surface; (e) ring-banded
ring-banded spherulite
spherulite surface; (f) amorphous surface. The scale bar represents
surface; (f) amorphous surface. The scale bar represents 100 µm [60]. 100 μm [60].

They used microscopy and image analysis to ascertain that the MC3T3-E1 cells spread out in a
They used microscopy and image analysis to ascertain that the MC3T3-E1 cells spread out in
random fashion on the amorphous PLA. At 24 h post-seeding, MC3T3-E1 cells on both types of
a random fashion on the amorphous PLA. At 24 h post-seeding, MC3T3-E1 cells on both types of
spherulite surfaces were elongated and aligned along the spherulite radius direction. For the banded
spherulite surfaces were elongated and aligned along the spherulite radius direction. For the banded
spherulite surface with radial stripes and coupling annular grooves, the cell orientation and cell
spherulite surface with radial stripes and coupling annular grooves, the cell orientation and cell nuclear
nuclear localization were related to the groove structure. With increasing time, this orientation
localization were related to the groove structure. With increasing time, this orientation preference
preference was weaker. They further demonstrated that the patterning of the polymer crystallization
was weaker. They further demonstrated that the patterning of the polymer crystallization structure
structure provides important indicators for guiding cells to exhibit characteristic orientation and
provides important indicators for guiding cells to exhibit characteristic orientation and morphology
morphology especially in the early stages of regeneration.
especially in the early stages of regeneration.
Figure 9 shows the three PLLA substratum samples for three time points. On non-patterned
Figure 9 shows the three PLLA substratum samples for three time points. On non-patterned
amorphous PLLA surface, MC3T3-E1 cells spread extensively with overall cell morphology showing
amorphous PLLA surface, MC3T3-E1 cells spread extensively with overall cell morphology showing no
no preferred direction. The actin arranged mainly around the periphery of the cells. On normal
preferred direction. The actin arranged mainly around the periphery of the cells. On normal spherulite
spherulite and banded spherulite films, MC3T3-E1 cells exhibited an elongated morphology. The
and banded spherulite films, MC3T3-E1 cells exhibited an elongated morphology. The spread direction
spread direction of cells cytoskeleton was basically consistent with the radial direction of the
of cells cytoskeleton was basically consistent with the radial direction of the spherulite. In particular,
spherulite. In particular, for the banded spherulite surface having radial stripes and coupling annular
for the banded spherulite surface having radial stripes and coupling annular grooves, there were
grooves, there were two competing factors to guide the cells’ behavior. The radical guidance
two competing factors to guide the cells’ behavior. The radical guidance preference decreased with
preference decreased with increasing valley width. On the other hand, the percentage of cell nucleus
increasing valley width. On the other hand, the percentage of cell nucleus located in the valley of the
located in the valley of the banded spherulitic structure increased with increasing valley width.
banded spherulitic structure increased with increasing valley width.
 Materials 2017, 10, 748 10 of 33
Materials 2017, 10, 748 10 of 33

Figure
Figure9.9.Morphology
MorphologyofofMC3T3-E1
MC3T3-E1cells cellscultured
culturedon onPLLA
PLLAsurfaces.
surfaces.Cells
Cellswere
werestained
stainedwith
withAlexa
Alexa
Fluor
Fluor568
568(red)
(red)and
and DAPI (40 ,[6]-diamidino-2-phenylindole;
DAPI (4′,6-diamidino-2-phenylindole; blue). The
blue). Thescale barbar
scale represents 5050μm.
represents µm.
Crystallization temperatures for normal spherulite samples (NSp) and ring-banded spherulite
Crystallization temperatures for normal spherulite samples (NSp) and ring-banded spherulite samples samples
(BSp)
(BSp)were
were150
150and
and143–147
143–147 ◦ C,
°C, respectively.
respectively.For
Forpreparing
preparingamorphous
amorphousPLLAPLLAfilms
films(Amo),
(Amo),the
themolten
molten
state
statesamples
sampleswere
werequenched
quenchedintointoice
icewater
water[60].
[60].

Using
Using aa novel
novel high-throughput
high-throughput method method for for creating
creating gradients
gradientsininpolymer
polymercrystallinity,
crystallinity,
Washburn
Washburn et al. [61] demonstrated that cells (MC3T3-E1 osteoblastic cells) were exquisitelysensitive
et al. [61] demonstrated that cells (MC3T3-E1 osteoblastic cells) were exquisitely sensitive
totovariations
variationsininnanometer-scale
nanometer-scalePLLA PLLAsurface
surfacetopography.
topography.They Theyfound
foundthatthatcells
cellswere
weresensitive
sensitivetoto
topographic
topographicfeatures
features of the order
of the orderof of55nm
nmandandthat
that
thethe observed
observed inhibition
inhibition of proliferation
of proliferation did seem
did not not
seem
to beto be mediated
mediated throughthrough
changes changes in adherent
in adherent proteinsproteins butseemed
but rather rather to
seemed to berelated
be directly directly
to related
changes
toinchanges in substrate roughness. Their results suggested that cells were much
substrate roughness. Their results suggested that cells were much more sensitive to topography than more sensitive to
topography than previously
previously expected, and theexpected,
inhibitionand the inhibitioncould
of proliferation of proliferation
be mediated could be mediated
through through
other mechanisms.
other
The down-regulation of cell division on crystalline surfaces compared with the up-regulationthe
mechanisms. The down-regulation of cell division on crystalline surfaces compared with on
up-regulation
phase-separated onpolymer
phase-separated polymer
blends indicated that blends indicated
the details that the
of topographic details ofwere
organization topographic
capable of
organization
exerting bothwere capable
positive andof exertinginfluences
negative both positive and negative influences on proliferation.
on proliferation.
Figure 10 indeed shows their results as
Figure 10 indeed shows their results as increasing increasing roughness causing
roughness significant
causing decline
significant in thein
decline
cell proliferation (from left to right). They also conducted fluorescence microscopy
the cell proliferation (from left to right). They also conducted fluorescence microscopy imaging imaging of anti-
paxillin-stained cells on amorphous
of anti-paxillin-stained PLLA, PLLA
cells on amorphous withPLLA
PLLA, intermediate crystallinity,crystallinity,
with intermediate and fully crystalline
and fully
PLLA
crystalline PLLA and reported that no significant variation in the number, positionoforthe
and reported that no significant variation in the number, position or shape adhesion
shape of the
plaques was observed across the library [61].
adhesion plaques was observed across the library [61].
Materials 2017, 10, 748 11 of 33
Materials 2017, 10, 748 11 of 33

Figure 10. Montage of representative images of PLLA morphology from AFM data (top panels, field
Figure 10. Montage of representative images of PLLA morphology from AFM data (top panels, field
of view in each image is 20 μm), and corresponding cell count from fluorescent microscopy (bottom
of view in each image is 20 µm), and corresponding cell count from fluorescent microscopy (bottom
panels, field of view in each image is 1500 μm) [61].
panels, field of view in each image is 1500 µm) [61].
4. Effect of Graphene on the Crystallization of PLA Polymers
4. Effect of Graphene on the Crystallization of PLA Polymers
PLA polymer-graphene nanocomposites have been increasingly gaining in popularity due to
PLA polymer-graphene
attractive features offered bynanocomposites have Norazlina
both materials [62,63]. been increasingly
and Kamal gaining in popularity
[64] reviewed advances duein to
attractive features offered by both materials [62,63]. Norazlina and Kamal
the fabrication of PLA/graphene nanocomposite in detail including requirements for chemical [64] reviewed advances
in modification
the fabrication of PLA/graphene
of graphene nanocomposite
for proper dispersion. in detail
However, their including
review didrequirements
not present any for chemical
potential
modification of graphene
biomedical attributes for proper nanocomposites.
of PLA/graphene dispersion. However,Fabricationtheir review did nanocomposites
of PLA-graphene not present any
potential biomedical
were described and attributes of PLA/graphene
classified according nanocomposites.
to in situ polymerization, Fabrication
solution, and melt ofblending;
PLA-graphene and
the properties were
nanocomposites of these nanocomposites
described and classifiedwere reviewed.
according The
to in situmain conclusion was
polymerization, that and
solution, unless
melt
graphene
blending; and could be chemically
the properties modified
of these to enable good
nanocomposites were dispersion
reviewed. The withinmain theconclusion
PLA matrix, wasno that
enhancements in mechanical properties would be possible. On the
unless graphene could be chemically modified to enable good dispersion within the PLA matrix, other hand, there is intense
nobiomedical
enhancements interestin in PLA-graphene
mechanical nanocomposites
properties would bebased on electrospinning
possible. On the other fabrication
hand, theremethods,is intense
i.e., nanofiber mats of PLA or PLA copolymers containing graphene or
biomedical interest in PLA-graphene nanocomposites based on electrospinning fabrication methods, graphene oxide; as they are
considered to be good candidates for biomedical scaffolds. Advances in
i.e., nanofiber mats of PLA or PLA copolymers containing graphene or graphene oxide; as they are this area will be reviewed
after this section, which will cover the effect of graphene on the crystallinity of PLA polymers.
considered to be good candidates for biomedical scaffolds. Advances in this area will be reviewed
Wu et al. [65] conducted a systematic study on the crystallization of PLA in the presence of
after this section, which will cover the effect of graphene on the crystallinity of PLA polymers.
graphene nano-platelets or nanosheets. Concentration of graphene nanosheets was maintained at
Wu et al. [65] conducted a systematic study on the crystallization of PLA in the presence of
1 wt% with respect to the polymer. They used solution mixing to disperse graphene in the PLA
graphene nano-platelets or nanosheets. Concentration of graphene nanosheets was maintained at
matrix. The presence of graphene nanosheets had a significant influence on both the cold and melt
1 wt% with respect to the polymer. They used solution mixing to disperse graphene in the PLA
crystallization dynamics of PLA polymer. Upon addition of graphene nanosheets, the overall rate of
matrix. The presence decreased,
cold crystallization of graphene andnanosheets
the graphene hadnanosheets
a significant influence
acted merelyon as both
inert the coldFor
fillers. and melt
melt
crystallization
crystallization, dynamics
however, of the
PLApresence
polymer.ofUpon addition
graphene of graphene
nanosheets gavenanosheets, the overall rate
rise to a heterogeneous
of nucleating
cold crystallization
effect and, as a result, accelerated the overall crystallization process despitefillers.
decreased, and the graphene nanosheets acted merely as inert For melt
the physical
crystallization, however, the presence of graphene nanosheets gave rise to
hindrance to PLA chain diffusion. Wu et al. [65] further stated that although both PLA and the a heterogeneous nucleating
effect and, aswere
composite a result,
hardaccelerated
to crystallizethein aoverall crystallization
non-isothermal process
quenching despite
process, theythe physical
could hindrance
be crystallized
to PLA chain diffusion.
by isothermal quenching Wu et al. [65]
from the further
molten stated
state, that although
as can be seen bothin PLA
Figureand11.theThecomposite
spheruliticwere
hard to crystallize
morphology in aclear
is very non-isothermal
in the figure forquenching
both PLAprocess,
and the they could be crystallized
graphene-composite, by isothermal
indicating that the
three-dimensional
quenching from the moltengrowthstate,
of PLA crystals
as can is unchanged
be seen in Figure 11.byThe
the spherulitic
addition ofmorphology
graphene nanosheets.
is very clear
However,
in the the both
figure for presence
PLA of and graphene nanosheets prevents
the graphene-composite, large crystalline
indicating domains from forming,
that the three-dimensional growth
resulting in smaller crystalline domains and/or a degraded crystallite
of PLA crystals is unchanged by the addition of graphene nanosheets. However, the presence structure (Figure 11b). This is of
indicative of a heterogeneous nucleating effect.
graphene nanosheets prevents large crystalline domains from forming, resulting in smaller crystalline
domains and/or a degraded crystallite structure (Figure 11b). This is indicative of a heterogeneous
nucleating effect.
Materials 2017, 10, 748 12 of 33
Materials 2017, 10, 748 12 of 33

Figure 11. Polarization photomicrographs of the final morphologies of (a,b) neat PLA and (c,d) PLAC
Figure 11. Polarization photomicrographs of the final morphologies of (a,b) neat PLA and (c,d) PLAC
(PLA-graphene composite) after melt crystallization (120 °C); The scale bars are (a,c) 50 and (b,d)
(PLA-graphene composite) after melt crystallization (120 ◦ C); The scale bars are (a,c) 50 and (b,d)
20 μm [65].
20 µm [65].
Wang et al. [66] prepared a series of biodegradable PLLA/GO nanocomposites using various
graphene
Wang et al. oxide
[66](GO) loadings
prepared ranging
a series of from 0.5 to 2 wt%
biodegradable using DMF
PLLA/GO as a mutual solvent.
nanocomposites using They
various
graphene oxide (GO) loadings ranging from 0.5 to 2 wt% using DMF as a mutual GO
showed that non-isothermal melt crystallization peak temperatures were slightly higher in the solvent.
nanocomposites than in neat PLLA; moreover, the overall isothermal melt crystallization rates were
They showed that non-isothermal melt crystallization peak temperatures were slightly higher in
significantly greater in the nanocomposites than in neat PLLA. They postulated that GO may act as
the GO nanocomposites than in neat PLLA; moreover, the overall isothermal melt crystallization rates
a nucleating agent for PLLA. With increasing crystallization temperature, the overall isothermal melt
were crystallization
significantly greater
rates werein found
the nanocomposites
to be reduced forthan bothin neat
neat PLLAPLLA.and They postulated
the PLLA/GO that GO may act
nanocomposites
as a nucleating
at differentagent
GO for PLLA. More
loadings. With increasing
importantly, crystallization
they showedtemperature,
that both thethenon-isothermal
overall isothermal melt melt
crystallization rates were found to be reduced for both neat PLLA and the PLLA/GO
crystallization peak temperature and the overall isothermal melt crystallization rate of PLLA first nanocomposites at
different GO loadings. More importantly, they showed that both the non-isothermal
increased and then decreased with increasing the GO loading from 0.5 to 2 wt% in the PLLA/GO melt crystallization
peak nanocomposites,
temperature and showing a maximum
the overall at a 1 wt%
isothermal melt GO loading. Polarized
crystallization ratemicroscope
of PLLA firstanalysis results and
increased
then were similarwith
decreased to theincreasing
results of WutheetGO
al. [65] in the from
loading sense 0.5
thattoGO 2 forced
wt% information
the PLLA/GOof smaller spherulitic
nanocomposites,
features.
showing a maximum at a 1 wt% GO loading. Polarized microscope analysis results were similar to the
Manafi et al. [67] prepared nanocomposites based on PLA/graphene nanoplatelets (GnPs) by
results of Wu et al. [65] in the sense that GO forced formation of smaller spherulitic features.
solution mixing. To improve the dispersion of GnPs in the matrix, functionalization using acid
Manafi et al. [67] prepared nanocomposites based on PLA/graphene nanoplatelets (GnPs) by
treatment and acylation reaction was performed. Characterization of functionalization reaction and
solution mixing.
grafting reactionTobetween
improve PLAtheanddispersion of GnPs
functionalized GnPs in wasthe matrix,
verified functionalization
by spectroscopic analysisusing
and acid
treatment
thermogravimetry. Transmission electron microscopy (TEM) results demonstrated that a relatively and
and acylation reaction was performed. Characterization of functionalization reaction
grafting
fine reaction
dispersion between
of GnPsPLA was and functionalized
achieved in the PLAGnPs matrix. was verified by spectroscopic
Non-isothermal tests revealed analysis
that the and
thermogravimetry. Transmission
crystallization temperature (Tc)electron microscopy
was increased (TEM)
in samples due results demonstrated
to better dispersion and thatgrafting
a relatively
reaction between
fine dispersion of GnPs GnPswasand PLA. Isothermal
achieved in the PLAtests also showed
matrix. that commencement
Non-isothermal tests revealed of that
the the
crystallization time was affected by the functionalization and then decreased.
crystallization temperature (Tc ) was increased in samples due to better dispersion and grafting reaction They also concluded
that the
between growth
GnPs andmechanism of crystallization
PLA. Isothermal tests also didshowed
not change thatsignificantly.
commencement of the crystallization
The effects of GO with polar groups and functionalized graphene oxide (fGO) with non-polar
time was affected by the functionalization and then decreased. They also concluded that the growth
groups on the isothermal crystallization of PLLA were studied by Zhao et al. [68]. Functionalized GO
mechanism of crystallization did not change significantly.
was obtained by grafting octadecylamine and characterized by Fourier transform infrared spectroscopy,
The
X-rayeffects of GOand
diffraction, with polar groups and
thermogravimetry. Thefunctionalized graphene kinetics
isothermal crystallization oxide (fGO) with non-polar
of PLLA/GO and
groups on the isothermal
PLLA/fGO nanocompositescrystallization of PLLA
was investigated bywere studied
combining by Zhao et
differential al. [68].calorimetry
scanning Functionalizeddata GO
was obtained by grafting
and the Avrami octadecylamine
equation and characterized
as shown in Figure 12. Note that the by Avrami
Fourierequation
transform infrared
describes howspectroscopy,
solids
X-raytransform
diffraction,
from and
onethermogravimetry.
phase (state of matter) Thetoisothermal crystallization
another at constant kinetics
temperature. of PLLA/GO
It can specifically and
describenanocomposites
PLLA/fGO the kinetics of crystallization.
was investigated The results showed differential
by combining that fGO could improve
scanning the PLLA data
calorimetry
and the Avrami equation as shown in Figure 12. Note that the Avrami equation describes how solids
transform from one phase (state of matter) to another at constant temperature. It can specifically
describe the kinetics of crystallization. The results showed that fGO could improve the PLLA
Materials 2017, 10, 748 13 of 33

Materials 2017, 10, 748 13 of 33

crystallization rate more apparently than GO. By analyzing the morphology obtained from polarized
light crystallization
microscopy, SEM rate more
andapparently
TEM, it wasthanfound
GO. Bythat
analyzing the morphology
fGO with large layerobtained from polarized
space dispersed better in
light microscopy, SEM and TEM, it was found that fGO with large layer space dispersed better in
PLLA and supplied more nucleation sites than GO. Therefore, for the multilayer graphene, increasing
PLLA and supplied more nucleation sites than GO. Therefore, for the multilayer graphene, increasing
the layer spaces is important to improve its dispersion in polymers, which will cause the modification
the layer spaces is important to improve its dispersion in polymers, which will cause the modification
of crystallization kinetics.
of crystallization According
kinetics. AccordingtotoFigure
Figure12,
12,PLLA/0.5fGO (0.5%
PLLA/0.5fGO (0.5% fGOfGO dispersion
dispersion inpolymer
in the the polymer
matrix) has the
matrix) hasbest crystallization
the best half-time
crystallization results
half-time atateach
results eachcrystallization
crystallization temperature
temperature (T(Tc)c )studied
studied by
Zhaoby [68].
Zhao [68].

FigureFigure 12. Avrami

12. Avrami plotsplots of neat
of neat PLLA
PLLA (a),
(a), andPLLA/0.5fGO
and PLLA/0.5fGO (b).
(b).Isothermal crystallization
Isothermal kinetics
crystallization kinetics
parameters of neat PLLA, PLLA/0.5GO and PLLA/0.5fGO obtained by Avrami
parameters of neat PLLA, PLLA/0.5GO and PLLA/0.5fGO obtained by Avrami fit are tabulatedfit are tabulated in in
(c) [68].
(c) [68].

Sun and He [69] prepared PLA–graphene oxide (GO) nanocomposites by blending commercial
Sun
PLLA and HePDLA
with [69] prepared
grafted GOPLA–graphene
(GO-g-PDLA), whereoxide GO-g-PDLA
(GO) nanocomposites by blending
was synthesized commercial
via ring-opening
PLLApolymerization
with PDLA grafted GO (GO-g-PDLA),
using modified where(see
GO as the initiator GO-g-PDLA was synthesized
Figure 13). Their via ring-opening
Fourier transform infrared
spectroscopy,using
polymerization differential
modifiedscanning
GO as calorimetry, and (see
the initiator X-rayFigure
diffraction
13). studies
Their showed
Fourier that a
transform
stereo-complex crystal could be formed between PLLA and GO-g-PDLA. The incorporation
infrared spectroscopy, differential scanning calorimetry, and X-ray diffraction studies showed that a of GO
nanosheets led to a lower crystallization activation energy of stereo-complex and a higher
stereo-complex crystal could be formed between PLLA and GO-g-PDLA. The incorporation of GO
crystallinity in solution casted samples, mainly due to the heterogeneous nucleating effect of the well-
nanosheets led to a lower crystallization activation energy of stereo-complex and a higher crystallinity
dispersed covalently bonded GO sheets, while in cold crystallized samples, the crystallinity was
in solution casted samples, mainly due to the heterogeneous nucleating effect of the well-dispersed
found to be low owing to exfoliated GO sheets which, they argued, could reduce chain mobility and
covalently bonded GO sheets, while in cold crystallized samples, the crystallinity was found to be
hinder crystal growth.
low owing to exfoliated
Liang GO sheets
et al. [70] studied which,
the role of sizethey argued, could
and structural reduce
integrity chain reduced
of thermally mobilitygraphene
and hinder
crystal growth.
oxide (rGO) in PLA crystallization. Reduced graphene oxide nanoplatelets with different
architectures
Liang were
et al. [70] obtained
studied thevia bath
role of and
size probe ultrasoundintegrity
and structural processing. Isothermalreduced
of thermally crystallization
graphene
kinetics of PLA nanocomposites containing bath (rGOw) and probe ultrasound
oxide (rGO) in PLA crystallization. Reduced graphene oxide nanoplatelets with different architecturesprocessed (rGOp)
were was determined
obtained via bathby and
in situ synchrotron
probe ultrasound wide-angle X-rayIsothermal
processing. diffraction.crystallization
The induction kinetics
period andof PLA
overall crystallization rate of PLA-rGO nanocomposites were strengthened with diminishing platelet
nanocomposites containing bath (rGOw) and probe ultrasound processed (rGOp) was determined by in
size because of more nucleation sites encouraged by redistribution of functional groups. Figure 14
situ synchrotron wide-angle X-ray diffraction. The induction period and overall crystallization rate of
illustrates the representative 2D-WAXD patterns of PLA-rGOw and PLA-rGOp nanocomposites
PLA-rGO nanocomposites were strengthened with diminishing platelet size because of more nucleation
isothermally crystallized at Tc of 135 °C. They observed a diffuse scattering ring in the first pattern
sites (t
encouraged bythe
= 0 min) for all redistribution of functional
samples, indicating no crystalsgroups.
could have Figure 14 in
survived illustrates the representative
the melt. With increasing
2D-WAXD patterns of PLA-rGOw and PLA-rGOp nanocomposites isothermally
crystallization time, the isotropic lattice planes, i.e., (200)/(110) reflection, can be observed crystallizedandat Tc
◦
of 135 C. They observed a diffuse scattering ring in the first pattern (t = 0 min) for all the samples,
indicating no crystals could have survived in the melt. With increasing crystallization time, the
isotropic lattice planes, i.e., (200)/(110) reflection, can be observed and gradually sharpen until the
Materials 2017, 10, 748 14 of 33
Materials 2017, 10, 748 14 of 33

gradually sharpen until the completion of crystallization, correlating with the formation and growth
completion of crystallization, correlating with the formation and growth of the PLA crystals. In contrast
of the PLA
Materials crystals. In contrast to neat PLA, the advent of ultrasonically treated rGO shortens the
to neat PLA,2017,
the10, 748
advent of ultrasonically treated rGO shortens the appearance time of the 14 of 33
diffraction
appearance time of the diffraction peak remarkably (from 30 min to lower values), demonstrating the
peak superb
remarkably
gradually (from
nucleation
sharpen 30 min
ability
until to lower values),
of completion
the rGO. demonstrating
of crystallization, the superb
correlating with thenucleation ability
formation and of rGO.
growth
of the PLA crystals. In contrast to neat PLA, the advent of ultrasonically treated rGO shortens the
appearance time of the diffraction peak remarkably (from 30 min to lower values), demonstrating the
superb nucleation ability of rGO.

Figure 13. Schematic representation of GO grafting to PDLA and blending in solution for casting
Figure 13. Schematic representation of GO grafting to PDLA and blending in solution for casting
stereocomplex homocomposites [69].
stereocomplex homocomposites [69].
Their
Figuremain conclusion
13. Schematic was that both
representation rGOw
of GO and to
grafting rGOp could
PDLA and serve as heterogeneous
blending nucleation
in solution for casting
Their
agents main conclusion
for PLA,
stereocomplex wasthe
decreasing
homocomposites thatinduction
both rGOw
[69]. period andofrGOp could serve
crystallization andasaccelerating
heterogeneous nucleation
the overall
agentscrystallization rate. Prolonged
for PLA, decreasing theultrasound
inductiontime, periodhowever, gradually enhanced
of crystallization the nucleation the
and accelerating ability
overall
of rGOw
Their but suppressed
main conclusion that of
was RGOp.
that both rGOw and rGOp could serve
crystallization rate. Prolonged ultrasound time, however, gradually enhanced the nucleation ability ofas heterogeneous nucleation
rGOw butXusuppressed
agents et al.
for PLA,[71]decreasing
fabricated mechanically
the
that of RGOp. induction strong
period and thermally stable
of crystallization andPLA films by integrating
accelerating the overall
stereocomplex crystals
crystallization rate. (SCs)-decorated
Prolonged ultrasound graphene oxide (GO)
time, however, nanosheets
gradually in enantiomeric
enhanced PLA.ability
the nucleation In the
Xu et al. [71] fabricated mechanically strong and thermally stable PLA films by integrating
PLA
of rGOwmatrix,
butGO nanosheets
suppressed thatwere
of RGOp.homogeneously dispersed and fully extended, principally due to
stereocomplex
the relatively
crystals (SCs)-decorated graphene oxide (GO) nanosheets in enantiomeric PLA. In the
Xu et al.low [71]GO loadings mechanically
fabricated and adequate stronginteractions between GO
and thermally and PLA
stable PLA. films
Such morphological
by integrating
PLA features
matrix, GO nanosheets
resulted
stereocomplex were
in the construction
crystals homogeneously
(SCs)-decorated of interconnected
graphene oxide dispersed
networks and fully extended,
of GO nanosheets.
(GO) nanosheets principally
Their examination
in enantiomeric PLA. In the due to
the relatively
of
PLAisothermallowGO
matrix, GO andloadings and
wereadequate
non-isothermal
nanosheets interactions
crystallization
homogeneously between
showed
dispersed andthat GOthe
fully andselectively
PLA.principally
extended, Suchaccelerated
morphological
due to
features resulted in
stereocomplex
the relatively low theGO construction
crystallization
loadingsinand theofadequate
interconnected
presence networks
of GO rendered
interactions ofGO
GOand
a dominant
between nanosheets.
generation
PLA. Such ofTheir
SCs examination
directly
morphological
proportional
of isothermal and to
features resulted GOtheloadings.
non-isothermal
in At of the same showed
crystallization
construction time, networks
interconnected thethatgeneration
the of homocrystals
ofselectively
GO nanosheets. accelerated (HCs) was
stereocomplex
Their examination
suppressed,
of isothermal primarily
and due to the
non-isothermal limited availability
crystallization of homochiral
showed that
crystallization in the presence of GO rendered a dominant generation of SCs directly proportional chains
the and spatial
selectively hindrance
accelerated
to GO caused by the
stereocomplex
loadings. surrounding
Atcrystallization
the same time, nanosheets
in the
thepresence and of
generation SCs.
GO ofMore
renderedimportantly,
homocrystalsa dominant thegeneration
(HCs) decoration
was of of
SCsGO
suppressed, with
directly
primarily
ordered
proportionalPLA lamellae served asAt
to GO loadings. strong
the and
sameresilient
time, theligaments, whichofresulted
generation in low (HCs)
homocrystals gas barrier
was
due to the limited availability of homochiral chains and spatial hindrance caused by the surrounding
properties,
suppressed,high strength,
primarily duehigh ductility,
to the limitedand improved of
availability chemical resistance
homochiral chainsforand
GO/PLA composites.
spatial hindrance
nanosheets and SCs. More importantly, the decoration of GO with ordered PLA lamellae served
caused by the surrounding nanosheets and SCs. More importantly, the decoration of GO with
as strong and resilient ligaments, which resulted in low gas barrier properties, high strength, high
ordered PLA lamellae served as strong and resilient ligaments, which resulted in low gas barrier
ductility, and improved
properties, high strength,chemical resistance
high ductility, andforimproved
GO/PLA composites.
chemical resistance for GO/PLA composites.

Figure 14. Left panel: Selected 2D-WAXD patterns of (a) neat PLA; (b) PLA-rGOw 0.5; (c) PLA-rGOw
1.0; and (d) PLA-rGOw 2.0 isothermally crystallized at 135 °C. Right panel: selected 2D-WAXD
patterns of (a) neat PLA; (b) PLA-rGOp 0.3; (c) PLA-rGOp 0.6; and (d) PLA-rGOp 1.0 isothermally
crystallized at 135
Figure 14. Left °C [70].
panel: Selected 2D-WAXD patterns of (a) neat PLA; (b) PLA-rGOw 0.5; (c) PLA-rGOw
Figure 14. Left panel: Selected 2D-WAXD patterns of (a) neat PLA; (b) PLA-rGOw 0.5; (c) PLA-rGOw
1.0; and (d) PLA-rGOw 2.0 isothermally crystallized at 135 °C. Right panel: selected 2D-WAXD
1.0; and (d) PLA-rGOw 2.0 isothermally crystallized at 135 ◦ C. Right panel: selected 2D-WAXD
patterns of (a) neat PLA; (b) PLA-rGOp 0.3; (c) PLA-rGOp 0.6; and (d) PLA-rGOp 1.0 isothermally
patterns of (a) neat
crystallized PLA;
at 135 (b) PLA-rGOp 0.3; (c) PLA-rGOp 0.6; and (d) PLA-rGOp 1.0 isothermally
°C [70].
crystallized at 135 ◦ C [70].
 Materials 2017, 10, 748 15 of 33

Materials 2017, 10, 748 15 of 33

Figure 15a demonstrates that in the absence of GO nanosheets formation of SCs is very limited
Figure 15a demonstrates that in the absence of GO nanosheets formation of SCs is very limited
and instead PLLA and PDLA spherules form. In the presence of GO nanosheets, however, many
and instead PLLA and PDLA spherules form. In the presence of GO nanosheets, however, many
more stereocomplex crystals, SCs, form (Figure 15b). The microscope images in the bottom panel of
more stereocomplex crystals, SCs, form (Figure 15b). The microscope images in the bottom panel of
Figure 15 clearly demonstrate this transformation from spherule-like crystal domains to diamond-like
Figure 15 clearly demonstrate this transformation from spherule-like crystal domains to diamond-
shaped crystals in the presence of GO at different crystallization temperatures.
like shaped crystals in the presence of GO at different crystallization temperatures.

Figure 15. Schematic representation comparing the evolution of homochiral and stereocomplex
Figure 15. Schematic representation comparing the evolution of homochiral and stereocomplex
crystallization in (a) GO0 (no graphene oxide) and (b) GO reinforced composites. Preferential
crystallization in (a) GO0 (no graphene oxide) and (b) GO reinforced composites. Preferential nucleation
nucleation of SCs assisted by GO, both on the basal planes and at the edges, leads to the dominating
of SCs assisted by GO, both on the basal planes and at the edges, leads to the dominating development
development of SCs and spatially hindered homocrystals, whereas limited SCs are generated during
of SCs and spatially hindered homocrystals, whereas limited SCs are generated during the simultaneous
the simultaneous growth of HCs for GO0. (c) Polarized optical micrographs showing the spherulitic
growth of HCs for GO0. (c) Polarized optical micrographs showing the spherulitic textures formed
textures formed in GO/racemic PLA composites after isothermal melt crystallization. The nucleation
in GO/racemic PLA composites after isothermal melt crystallization. The nucleation activity of PLA
activity of PLA was evidently enhanced in the presence of GO nanosheets, regardless of
was evidently enhanced in the presence of GO nanosheets, regardless of crystallization temperature.
crystallization temperature. The scale bar represents 100 μm for all images. GO0 stands for no
The scale bar represents 100 µm for all images. GO0 stands for no graphene oxide [71].
graphene oxide [71].

5. Effect of Graphene on Mechanical Properties of PLA Polymers

5. Effect of Graphene on Mechanical Properties of PLA Polymers
Review of the literature on PLA-graphene nanocomposites in the form of free standing films
Review of the literature on PLA-graphene nanocomposites in the form of free standing films
produced either by solvent processing or by melt molding/extrusion indicates that there is still a
produced either by solvent processing or by melt molding/extrusion indicates that there is still a
significant lack of established recipe or nanocomposite fabrication protocol that could be used to
significant lack of established recipe or nanocomposite fabrication protocol that could be used to
enhance the elastic modulus or elongation at break values of PLA polymers using graphene-based
enhance the elastic modulus or elongation at break values of PLA polymers using graphene-based
materials. This is due to the fact that graphene comes as filler in the form of many stacked layers
materials. This is due to the fact that graphene comes as filler in the form of many stacked layers
(known as GPs, GnPs etc.), which is influenced by the synthesis history. Different lateral size and
(known as GPs, GnPs etc.), which is influenced by the synthesis history. Different lateral size and
thickness distribution exist in all these “commercially available” graphene products. Separately,
thickness distribution exist in all these “commercially available” graphene products. Separately,
graphene oxide or reduced graphene oxide introduces other parameters such as surface reactive sites
Materials 2017, 10, 748 16 of 33

graphene oxide or reduced graphene oxide introduces other parameters such as surface reactive sites
and polarity effects. However, most recent reports highlight the necessity to functionalize graphene
surfaces or use graphene oxide with graft chemistry [72] in order to compound in PLA polymers.
For instance, Fu et al. [73] used an approach to modify the interfacial compatibility between graphene
and polylactic acid (PLA) by manipulating the functionalization of graphene and introducing an
epoxy-containing elastomer modifier. Curing between the functional groups of the modified graphene
and the epoxy groups of the elastomer modifier resulted in controlled dispersion and distribution of
graphene in the composite system and hence improved the interfacial adhesion between PLA and
graphene. Effects of different graphene functionalization on morphology, viscoelasticity, and thermal
properties of the resulting PLA nanocomposites were thoroughly examined. The resulting percolated
structures were the origin of the improved properties of PLA/graphene nanocomposites. Storage
modulus and complex viscosity measurements showed that due to crosslinking reaction between
functionalized graphene oxide and the epoxy-modified rubber phase/PLA matrix, a three-dimensional
percolated structure formed which was the origin of the improved viscoelasticity and thermal stability.
Cataldi et al. [74] fabricated biocomposites by dispersing different graphene nanoplatelets
(GnPs) as well as other non-graphitic 2D and 3D nanoscale particles (See table in Figure 16) in
two representative bio-polyesters classified as soft (Mater-Bi, polycaprolactone based) and hard
(PLA) matrices as shown in Figure 16a. Films were produced by solvent casting and hot-pressing.
Hot-pressing was found to align the GnPs within the polymer matrices. The alignment had a more
significant effect on the amorphous soft bio-polyester rather than PLA. In the case of solvent cast soft
matrix biocomposites, GnPs did not induce any superior enhancements in elastic moduli compared to
other non-graphitic 2D and 3D fillers.
In the case of PLA, however, both large multi- and few-layer GnPs caused up to 35% increase in
elastic (Young’s) modulus compared to other model 2D or 3D fillers; see Figure 16b. As a result
of hot-pressing, GnP flakes significantly increased the elastic modulus of the soft bio-polyester.
In particular, large multi-layer GnPs induced up to 200% stiffness enhancement compared to few-layer
GnPs and non-graphitic 2D fillers. Upon hot-pressing, about 15% stiffness enhancement levels were
measured in the nanoparticle filled soft bio-polyester. Compared to solvent casting, hot-pressing of the
PLA matrix nanocomposites did not yield better elastic modulus enhancements neither by large multi-
nor few-layer GnPs. Stiffening levels remained the same.
Cao et al. [75] produced solvent-free graphene nanosheets, which were in the form of chemically
reduced graphite oxide nanosheets, using an environmentally friendly freeze drying (lyophilization)
process. Compared to those produced through the vacuum filtration method, they claimed that the
lyophilized graphene powders (GNS) were extremely light, loosely packed, and could be readily
re-dispersed in organic solvents like N,N-dimethylformamide as individual sheets with the aid of
sonication. This facilitated GNS powder incorporation into PLA through a solution-based processing
method. In the resulting nanocomposites, GNSs were uniformly dispersed in the matrix and enhanced
the mechanical and thermal properties of the host PLA polymer as shown in Figure 17a. They showed
that GNS loading of 0.2 wt% (0.2G-PLA in Figure 17) was sufficient to enhance mechanical properties.
They reported that even for such a small percentage of GNS the reinforcing effect is rather
significant: In other words, 0.2 wt% GNS incorporation leads to a 26% increase in tensile strength and
a 18% raise in Young’s modulus. In their work [75], Young’s modulus was determined from DMA
traces as storage modulus at 35 ◦ C, at which PLA and 0.2G-PLA are both in the glassy state, as shown
in Figure 17b.
Materials 2017, 10, 748 17 of 33
Materials2017,
Materials 2017,10,
10,748
748 1717ofof3333

Figure 16.
Figure 16. (a)
(a) Photograph
Photograph ofof different
different solvent-cast
solvent-cast composite
composite films
films from
from PLA
PLA and
and Mater-Bi
Mater-Bi
®®
Figure 16. (a) Photograph of different solvent-cast composite films from PLA and Mater-Bi®
(Novamont,Italy)
(Novamont, Italy)biopolymers.
biopolymers.Transparent
Transparentand andwhitish
whitishfilms
filmsfeatured
featuredon onthe
thetop
topare
aretwo
twounfilled
unfilled
(Novamont, Italy) biopolymers. Transparent and whitish films featured on the top are two unfilled
matrices; (b)
matrices; (b) Elastic
Elastic modulus
modulus versus
versus filler
filler weight
weight percent
percent measurements
measurements for for solvent-cast
solvent-cast PLA
PLA
matrices; (b) matrix
polymer Elasticcomposites;
modulus versus
(c)The
Thefiller weight
table percent measurements
listsdimensional
dimensional forofsolvent-cast
characteristics allthe PLA fillers
thenanoscale
nanoscale polymer
polymer matrix composites; (c) table lists characteristics of all fillers
matrix composites;
used.
used. [74].
[74].
(c) The table lists dimensional characteristics of all the nanoscale fillers used [74].

Figure
Figure
Figure 17.(a)
17.17.
(a) (a)Representative
Representative
Representative stress–strain
stress–strain
stress–strain curves;
curves;
curves; and
and
and (b)
(b)
(b) plotsof
plots
plots ofofstorage
storagemodulus
storage modulusvs.
modulus vs.temperature
temperature for
temperature
PLA for
for
andPLA
PLA and0.2GNS-PLA.
and 0.2GNS-PLA.
0.2GNS-PLA. Theinset
TheThe
inset inset
inin(a)
in (a) (a)contains
contains
contains tensile
tensile
tensile strengthvalues
strength
strength valuesofof
values ofPLA
PLA
PLA and
and 0.2GNS-PLA
0.2GNS-PLA
and 0.2GNS-PLA [75].
[75].
[75].

Marquesetetal.
Marques al.[76]
[76]reported
reportedaanovel
noveltwo-step
two-stepmethod
methodfor forthe
thepreparation
preparationofofPLLA/hydroxyapatite
PLLA/hydroxyapatite
Marquesnanocomposites
(Hap)/GO
(Hap)/GO
et al. [76] reported
nanocomposites with
a novel
with two-step
augmented
augmented
method properties
mechanical
mechanical
for the preparation
properties of PLLA/hydroxyapatite
whencompared
when compared totoPLLA/Hap
PLLA/Hap
(Hap)/GO
and neatnanocomposites
andneat PLLA.The
PLLA. Thepresence withofof
presence augmented
grapheneoxide
graphene mechanical
oxide (GO)had
(GO) properties
had aapositive when
positive effect
effect compared
ononthe to PLLA/Hap
thedispersion
dispersion ofof
andhydroxyapatite
neat PLLA. The
hydroxyapatite presence
particles
particles inin theof polymeric
the graphene oxide
matrix(GO)
polymeric matrix withhad
with good
good a homogenous
positive
homogenous effect on nanocomposite
final
final the dispersion of
nanocomposite
hydroxyapatite
structure.PLLA
structure. particles
PLLA in the polymeric
nanocomposites
nanocomposites preparedmatrix
prepared withwith
with 30%good
30% (w/w)
(w/w) homogenous
ofofHap
Hapand and1%final
1% nanocomposite
(w/w)
(w/w) ofofGOGOshowed
showedstructure.
the
the
PLLA nanocomposites
highest hardness and prepared
storage with
modulus 30% (w/w)
values of
indicatingHap anand 1%
efficient (w/w)
load of GO
transfer
highest hardness and storage modulus values indicating an efficient load transfer between the fillers showed
between the
the highest
fillers
andthe
hardness
and the
andPLLA
storage
PLLA matrix.
matrix. Theypostulated
modulus
They postulated
values thatthese
indicating
that these nanocomposites
an efficient couldbetween
load transfer
nanocomposites could beused
be usedtheininbiomedical
biomedical
fillers and the
applications
applications
PLLA suchas
such
matrix. They asbone
bonescrews.
postulated screws. Theysynthesized
that They
these synthesizedGO
nanocomposites GOby by the
couldthechemical
chemical exfoliation
be usedexfoliation
in biomedical ofofgraphite
graphite inin
applications
such aqueous
aqueous
as bone media
media followed
followed
screws. They by byultrasonic
ultrasonicprocessing,
synthesized GOprocessing, resultingin
by the resulting
chemical inthin
thinsheets
exfoliationsheets
ofofofGOGOthat
graphite thatreadily
inreadily formed
formed
aqueous media
stablecolloidal
stable
followed colloidal
by suspensions
suspensions
ultrasonic ininwater.
processing, water. TheirGO
Their
resulting GO nanosheets
in nanosheets
thin were
sheetswere
of GO described
described asaslarge
that readily large flakesstable
flakes
formed withlateral
with lateral
colloidal
dimensionshigher
dimensions higherthan than44μm μm(see(seeSEM
SEMimage
imageininFigure
Figure18a).
18a).
suspensions in water. Their GO nanosheets were described as large flakes with lateral dimensions
Highlypure
Highly purenanocrystals
nanocrystalsofofHap Hapwere
wereused
used in the form of spray dried aggregated micron-size
higher than 4 µm (see SEM image in Figure 18a). in the form of spray dried aggregated micron-size
powdersasascan
powders canbe beobserved
observedininFigure
Figure18b.
18b.ToTounderstand
understandhow howGO GOinfluences
influencesthe thecrystallinity
crystallinityofof
Highly pure nanocrystals of Hap were used in the form of spray dried aggregated micron-size
PLLA/Hap, they conducted X-ray diffraction (XRD) analysis.
PLLA/Hap, they conducted X-ray diffraction (XRD) analysis. Figure 18c displays the Figure 18c displays thediffraction
diffraction
powders as can be observed in Figure 18b. To understand how GO influences the crystallinity of
PLLA/Hap, they conducted X-ray diffraction (XRD) analysis. Figure 18c displays the diffraction
patterns of PLLA/Hap and PLLA/Hap with 0.1, 1, and 5% GO nanocomposites. The diffraction from
Materials 2017, 10, 748 18 of 33
Materials 2017, 10, 748 18 of 33

patterns of regions
the crystalline PLLA/Hap thatand
arePLLA/Hap
present evenwith in
0.1,amorphous
1, and 5% GO nanocomposites.
PLLA has severalThe diffraction from
characteristic maxima,
the crystalline regions that are◦present even
◦ in amorphous PLLA has several characteristic maxima,
the two strongest are at 2θ of 17 and 19 corresponding to a typical PLLA pseudo-orthorhombic α
the two strongest are at 2θ of 17° and 19° corresponding to a typical PLLA pseudo-orthorhombic α
structure. These peaks are also present in the XRD pattern of the nanocomposites (Figure 18c) but
structure. These peaks are also present in the XRD pattern of the nanocomposites (Figure 18c) but
with with
lower intensity
lower in the
intensity presence
in the presenceGO GOwhen
whencompared withPLLA/Hap.
compared with PLLA/Hap. They
They argued
argued that that
this this
indicated a decreasing crystallinity of the polymeric phase of the nanocomposites with GO
indicated a decreasing crystallinity of the polymeric phase of the nanocomposites with GO addition. addition.
Changes in the
Changes inmechanical properties
the mechanical of the
properties PLLA
of the PLLA nanocomposites
nanocompositesincluding
including different amountsofof GO
different amounts
are summarized in the table
GO are summarized in table
in the Figurein 18. As seen,
Figure 18. Asthe overall
seen, mechanical
the overall properties
mechanical of theofresulting
properties the
resulting nanocomposites were enhanced and present a maximum improvement
nanocomposites were enhanced and present a maximum improvement with the addition of 1% (w/w) with the addition
of 1% (w/w) of GO.
of GO.

Figure
Figure 18. SEM
18. SEM images
images of (a)
of (a) grapheneoxide
graphene oxide and
and (b)
(b) hydroxyapatide;
hydroxyapatide; (c)(c)
X-ray diffraction
X-ray patterns
diffraction patterns
of PLLA/Hap
of PLLA/Hap andand PLLA/Hapwith
PLLA/Hap with 0.1,
0.1, 1,
1, and
and 5%5%ofofGO.
GO.Inset:
Inset:schematic representation
schematic of polymer
representation of polymer
chains
chains withwith
andand without
without HapHapandand
GOGOfillers.
fillers. The
The table
tableshows
showshardness
hardnessand elastic
and modulus
elastic values
modulus of
values of
PLLA, PLLA/Hap, and PLLA/Hap with different GO percentages, measured by nano-indentation [76].
PLLA, PLLA/Hap, and PLLA/Hap with different GO percentages, measured by nano-indentation [76].
Bao et al. [77] produced graphene from graphite by pressurized oxidation and multiplex
Bao et al. [77]
reduction. They produced
executedgraphene from graphite
this pressurized oxidationby process
pressurized
with oxidation
multiplex and multiplex
reduction basedreduction.
on
ammonia and hydrazine and produced single-atom-thick graphene (0.4–0.6
They executed this pressurized oxidation process with multiplex reduction based on ammonia and nm thick; see Figure 19a).
They then
hydrazine anddispersed
producedtheir graphene in PLAgraphene
single-atom-thick by melt blending.
(0.4–0.6 Thenm graphene
thick; seewas found
Figure to beThey
19a). well then
dispersed and the obtained nanocomposites had markedly improved
dispersed their graphene in PLA by melt blending. The graphene was found to be well dispersed crystallinity, rate of
crystallization, and mechanical properties. The properties were found to be dependent on the
and the obtained nanocomposites had markedly improved crystallinity, rate of crystallization, and
dispersion and loading content of graphene, showing a percolation threshold at 0.08 wt%. They
mechanical properties. The properties were found to be dependent on the dispersion and loading
argued that graphene had reinforced the PLA resin but after a critical concentration perturbed the
content of graphene, showing a percolation threshold at 0.08 wt%. They argued that graphene had
interactions among the polymer chains, which led to somewhat reduced mechanical properties (see
reinforced
Figurethe PLA
19b). resin
Their but after
tensile a critical
strength concentration
measurements perturbed
indicated the interactions
that tensile among
strength increases upthe polymer
to 0.08
chains, which led to somewhat reduced mechanical properties (see Figure 19b).
wt% (a 35% increase) and then declines. The hardness of the PLA/graphene nanocomposites increases Their tensile strength
measurements indicated
with the graphene thatand
content tensile strength
the increase increases
is about 30 N·mmup toat0.08
−2 2 wt%wt% (a 35%loading.
graphene increase) and then
Sabzihardness
declines. The et al. [78]ofprepared solvent cast PLA
the PLA/graphene nanocompositeincreases
nanocomposites samples with
with two
the types
grapheneof graphene
content and
nanoplatelets.
the increase is about They30 N ·mm−2 at
observed that one graphene
2 wt% of the graphene samples was homogenously dispersed
loading.
throughout
Sabzi et al. the
[78]PLA matrix, solvent
prepared mainly in single
cast PLA sheets (see Figure 20a,b),
nanocomposite sampleswhereas
withthe other
two wasofpoorly
types graphene
delaminated and dispersed (Figure 20c,d). Well-dispersed GnPs had a surface area of ~600 m2/g,
nanoplatelets. They observed that one of the graphene samples was homogenously dispersed
thickness of 1 nm with a diameter less than 10 microns; whereas poorly-dispersed GnPs had a surface
throughout the PLA matrix, mainly in single sheets (see Figure 20a,b), whereas the other was poorly
area of ~140 m2/g, thickness of 7 nm with a diameter more than 25 microns. Dynamic rheological tests 2
delaminated and dispersed
and theoretical modeling were (Figure 20c,d).toWell-dispersed
conducted characterize the GnPs
structureshadanda surface areabehavior
viscoelastic of ~600ofm /g,
thickness of 1 nm with a diameter
the graphene percolation networks. less than 10 microns; whereas poorly-dispersed GnPs had a surface
area of ~140 2
m /g, thickness
Well-dispersed GnPs ofhad7 nm withpercolation
a lower a diameter more than
threshold than25poorly-dispersed
microns. Dynamic GnPsrheological
because of tests
and theoretical
the former’smodeling
larger aspect were conducted
ratio, to characterize
better dispersion, favorablethe structures
surface and
structure, andviscoelastic
chemistry. A behavior
well- of
dispersedpercolation
the graphene GnP networknetworks.
with high elasticity and strength was established through high density direct
Well-dispersed GnPs had a lower percolation threshold than poorly-dispersed GnPs because
of the former’s larger aspect ratio, better dispersion, favorable surface structure, and chemistry.
A well-dispersed GnP network with high elasticity and strength was established through high density
Materials 2017, 10, 748 19 of 33
Materials 2017, 10, 748 19 of 33
Materials 2017, 10, 748 19 of 33
direct inter-particle
inter-particle connections,
connections, whereas
whereas the the poorly-dispersed
poorly-dispersed GnPGnP network
network waswas
a apolymer-bridged
polymer-bridged
inter-particle
network which connections,
was formed whereas
at high the
GnP poorly-dispersed
content and showedGnP
lownetwork
elasticitywas a polymer-bridged
(Figure
network which was formed at high GnP content and showed low elasticity (Figure 20). 20).
network which was formed at high GnP content and showed low elasticity (Figure 20).

Figure
Figure 19.19.(a)(a)Illustration
Illustrationfor
forthe
thepreparation
preparation of of graphene
graphene and
and PLA/graphene
PLA/graphenenanocomposites.
nanocomposites.
Figure 19. (a)is Illustration for the preparation of graphene and PLA/graphene nanocomposites.
The graphite is oxidized by pressurized oxidization and then reduced totosingle-atom-thick
The graphite oxidized by pressurized oxidization and then reduced single-atom-thick grapheneby
graphene
The
by agraphite
multiplex is oxidized
reduction bymethod.
pressurized
The oxidization
graphene/PLA andmasterbatch
then reduced(20%
to single-atom-thick
graphene) is graphene
prepared by
a multiplex reduction method. The graphene/PLA masterbatch (20% graphene) is prepared by solvent
by a multiplex
solvent blending reduction
from PLA method. The graphene/PLA
and graphene masterbatch
in THF media. (20%
The ‘‘x’’ graphene) is prepared
in ‘‘PLA/Graphene-x’’ by
is the
blending from PLA and graphene in THF media. The “x” in “PLA/Graphene-x” is the percentage of
solvent blending
percentage from PLAThe
of the graphene. andblack
graphene
sampleinis THF media. The ‘‘x’’
PLA/graphene-0.02 whichin ‘‘PLA/Graphene-x’’ is the
contains 0.02% grapheme;
thepercentage
graphene.ofThe the black sample
graphene. The is PLA/graphene-0.02
black which contains 0.02% grapheme; (b) Tensile
(b) Tensile strength and hardness [77].sample is PLA/graphene-0.02 which contains 0.02% grapheme;
strength and strength
(b) Tensile hardnessand [77].
hardness [77].

Figure 20. High resolution transmission electron microscope images of well-dispersed GnPs (a,b);
Figure 20. High resolution
and poorly-dispersed transmission
GnPstransmission electron
(c,d) in the PLA microscope
matrix. images
The graphene of well-dispersed
concentration in eachGnPs
figure(a,b);
Figure 20. High resolution electron microscope images of well-dispersed GnPswas(a,b);
and poorly-dispersed GnPs (c,d) in the PLA matrix. The graphene concentration in each figure was
and0.56 vol% [78].
poorly-dispersed GnPs (c,d) in the PLA matrix. The graphene concentration in each figure was
0.56 vol% [78].
0.56 vol% [78].
Zhang et al. [79] prepared PLA/GO nanocomposites with enhanced thermomechanical and
Zhang
electrical et al. [79]
properties by prepared PLA/GO
a solvent-casting nanocomposites
method withcommercial
while blending enhanced thermomechanical
PLLA resin with GO-g- and
Zhangproperties
electrical et al. [79]by prepared
a PLA/GO
solvent-casting nanocomposites
method while with
blending enhancedPLLA
commercial thermomechanical
resin with GO-g- and
PDLA, which was synthesized via ring-opening polymerization in which GO acted as an initiator.
electrical
PDLA, properties
which was by a solvent-casting
synthesized method while blending
via ring-opening commercial PLLA resin with
The percolation concentration was shown to be polymerization
1.0 wt% and 0.5inwt% which in GO
PLLA acted as anfilled
matrix initiator.
with
GO-g-PDLA,
pristine GO and GO-g-PDLA, respectively. Their in-situ FTIR showed that the SCs grew from the GOan
The which
percolation was synthesized
concentration was via
shown ring-opening
to be 1.0 wt% polymerization
and 0.5 wt% in in
PLLAwhich GO
matrix acted
filled as
with
initiator.
surface The
pristine GO
andand percolation
GO-g-PDLA,
stereocomplex concentration
respectively.
crystallite was shown
Their
networks weretoformed
in-situ be 1.0
FTIR wt%that
showed
with andenhanced
the 0.5SCs
the wt% infrom
grew
GO PLLA thematrix
network GO in
surface
PLLA/GO-g-PDLA composites, while only stereocomplex crystallite networks consisted in
filled withand stereocomplex
pristine GO and crystallite
GO-g-PDLA, networks were
respectively. formed
Theirwith the
in-situ enhanced
FTIR GO
showed network
that the SCs
of
grewPLLA/GO-g-PDLA
from the GO composites,
surface and while
stereocomplexonly stereocomplex
crystallite networkscrystallite
were networks
formed
spherocrystal (spherical crystalline mass) and PLA chains in PLLA/PDLA composites. Remarkable withconsisted
the of
enhanced
GOspherocrystal
network in(spherical
improvement in crystalline
PLLA/GO-g-PDLA
crystallinity mass) and PLAnanocomposites
composites,
of stereocomplex chains only
while in PLLA/PDLA
stereocomplex composites.
was observed Remarkable
crystallite
because networks
of the
improvement
consisted
heterogeneous in crystallinity
of spherocrystal of
nucleating(spherical stereocomplex
effect of crystalline
GO-g-PDLA. nanocomposites
mass)
Theyand was
PLA chains in
demonstrated observed
PLLA/PDLA
that because of the
composites.
the high-temperature
heterogeneous
Remarkable
stereocomplex nucleatingin
improvement
crystallite effect
platform of GO-g-PDLA.
crystallinity They
of stereocomplex
appeared when the demonstrated
nanocomposites
content of PDLA and thatGO-g-PDLA
the observed
was high-temperature
because
reached 15 of
stereocomplex crystallite platform appeared when the content of PDLA and
the heterogeneous nucleating effect of GO-g-PDLA. They demonstrated that the high-temperature GO-g-PDLA reached 15
Materials 2017, 10, 748 20 of 33

stereocomplex crystallite
Materials 2017, 10, 748 platform appeared when the content of PDLA and GO-g-PDLA reached 20 of 33
15 and 10 wt%, respectively. The formation of stereocomplex crystallite on the surface of GO increased
theand 10 wt%,
strength respectively. The
of stereocomplex formation
crystallite of stereocomplex
networks crystalliteimproved
and significantly on the surface of GO modulus
the storage increased at
the
◦ strength of stereocomplex crystallite networks
195 C for PLLA/GO-g-PDLA mixtures at various compositions. and significantly improved the storage modulus
at Figure
195 °C 21a
for PLLA/GO-g-PDLA mixtures at various compositions.
displays GO flake which is about 1 micron in the size later used by Zhang et al. [79], and
Figure
in Figure 21b PDLA 21a displays GOGO
a grafted flake which
crystal is is about 1 micron
displayed. As shownin theinsize later21c,
Figure used by Zhang
storage et al. [79],
modulus (E’) at
and in Figure 21b PDLA a grafted GO crystal is displayed. As shown in Figure 21c, storage modulus
◦
30 C increased from 2.2 GPa to 4.1 GPa with the addition of 20 wt% GO-g-PDLA (containing 1 wt%
(E’) at 30 °C increased from 2.2 GPa to 4.1 GPa with the addition of 20 wt% GO-g-PDLA (containing
GO). The large mechanical enhancement was ascribed to the reinforcement of GO on the PLLA–PDLA
1 wt% GO). The large mechanical enhancement was ascribed to the reinforcement of GO on the
stereocomplex networks. Similarly, this effect is more significant at 195 ◦ C, particularly after 10 wt%
PLLA–PDLA stereocomplex networks. Similarly, this effect is more significant at 195 °C, particularly
PDLA inclusion. They argued that existence of GO can be regarded as the landscape of the ordering of
after 10 wt% PDLA inclusion. They argued that existence of GO can be regarded as the landscape of
SC the
helices surrounding
ordering the PLA/GO
of SC helices surroundinginterface. At last,interface.
the PLA/GO the stereocomplex
At last, the crystallite, SC, crystallite,
stereocomplex could begin
to grow from the GO surface with the hydrogen bonding inter-chain interactions
SC, could begin to grow from the GO surface with the hydrogen bonding inter-chain interactions between PLLA and
PDLA and finally an SC network would form with the link of PLA chains
between PLLA and PDLA and finally an SC network would form with the link of PLA chains and enhancement of GOandas
schematically
enhancement shown
of GOinasFigure 21e.
schematically shown in Figure 21e.

Figure
Figure 21.21. TEMimages
TEM imagesofof(a)
(a)GO
GO and
and (b)
(b) purified
purified GO-g-PDLA.
GO-g-PDLA.Temperature
Temperaturedependency
dependencyofof storage
storage
modulus (E’) for PLLA mixed with various concentrations of (c) GO-g-PDLA. (d) Variation
modulus (E’) for PLLA mixed with various concentrations of (c) GO-g-PDLA. (d) Variation in storage in storage
modulus
modulus at at
195195
◦ C°C
as as a functionofofthe
a function thePDLA
PDLAcontent
contentfor
forPLLA/PDLA
PLLA/PDLA andand PLLA/GO-g-PDLA
PLLA/GO-g-PDLA
mixtures, respectively. (e) Schematic showing the synthesis of GO-g-PDLA by the direct melt-
mixtures, respectively. (e) Schematic showing the synthesis of GO-g-PDLA by the direct
polycondensation approach and the formation of GO enhanced stereocomplex (Sc) network. p-TSA
melt-polycondensation approach and the formation of GO enhanced stereocomplex (Sc) network.
stands for p-toluenesulfonic acid [79].
p-TSA stands for p-toluenesulfonic acid [79].

Xu et al. [80] demonstrated a very interesting way to use GO in enhancing thermomechanical

Xu et al.of
properties [80] demonstrated
PLA. In their study,a avery
thin interesting
layer of GO way to use was
nanosheets GO immobilized
in enhancingontothermomechanical
starch granule
properties
surfacesofbyPLA. In their
hydrogen study,to
bonding a thin
create layer of GO@starch
active GO nanosheets was Once
surfaces. immobilized onto into
incorporated starch
thegranule
PLA
surfaces
matrix,byGO@starch
hydrogen allowed
bondingformation
to create active GO@starchbetween
of nanointerfaces surfaces.PLAOnce incorporated
matrix and starch into the PLA
particles,
matrix, GO@starch
without migrationallowed formation
of GO into the PLAofmatrix.
nanointerfaces
Althoughbetween PLA was
the GO layer matrix and starch
ultrathin and GO particles,
was
present
without at ultralow
migration of GOconcentration
into the PLA (upmatrix.
to 0.03Although
wt%), these thenanointerfaces
GO layer wassignificantly
ultrathin andenhanced
GO was PLA–present
starch interfacial
at ultralow interactions
concentration (up towith
0.03uniform dispersion
wt%), these of GO@starch
nanointerfaces in PLA matrix.
significantly enhancedDriven by the
PLA–starch
high surface
interfacial activitywith
interactions of uniform
high-aspect-ratio
dispersion nanosheets,
of GO@starch50% inenrichment
PLA matrix. in Driven
crystallinity
by thewashigh
demonstrated for PLA/GO@starch films; clearly surpassing that of PLA/starch
surface activity of high-aspect-ratio nanosheets, 50% enrichment in crystallinity was demonstrated composites (below
for15%). They demonstrated
PLA/GO@starch that the
films; clearly combination
surpassing thatofofhigh tensile strength
PLA/starch (58.2(below
composites MPa), 15%).
improvedThey
elongation (6.1%) (see Figure 22A–D) and superior low gas barrier properties,
demonstrated that the combination of high tensile strength (58.2 MPa), improved elongation (6.1%) outperforming
(seePLA/starch30
Figure 22A–D) with an increase of ~280% in both strength and elongation, and a drastic decline in
and superior low gas barrier properties, outperforming PLA/starch30 with an
81.6% in oxygen gas barrier. Microscale morphologies of PLA/starch and PLA/GO@starch composites
increase of ~280% in both strength and elongation, and a drastic decline in 81.6% in oxygen gas barrier.
are presented in Figure 22E. PLA/GO@starch interfaces demonstrate much better bonding via fibrillar
Microscale morphologies of PLA/starch and PLA/GO@starch composites are presented in Figure 22E.
ligaments, which did not form in PLA/starch composites.
Materials 2017, 10, 748 21 of 33

PLA/GO@starch interfaces demonstrate much better bonding via fibrillar ligaments, which did not
form in PLA/starch
Materials 2017, 10, 748 composites. 21 of 33

Figure
Figure 22.22. Mechanical
Mechanical propertiesofofcomposite
properties compositefilms.
films.(A)
(A)Tensile
Tensilestrength;
strength;(B)
(B)tensile
tensilemodulus;
modulus;and
and(C)
(C) elongation at break demonstrating the superior tensile properties of PLA/GO@starch
elongation at break demonstrating the superior tensile properties of PLA/GO@starch in comparison in
comparison with PLA/starch; (D) Comparison of tensile strength for modified PLA/starch composites
with PLA/starch; (D) Comparison of tensile strength for modified PLA/starch composites using the
using the present method and various published covalent grafting techniques, including epoxidized
present method and various published covalent grafting techniques, including epoxidized itaconic acid
itaconic acid (EIA)-g-starch, epoxidized cardanol (Epicard)-g-starch, combined use of starch and
(EIA)-g-starch, epoxidized cardanol (Epicard)-g-starch, combined use of starch and starch-g-PLA (5 and
starch-g-PLA (5 and 10 wt%), starch-g-poly(ethylene glycol) (PEG), maleic anhydride (MA)-g-starch
10 wt%), starch-g-poly(ethylene glycol) (PEG), maleic anhydride (MA)-g-starch and starch-g-PLA.
and starch-g-PLA. See references in [80] related to each grafting method; (E) SEM images of fracture
See references in [80] related to each grafting method; (E) SEM images of fracture surfaces after
surfaces after tensile failure suggesting strong interfacial bonding and even formation of numerous
tensile failure suggesting strong interfacial bonding and even formation of numerous ligaments in
ligaments in PLA/GO@starch films [80].
PLA/GO@starch films [80].
6. Scaffolds Based on PLA-Graphene Nanocomposites: Porous Networks, Nanofibers, and Foams
6. Scaffolds Based on PLA-Graphene Nanocomposites: Porous Networks, Nanofibers, and Foams
In biomedicine, polymeric muscle or bone scaffolds must have an appropriate porosity range
In
with anbiomedicine, polymeric
interconnected and open muscle or bone
porosity, scaffolds
and have must haverate
a biodegradable an and
appropriate
mechanical porosity range
properties
with an interconnected and open porosity, and have a biodegradable rate and mechanical
that match the injured tissue. PLA polymers have been extensively studied for this purpose [81]. As properties
that match the
expected, injured
graphene ortissue.
graphenePLA polymershave
derivatives have been
also been extensively
incorporated studied
in PLAfor this scaffolds
based purpose to [81].
Astune
expected, graphene
and enhance or graphene
various derivativesattributes
thermomechanical have alsosuitable
been incorporated
for biomedical in engineering.
PLA based scaffolds to
tune and Nieto et al. various
enhance [82] demonstrated high strength
thermomechanical biocompatible
attributes suitable forscaffolds synthesized
biomedical by dipping
engineering.
graphene
Nieto etfoam (GrF)
al. [82] into a liquid solution
demonstrated containing
high strength PLA–poly-ε-caprolactone
biocompatible (PCL) copolymer,
scaffolds synthesized by dipping
designated
graphene foamas PLC.
(GrF)Exceptional
into a liquidwettability
solutionofcontaining
PLC on graphene foam resulted in PLC(PCL)
PLA–poly-ε-caprolactone forming a thin
copolymer,
designated as PLC. Exceptional wettability of PLC on graphene foam resulted in PLC formingand
uniform coating over the graphene foam. This effective wettability allowed the PLC to fill voids a thin
defectscoating
uniform that hadover
beenthepresent in the
graphene graphene
foam. foam. Thewettability
This effective healing ofallowed
defects in conjunction
the PLC to fillwith the
voids and
formation of PLC bridges caused enhanced strength and ductility in the GrF-PLC
defects that had been present in the graphene foam. The healing of defects in conjunction with the scaffold. They
conducted
formation ofnano-indentation
PLC bridges caused and macroscale
enhanced tensile
strengthtests
andand demonstrated
ductility in the that significant
GrF-PLC increase
scaffold. They
in strength in both compression and tension at multiple scales occurred, as shown in Figure 23 (see
conducted nano-indentation and macroscale tensile tests and demonstrated that significant increase
the left panel). Further, the effectiveness of PLC bridges bearing load was observed directly via in
in strength in both compression and tension at multiple scales occurred, as shown in Figure 23 (see
situ tensile testing in an SEM. Cellular studies conducted on both graphene foam and GrF-PLC
the left panel). Further, the effectiveness of PLC bridges bearing load was observed directly via
scaffolds demonstrate the ability of human mesenchymal stem cells (hMSCs) to survive and
proliferate throughout both scaffolds. The higher strength of the GrF-PLC scaffold enables the hMSCs
Materials 2017, 10, 748 22 of 33

in situ tensile testing in an SEM. Cellular studies conducted on both graphene foam and GrF-PLC
scaffolds demonstrate
Materials 2017, 10, 748 the ability of human mesenchymal stem cells (hMSCs) to survive and proliferate 22 of 33
throughout both scaffolds. The higher strength of the GrF-PLC scaffold enables the hMSCs to
to grow
grow normally
normally without
without undergoing
undergoing large
large deformations.The
deformations. Thepure
puregraphene
graphenefoamfoamdoes
doesnot
not have
have
sufficientstrength
sufficient strength to to withstand
withstand cell-induced
cell-induced strains
strains and toleads
and leads hMSCsto growing
hMSCs withgrowing with
highly highly
elongated
elongated morphology
morphology (Figure 23e),(Figure
whereas23e), whereas
in Figure in Figure
23f cells grown23f
on cells grown
GrF-PLC on GrF-PLC
scaffold scaffold
have larger have
and more
larger and
rounded more rounded
morphology morphology
indicating normalindicating
growth. normal growth.

Figure 23. (a) GrF-PLC sample

Figure 23. sample inin tensile
tensiletest
testsetup;
setup;(b)
(b)GrF-PLC
GrF-PLCsample
sampleinintension
tensionshowing
showing signs
signs of
of necking;
necking; (c)(c) GrF-PLC
GrF-PLC after
after tensile
tensile failure,
failure, loadload prior
prior to failure
to failure can can be seen
be seen to betoborne
be borne
by a by
fewa high
few
high strength
strength strands.
strands. (d,e) (d,e)
SEMSEMimagesimages of graphene
of graphene foamfoam scaffold
scaffold withwith
humanhuman mesenchymal
mesenchymal stemstem
cells
cells (hMSCs);
(hMSCs); (f,g) (f,g)
SEMSEM imagesimages of graphene
of graphene foam-PLC
foam-PLC hybrid hybrid scaffold
scaffold with with
human human mesenchymal
mesenchymal stem
stem
cells cells (hMSCs);
(hMSCs); (h) Fluorescence
(h) Fluorescence microscopy
microscopy image
image of of stemcells
stem cellson
ongraphene
graphene foam,
foam, cells appears
appears
severely
severelystretched
stretchedandandthinned;
thinned;(i)(i)
Fluorescence
Fluorescencemicroscopy
microscopy images of stem
images cellscells
of stem on graphene foam-PLC
on graphene foam-
scaffold [82].
PLC scaffold [82].

Chenet et
Chen al. produced
al. [83] [83] produced3D printed 3D elastic
printed elastic thermoplastic
thermoplastic polyurethane/PLA/GO polyurethane/PLA/GO
or TPU/PLA/GO or
TPU/PLA/GO nanocomposites by using a solvent mixing process as
nanocomposites by using a solvent mixing process as well as a fused deposition modeling technique, well as a fused deposition
modeling
as displayed technique,
in Figureas24a,b.
displayed in Figure 24a,b.
Nanocomposites wereNanocomposites
printed into complex were printed
shapes withinto complex shapes
high resolution.
with high resolution. Addition of GO significantly enhanced
Addition of GO significantly enhanced the mechanical properties of the polymer matrix, 167%the mechanical properties of the
in
polymer matrix,
compression 167% and
modulus, in compression
75.5% in tensile modulus,
modulusand(see75.5% in tensile
Figure 24c,d).modulus
Printing (see Figure 24c,d).
orientation led to
Printing mechanical
different orientation led to different
responses due tomechanical responsesstrength
the weak adhesion due to the weak adhesion
between strength
layers during between
3D printing.
layers during 3D printing. To investigate the effect of the addition of GO
To investigate the effect of the addition of GO on the mechanical property of the 3D printed parts on the mechanical property
of well
as the 3D printed
as the effectparts as wellorientation,
of printing as the effectcompression
of printing orientation,
testing and compression
tensile testingtesting and tensile
were performed.
testing were performed. For compression testing, all samples were
For compression testing, all samples were printed into a cuboid shape specimen. To investigate printed into a cuboid shape
specimen. To investigate the effect of printing orientation on mechanical
the effect of printing orientation on mechanical response, each type of sample (including TPU/PLA response, each type of
sample
blend (including
without TPU/PLA
GO, with 0.5, 2,blend
and 5without
wt% of GO) GO, was
withprepared
0.5, 2, and in 5two
wt% of GO) printing
different was prepared in two
orientations:
different printing orientations: standing specimen and lying specimen. For
standing specimen and lying specimen. For the standing specimen, the printing orientation is the same the standing specimen,
thethe
as printing
height orientation
direction whileis thefor
same as thespecimen;
the lying height direction whileorientation
the printing for the lying is specimen;
the same as the printing
the width
orientation
(length) is the Both
direction. sametypesas theof width (length)
specimens weredirection.
performedBoth under types of specimens
compression testingwere performed
in such a way
under compression testing in such a way that the height direction is parallel
that the height direction is parallel to the compression direction. Therefore, two kinds of compression to the compression
direction.
testing wereTherefore,
performed twoonkinds
eachof compression
sample: testing were
“S compression performed
testing”, whereon each
the sample:
standing “S compression
specimen is used
testing”, where the standing specimen is used in the testing, and “L compression
in the testing, and “L compression tests”, where the lying specimen is used in the testing. The data tests”, where the
lying specimen is used in the testing. The data are shown in Figure 24d,e. Compression modulus in
both L and S compression constantly increased with the loading of GO. Modulus increased 56% in L
compression and 167% in S compression at 5 wt% GO. They argued that addition of GO had
significantly improved the compression strength of TPU/PLA matrix.
Materials 2017, 10, 748 23 of 33

are shown in Figure 24d,e. Compression modulus in both L and S compression constantly increased
with the loading of GO. Modulus increased 56% in L compression and 167% in S compression at
5 wt% GO. They argued that addition of GO had significantly improved the compression strength of
Materials 2017, 10, 748 23 of 33
TPU/PLA matrix.
Thermal stability was also shown to be improved, with 90 ◦ C increase in degradation temperature
Thermal stability was also shown to be improved, with 90 °C increase in degradation temperature
as well as the new formation of better crystalline structures. They also conducted cell culturing
as well as the new formation of better crystalline structures. They also conducted cell culturing
experiments
experimentsand andresults
resultsrevealed
revealed excellent cell viability
excellent cell viability (Figure
(Figure24e–h)
24e–h)ofof3D3Dprinted
printed scaffolds,
scaffolds,
indicating
indicating limited
limitedaddition
additionofofGO
GOhas
has no
no obvious toxicityto
obvious toxicity tocell
cellgrowth,
growth,and
andsmall
small amount
amount of of
GOGO is is
beneficial for cell proliferation. They claimed that these 3D printed scaffolds can be good
beneficial for cell proliferation. They claimed that these 3D printed scaffolds can be good potential potential
candidates
candidates forfortissue
tissueengineering
engineeringapplications.
applications.

Figure
Figure 24.24.(a)
(a)3D
3Dprinted
printedmicrolattice
microlattice (5
(5 wt%
wt% of
of GO);
GO);(b)
(b)3D
3Dprinted
printedmicrolattice
microlatticeunder bending
under (5 wt%
bending (5 wt%
of GO); (c) S Compression testing curves of samples of different GO loadings; (d) L Compression
of GO); (c) S Compression testing curves of samples of different GO loadings; (d) L Compression
testing curves of samples of different GO loadings. 96 h cell culture results of NIH3T3 cells on 3D
testing curves of samples of different GO loadings. 96 h cell culture results of NIH3T3 cells on 3D
printed TPU/PLA with different GO loadings: (e) 0 wt% GO; (f) 0.5 wt% GO; (g) 2 wt% GO; (h) 5 wt%
printed TPU/PLA with different GO loadings: (e) 0 wt% GO; (f) 0.5 wt% GO; (g) 2 wt% GO; (h) 5 wt%
GO. Green color indicates live cells, whereas red color indicates dead cell [83].
GO. Green color indicates live cells, whereas red color indicates dead cell [83].
Chen et al. [83] further printed thin sheets of TPU/PLA blends containing 0.5, 2, and 5 wt% GO
Chen et cell
to support al. [83] further
adhesion, printed
growth, and thin sheets of TPU/PLA
proliferation. blends TPU/PLA
These 3D printed containing GO 0.5, 2, and 5 wt%
monolayers wereGO
to evaluated
support cellas adhesion, growth,using
seeding scaffolds and proliferation.
NIH3T3 mouse These 3D printed
embryonic TPU/PLA
fibroblast GO monolayers
cells with a LIVE/DEAD were
evaluated as seeding scaffolds using NIH3T3 mouse embryonic fibroblast cells
viability/cytotoxicity assay. The results of the LIVE/DEAD assay indicated that at all scaffolds, with a LIVE/DEAD
supported cell growth
viability/cytotoxicity as only
assay. Thelive cells was
results detected;
of the no deadassay
LIVE/DEAD cells were detected
indicated thatinatany
allscaffold
scaffolds,
(Figure 24e–h).
supported Additionally,
cell growth as onlythe NIH3T3
live cells detected;
cells was spread well noand proliferated
dead cells were ondetected
all scaffolds. Although
in any scaffold
all scaffolds supported cell growth and proliferation, the TPU/PLA with 0.5% GO
(Figure 24e–h). Additionally, the NIH3T3 cells spread well and proliferated on all scaffolds. Although (Figure 23f) showed
allthe highestsupported
scaffolds density ofcell
cells acrossand
growth all proliferation,
loadings of GO the and even when
TPU/PLA with compared
0.5% GO (Figureto the 23f)
TPU/PLA
showed
thecontrol.
highestThey attributed
density of cellsthis to the
across all configuration
loadings of GO ofand
GO even
in thewhen
TPU/PLA scaffold.
compared If the
to the loading level
TPU/PLA control.
of GO is too high, the GO may not “lay” flat in the polymer matrix and thus
They attributed this to the configuration of GO in the TPU/PLA scaffold. If the loading level of GO the enhancement in is
cellular growth may not advance. Taken together, they claimed that all scaffolds were nontoxic
too high, the GO may not “lay” flat in the polymer matrix and thus the enhancement in cellular growth
toward embryonic cells with 0.5% GO providing the greatest enhancement in cellular growth and
may not advance. Taken together, they claimed that all scaffolds were nontoxic toward embryonic cells
proliferation as indicated by the LIVE/DEAD assay.
with 0.5% GO providing the greatest enhancement in cellular growth and proliferation as indicated by
An et al. [84] produced porous films and nanofiber nanocomposites from PLA/Polyurethane
the LIVE/DEAD assay.
(PU) blends containing small concentrations of GO by simple liquid-phase mixing followed by
An et al. [84] produced porous films and nanofiber nanocomposites from PLA/Polyurethane
casting. The as-prepared ternary PLA/PU/GO composite films exhibited good antibacterial activity
(PU) blendsthe
against containing small concentrations
Gram-positive Staphylococcus of GO byand
aureus simple
the liquid-phase
Gram-negative mixing followed
Escherichia by They
coli. casting.
Theattributed this to the inherent antibacterial property of GO sheets with high specific surface area (seethe
as-prepared ternary PLA/PU/GO composite films exhibited good antibacterial activity against
Gram-positive Staphylococcus
Figure 25a). Addition of GO aureus andattachment
inhibited the Gram-negative Escherichia
and proliferation coli. Theyonattributed
of microbes this to the
the film surfaces,
inherent antibacterial property of GO sheets with high specific surface area
resulting in remarkably improved antibacterial activity compared to PLA/PU composite film. (see Figure 25a). Addition
The
inhibition efficiency was found to be proportional to the amount of GO (Figure 25a). Furthermore,
they fabricated PLA/PU/GO composite fibrous surfaces using electrospinning with good
biocompatibility. Incorporation of GO did not destroy normal cell’s proliferation and differentiation
as shown in Figure 25b,c. Even though they did not present any thermomechanical properties of the
Materials 2017, 10, 748 24 of 33

of GO inhibited attachment and proliferation of microbes on the film surfaces, resulting in remarkably
improved antibacterial activity compared to PLA/PU composite film. The inhibition efficiency was
found to be proportional to the amount of GO (Figure 25a). Furthermore, they fabricated PLA/PU/GO
composite fibrous surfaces using electrospinning with good biocompatibility. Incorporation of GO did
not destroy
Materials 2017, normal
10, 748 cell’s proliferation and differentiation as shown in Figure 25b,c. Even though 24they
of 33
did not present any thermomechanical properties of the composites they concluded that PLA/PU/GO
composites with they good antibacterial
concluded activity and composites
that PLA/PU/GO biocompatibility
with could
good render them attractive
antibacterial for
activity and
clinical applications
biocompatibility such
could as tissue
render themengineering.
attractive for clinical applications such as tissue engineering.

Figure 25.
Figure 25. Photographs
Photographs of
of (a)
(a)S.S.aureus
aureusand
and(b)
(b)E.E.coli
coligrown
grown onon PLA/PU/GO(3%)
PLA/PU/GO (3%)and
andPLA/PU/GO
PLA/PU/GO
(5%) for 4 h, respectively; (c) Fluorescence microscopy image of MC3T3-E1
(5%) for 4 h, respectively; (c) Fluorescence microscopy image of MC3T3-E1 cells grown on cells grown on the
the
electrospun PLA/PU/GO (5%) nanofibers for 48 h at 37 °C. The magnification is
electrospun PLA/PU/GO (5%) nanofibers for 48 h at 37 ◦ C. The magnification is 100× [84]. 100× [84].

Yoon et al. [85] constructed nanocomposite nanofibers of poly(D,L-lactic-co-glycolic acid)

Yoon et al. [85] constructed nanocomposite nanofibers of poly(D,L-lactic-co-glycolic acid) (PLGA)
(PLGA) embedded with GO nanosheets by electrospinning, and investigated their physiochemical
embedded with GO nanosheets by electrospinning, and investigated their physiochemical properties
properties including morphological, surface-chemical, mechanical, and thermomechanical properties.
including morphological, surface-chemical, mechanical, and thermomechanical properties. They
They showed a 2-fold increase in tensile modulus as compared to that of pristine PLGA nanofibers.
showed a 2-fold increase in tensile modulus as compared to that of pristine PLGA nanofibers. Storage
Storage modulus and glass transition temperatures of the PLGA/GO (2 wt%) nanocomposites were
modulus and glass transition temperatures of the PLGA/GO (2 wt%) nanocomposites were much
much higher than those of PLGA nanofibers and PLGA/GO (1 wt%) nanocomposite nanofibers, as
higher than those of PLGA nanofibers and PLGA/GO (1 wt%) nanocomposite nanofibers, as shown in
shown in Figure 26a,b.
Figure 26a,b.
Interestingly they claimed that the incorporation of GO nanosheets according to concentration
Interestingly they claimed that the incorporation of GO nanosheets according to concentration led
led to effective enhancement of their mechanical and thermomechanical properties compared to those
to effective enhancement of their mechanical and thermomechanical properties compared to those of
of pristine PLGA nanofibers. This enhancement was attributed to stronger interfacial interactions
pristine PLGA nanofibers. This enhancement was attributed to stronger interfacial interactions caused
caused by stronger chemical bonding between the PLGA and GO nanosheets and the axial alignment
by stronger chemical bonding between the PLGA and GO nanosheets and the axial alignment of the GO
of the GO nanosheets embedded nanocomposite nanofibers. They also showed that nanocomposite
nanosheets embedded nanocomposite nanofibers. They also showed that nanocomposite nanofibers
nanofibers were more hydrophilic than PLGA nanofibers. In order to investigate the effects of GO
were more hydrophilic than PLGA nanofibers. In order to investigate the effects of GO nanosheets
nanosheets on nanocomposite biocompatibility, PC 12 neuronal cells were cultured for two days on
on nanocomposite biocompatibility, PC 12 neuronal cells were cultured for two days on various
various nanofibrous scaffolds, and the proliferation and viability of the cells were measured.
nanofibrous scaffolds, and the proliferation and viability of the cells were measured. Biocompatibility
Biocompatibility tests (Figure 26c–f) showed that the enhanced surface chemical properties of the
tests (Figure 26c–f) showed that the enhanced surface chemical properties of the PLGA/GO (2 wt%)
PLGA/GO (2 wt%) nanocomposites effectively improved neuronal cell proliferation and viability
nanocomposites effectively improved neuronal cell proliferation and viability (Figure 26f), indicating
(Figure 26f), indicating enhanced biocompatibility of PLGA nanocomposite nanofibers by the
enhanced
addition ofbiocompatibility
2-D GO nanofillers.of PLGA nanocomposite nanofibers by the addition of 2-D GO nanofillers.
Zhang
Zhang etet al.
al. [86]
[86] surface
surface grafted
grafted GO
GO with
with polyethylene
polyethylene glycol
glycol (PEG)
(PEG) toto improve
improve its
its interfacial
interfacial
adhesion with PLA. Both GO and GO-g-PEG were incorporated in PLA polymer
adhesion with PLA. Both GO and GO-g-PEG were incorporated in PLA polymer as reinforcing as reinforcing fillers to
fillers
fabricate nanofibrous scaffolds of PLA, PLA/GO, and PLA/GO-g-PEG, which were
to fabricate nanofibrous scaffolds of PLA, PLA/GO, and PLA/GO-g-PEG, which were prepared by prepared by means
of electrospinning.
means They measured
of electrospinning. They decreases
measuredindecreases
complex viscosity
in complexfor PLA/GO-g-PEG electrospinning
viscosity for PLA/GO-g-PEG
suspensions and attributed these observations to the plasticizing effect of the
electrospinning suspensions and attributed these observations to the plasticizing effectgrafted PEG molecules
of the grafted
on the surface of GO.
PEG molecules on the surface of GO.
Materials 2017, 10, 748 25 of 33
Materials 2017, 10, 748 25 of 33

Figure 26.
Figure 26. Dynamic
Dynamic mechanical
mechanical analyzer
analyzercurves
curvesasas a function
a function of measurement
of measurement temperature.
temperature. (a)
(a) Storage modulus and (b) loss modulus. For PLGA, PLGA/GO (1
Storage modulus and (b) loss modulus. For PLGA, PLGA/GO (1 wt%), and PLGA/GO (2wt%), and PLGA/GO (2 wt%)
wt%)
nanofibers. Optical
nanofibers. Optical microscope
microscope images
images of
of PC
PC 12
12 cells
cells on
on (c)
(c) PLGA
PLGA nanofibers;
nanofibers; (d)
(d)PLGA/GO
PLGA/GO (1 (1 wt%)
wt%)
nanocomposites; (e) PLGA/GO (2 wt%) nanocomposites; and (f) cell proliferation and viability
nanocomposites; (e) PLGA/GO (2 wt%) nanocomposites; and (f) cell proliferation and viability data data
obtained by WST-1 assay of the cells cultured for 2 days (n = 12, p <0.05)
obtained by WST-1 assay of the cells cultured for 2 days (n = 12, p <0.05) [85]. [85].

This indicated good interfacial adhesion between GO-g-PEG and PLA and accounted for the
This indicated good interfacial adhesion between GO-g-PEG and PLA and accounted for the
decrease in the average fiber diameter from PLA to PLA/GO-g-PEG. Based on the TGA results, GO-
decrease in the average fiber diameter from PLA to PLA/GO-g-PEG. Based on the TGA results,
g-PEG had a greater influence than GO in improving the thermal stability of PLA. Tensile tests
GO-g-PEG had a greater influence than GO in improving the thermal stability of PLA. Tensile tests
showed that the addition of GO and GO-g-PEG in PLA achieved significantly improved tensile stress,
showed that the addition of GO and GO-g-PEG in PLA achieved significantly improved tensile stress,
as shown in Figure 27a,b. However, GO-g-PEG was more effective in reinforcing PLA than GO due
as shown in Figure 27a,b. However, GO-g-PEG was more effective in reinforcing PLA than GO due
to the grafted PEG chains, which improved the filler dispersion as well as the adhesion between GO
to the grafted PEG chains, which improved the filler dispersion as well as the adhesion between
and PLA. Moreover, PLA/GO and PLA/GO-g-PEG composite nanofibrous scaffolds were found to be
GO and PLA. Moreover, PLA/GO and PLA/GO-g-PEG composite nanofibrous scaffolds were found
non-toxic to NIH 3T3 cells and were capable of supporting cell attachment and growth (see Figure 27c,d).
to be non-toxic to NIH 3T3 cells and were capable of supporting cell attachment and growth (see
They claimed that their PLA/GO-g-PEG scaffolds with enhanced mechanical strength and good
Figure 27c,d). They claimed that their PLA/GO-g-PEG scaffolds with enhanced mechanical strength
cytocompatibility could be promising for applications in tissue engineering. This study is unique in
and good cytocompatibility could be promising for applications in tissue engineering. This study is
the sense that it demonstrated both improved thermomechanical properties due to GO as well as
unique in the sense that it demonstrated both improved thermomechanical properties due to GO as
promising cytocompatibility.
well as promising cytocompatibility.
Pal et al. [87] formulated PLA scaffolds loaded with different percentage of cellulose nanocrystals
Pal et al. [87] formulated PLA scaffolds loaded with different percentage of cellulose nanocrystals
(CNC) and reduced GO, rGO, as reinforcing nanofillers by a solution casting method. The modification
(CNC) and reduced GO, rGO, as reinforcing nanofillers by a solution casting method. The modification
and effects of the incorporated CNC and rGO on the morphology, crystallinity, thermal, mechanical,
and effects of the incorporated CNC and rGO on the morphology, crystallinity, thermal, mechanical,
and bio-medical properties were analyzed. Scaffolds with the percentage of CNC (1%) and rGO
and bio-medical properties were analyzed. Scaffolds with the percentage of CNC (1%) and rGO
(0.5%) were displayed improved biocompatibility, thermal and mechanical properties. For instance,
(0.5%) were displayed improved biocompatibility, thermal and mechanical properties. For instance,
an increase of 23.35% in tensile strength and 28.71% in Young’s Modulus, with respect to neat PLA
was observed in the scaffolds along with increase in thermal degradation threshold temperatures.
Materials 2017, 10, 748 26 of 33

an increase of 23.35% in tensile strength and 28.71% in Young’s Modulus, with respect to neat PLA
Materials 2017,
was observed in10,the
748 scaffolds along with increase in thermal degradation threshold temperatures. 26 of 33

Cytocompatibility tests proved that the PLA/CNC/rGO scaffolds were not toxic to NIH-3T3 cells.
Cytocompatibility tests proved that the PLA/CNC/rGO scaffolds were not toxic to NIH-3T3 cells.
This was also supported by the morphological examination of treated cells. Moreover, the scaffolds
This was also supported by the morphological examination of treated cells. Moreover, the scaffolds
were shown
were shownto have antibacterial
to have potential
antibacterial potentialagainst
against Gram Staphylococcus
positiveStaphylococcus
Gram positive aureus
aureus (S. aureus)
(S. aureus) and and
GramGram
negative bacteria
negative Escherichia
bacteria coli
Escherichia (E.coli)
coli(E. coli)as
as well. Althoughpromising
well. Although promising results
results were
were reported
reported by by
Pal etPal
al. et
[87],
al. [87], further in vivo tests along with biodegradation dynamics need to be studied in order to
further in vivo tests along with biodegradation dynamics need to be studied in order
ascertain applicability
to ascertain for biomedical
applicability uses.uses.
for biomedical

Figure 27. (a) Typical stress–strain curves of electrospun PLA, PLA/GO, and PLA/GO-g-PEG
Figure 27. (a) Typical stress–strain curves of electrospun PLA, PLA/GO, and PLA/GO-g-PEG
composite nanofiber mats; (b) Table demonstrating elastic modulus (σmax) and elongation at break
composite nanofiber mats; (b) Table demonstrating elastic modulus (σmax ) and elongation at break
values (εb); (c) Proliferation cell counts of NIH 3T3 cells on scaffolds after 3 and 10 days of culture.
valuesFluorescence
(εb ); (c) Proliferation cell counts of NIH 3T3 cells on scaffolds after 3 and 10 days of culture.
micrographs of stained cells showing live (green) and dead (red) cells on PLA/GO-g-
Fluorescence
PEG (2%)micrographs of(d)
scaffolds after stained
3 and cells
(e) 10showing live (green)
days of culture [86]. and dead (red) cells on PLA/GO-g-PEG
(2%) scaffolds after (d) 3 and (e) 10 days of culture [86].
Luo et al. [88] fabricated GO-incorporated PLGA nanofibrous mats via an electrospinning
Luo et al.and
approach explored
[88] the materials
fabricated use as scaffold
GO-incorporated materials
PLGA for tissue engineering.
nanofibrous mats via an GOelectrospinning
was readily
electrospun into PLGA nanofibers without changing the 3D porous structure. The impact of the GO
approach and explored the materials use as scaffold materials for tissue engineering. GO was readily
incorporation on the mechanical strength of the PLGA nanofibrous mat was also probed. They
electrospun into PLGA nanofibers without changing the 3D porous structure. The impact of the
concluded that all the materials possess excellent mechanical properties. But the breaking strength
GO incorporation on the decreased
and Young’s modulus mechanical strength
after of the
the addition PLGA
of 1% GO. nanofibrous mat was
This was attributed to itsalso probed. They
2D topological
concluded
plane that all theGO
structure, materials
might tendpossess
to beexcellent
vertical tomechanical properties.
the fibers. They further But
statedthethat
breaking
when thestrength
fibers and
Young’s
are modulus decreased
under stress, GO cannotafter the addition
transfer the partofforce,
1% GO. Thistowas
leading attributed
the decrease of to its 2D topological
breaking strength. Theplane
structure, GO might
GO-doped PLGA tend to be vertical
nanofibrous matsto could
the fibers. They
sustain thefurther stated that
extracellular matrixwhen the fibers
(ECM) are under
biomimetic
microenvironment for human mesenchymal stem cells (hMSCs) adhesion and
stress, GO cannot transfer the part force, leading to the decrease of breaking strength. The GO-doped proliferation. They
claimed that these GO-incorporated PLGA nanofibers could serve as
PLGA nanofibrous mats could sustain the extracellular matrix (ECM) biomimetic microenvironment novel tissue engineering
scaffolds
for human with good biocompatibility.
mesenchymal stem cells (hMSCs) Furthermore,
adhesion theyanddemonstrated
proliferation.that They
the GO-doped
claimed PLGA
that these
substrate induced expression of osteogenic marker genes such as ALP, Col I, and Ocn. Meanwhile, it
GO-incorporated PLGA nanofibers could serve as novel tissue engineering scaffolds with good
promoted ALP activity and osteocalcin secretion. The PLGA nanofibers incorporated with GO not
biocompatibility. Furthermore, they demonstrated that the GO-doped PLGA substrate induced
only promoted the attachment and proliferation of hMSCs but also enhance the hMSCs
expression of osteogenic
differentiation towardmarker genes
osteoblast, whichsuchisasimportant
ALP, Colfor I, and Ocn. Meanwhile,
biomedical applications itrequiring
promoted theALP
activity and osteocalcin secretion. The
biomimetic of extracellular matrix (ECM). PLGA nanofibers incorporated with GO not only promoted the
attachment and proliferation of hMSCs but also enhance the hMSCs differentiation toward osteoblast,
which is important for biomedical applications requiring the biomimetic of extracellular matrix (ECM).
Materials 2017, 10, 748 27 of 33

Kuang et al. [89] investigated the influence of graphene oxide (GO) on the unidirectional foaming
of PLA using supercritical CO2 as blowing agent. PLA/GO nanocomposite foams with oriented
and highly elongated cell structure were prepared for the first time in this study. They prepared the
nanocomposites by a solution mixing method at various GO contents. The thermal and rheological
properties, and CO2 absorption ability of the composites, were investigated and compared with
neat PLA. It was found that the incorporation of GO improved the crystallinity of the PLA matrix.
The addition of GO enhanced the storage modulus, loss modulus, and complex viscosity of the PLA
matrix as well, and the improvements increased with increase of GO content. The unidirectional
microcellular foaming revealed that the PLA/GO nanocomposites had the ability to form highly
elongated cell structure. Compared to neat PLA, the relatively high GO content (i.e., >0.6 wt%) in
PLA/GO nanocomposites showed significantly higher expansion ratio, average foam cell size, and
cell diameter ratio in the long axis and minor axis. These behaviors were caused by the enhanced
CO2 absorption rate, PLA/GO matrix viscosity, and the unidirectional foaming process. They did not
demonstrate biocompatibility or discuss any potential biomedical applications although their cellular
architectures were claimed to be very suitable as 3D biomedical scaffolds.

7. Conclusions and Outlook

Although PLA polymer is regarded as one of the most suitable biopolymers for biomedical
applications with many successful in vitro and in vivo demonstrations to date, it is now understood
that homo-composites of PLA polymers are not only thermally and mechanically superior to pure
PLA but also perform better as drug release platforms [90]. In other words, by homo-composite
blending PLLA and PDLA or synthesis of stereo block PLA, a stereocomplex (SC), is formed. PLA
SC polymers have higher melting temperatures (or heat resistance), mechanical performance, and
hydrolysis-resistance compared to those of neat PLLA and PDLA. As such, they have been extensively
studied in terms of biomedical and pharmaceutical applications as well as commodity, industrial,
and environmental applications [32,91]. Stereocomplexation stabilizes and strengthens PLA-based
hydrogel or nanoparticles for biomedical applications, as well. Stereocomplexation increases the
barrier property of PLA-based materials and thereby prolongs drug release from PLA based materials.
Table 1 presents a summary of the work reviewed herein. The table indicates that more work should
be devoted to SC blends, and the majority of the works have been conducted by solvent instead of
melt processing and somewhat lack proper thermomechanical characterization.
On the other hand, clearly, graphene has the ability to modulate crystallinity and the final
thermomechanical properties of PLA polymers including SCs. However, the effectiveness lies in the
proper surface functionalization of graphene of GO, a priori. As such, compatibilization of graphene or
graphene derivatives with PLA SCs can yet produce significant improvements in the thermomechnical
properties of SCs [92]. To achieve this PLA-graphene interface engineering is needed in order to be
able to enhance the thermomechanical properties of PLA polymers by graphene incorporation [93].
In addition, more systematic works need to be conducted in order to establish the effect of the type of
graphene nanosheets in terms of lateral size and number of layers on the thermomechanical properties
of PLA polymers and at the same time to maintain acceptable biocompatibility levels for potential
clinical applications [94].
An outstanding biomedical issue that still needs to be addressed is the fact that various chemical
pathways and agents used or proposed for surface functionalization or grafting graphene or graphene
oxide nanosheets so that they can be inserted into PLA matrices must not yield cytotoxicity.
Furthermore, their fate after passing through the cell membranes should be carefully evaluated.
This issue is still open; however, encouraging new results are being published [95,96]. Similarly, the
biological applications of graphene derivatives have significant potential because many attempts have
shown promising results regarding bio-functionalization and standardization of graphene derivatives
by fractionation based on size, number of layers, and chemical functionalities [33,96]. Therefore, it is
Materials 2017, 10, 748 28 of 33

not surprising that PLA/graphene material systems offer many unique advantages and chances to
discover
Materialsnew
2017, fascinating
10, 748 properties and potential applications. 28 of 33

Table 1. Summary
Table 1. Summary ofof
literature
literaturereviewed.
reviewed.The
The table
table lists the type
lists the typeof
ofPLA
PLApolymer
polymerand
andgraphene
graphene used
used
as well as the processing conditions and characterization methods employed.
as well as the processing conditions and characterization methods employed.
Type of PLA Type of Graphene Processing Crystallinity Annealing Mechanical Cell Morphology Reference
Graphene* Functionalization Analysis Properties Viability (2D or 3D)
Stereoblock None None Solvent Yes Yes Storage None Film 53
PLAs modulus
PLLA/PDLA
PLLA, PLDA, None None Solvent Yes Yes None None Film 54
blends with
PDLA
PLLA, PDLA None None Extrusion, No Yes Stress- None Thick 55
Injection strain specimen
molding
PLA None None Extrusion, Yes Yes Stress- None Thick 56
Injection strain specimen
molding
PLLA/PDLLA None None Solvent Yes Yes None Yes Film 58
PLLA None None Solvent Yes Yes None Yes Film 59
PLLA None None Solvent Yes Yes None Yes Film 60
PLLA None None Solvent Yes Yes None Yes Film 61
PLA Nanoflakes None Solvent Yes Yes None None Film 65
PLLA GO None Solvent Yes Yes None None Film 66
PLA Nanoflakes Acid/acylation Solvent Yes Yes None None Film 67
PLLA GO Octadecylamine Solvent Yes Yes None None Film 68
PLLA GO PDLA-graft Solvent Yes Yes None None Film 69
PLA rGO* None Solvent Yes Yes None None Film 70
PLA GO SC-graft Solvent Yes Yes None None Film 71
PLA Nanoflakes Epoxy Solvent/melt No Yes Storage None Film 73
modulus
PLA Nanoflakes None Solvent/melt No Yes Stress- None Film 74
strain
PLA Nanoflakes Liophilization Solvent No Yes Stress- None Film 75
strain
PLLA GO Hydroxyapatite Solvent Yes No Stress- None Film 76
strain
PLA Nanosheets Hydrazine/ammonia Melt Yes No Stress- None Film 77
strain
TypePLA
of PLA Type of
Nanosheets Graphene
None Processing
Solvent Crystallinity
Yes Annealing
No Mechanical
Storage Cell
None Morphology
Film Reference
78
modulus
PLA GO PDLA-graft Solvent Yes Yes Storage None Film 79
modulus
PLA GO Starch-graft Solvent Yes Yes Stress- None Film 80
strain
PLA-PCL* Graphene None Solvent No No Stress- Yes Foam 82
strain
PLA-PU* GO None Solvent No No Stress- Yes 3D printing 83
strain
PLA-PU GO None Solvent No No None Yes Fiber mats 84
PLGA* GO None Solvent No No Storage Yes Fiber mats 85
modulus
PLA GO PEG* Solvent No No Stress- Yes Fiber mats 86
strain
PLA GO/rGO Cellulose Solvent Yes No Stress- Yes Film 87
strain
PLGA GO None Solvent No No Stress- Yes Fiber mats 88
strain
PLA GO None Solvent/CO2 Yes No Storage Yes Foam 89
blowing modulus

* rGO:
* rGO:reduced
reducedgraphene
grapheneoxide; PCL: Polycaprolactone;
oxide; PCL: Polycaprolactone; PU:
PU:Polyurethane;
Polyurethane;PEG:
PEG: Polyethylene
Polyethylene glycol;
glycol;
PLGA: Poly(lactic-co-glycolic acid).
PLGA: Poly(lactic-co-glycolic acid).

SoSo
farfar
many
many published
publishedworksworkson onPLA/graphene compositesexclusively
PLA/graphene composites exclusivelyfocus
focuseither
either
onon thethe
thermomechanical
thermomechanicalproperties
propertiesor oron
oncell
cell compatibility and behavior.
compatibility and behavior. Hence,
Hence,nonenoneofofthem
themcancanbebe
explicitly
explicitly singledout
singled outasasananideal
idealcandidate
candidate for
for clinical
clinical applications.
applications. InInfact,
fact,both
bothproperties
properties areare
interrelated
interrelated as was
as was shown
shown by someby some
recentrecent
studiesstudies on theof effect
on the effect of PLA crystallinity
PLA crystallinity on cell
on cell proliferation.
Therefore, it is suggested that future studies on PLA-graphene systems should focus on focus
proliferation. Therefore, it is suggested that future studies on PLA-graphene systems should surface
on surface functionalization
functionalization of graphene and of graphene
graphene andoxidegraphene oxide with biomacromolecules
with biomacromolecules and incorporation and of
incorporation
these of these modified
modified graphene nanoflakesgraphene
into PLAnanoflakes
polymers,into particularly
PLA polymers,intoparticularly into PLA
PLA stereocomplexes.
stereocomplexes. The characterization should clearly demonstrate changes in
The characterization should clearly demonstrate changes in crystallinity, thermomechanical propertiescrystallinity,
thermomechanical properties due to functionalized graphene addition, and how this translates into
cell proliferation and cytocompatibility compared to simple PLA polymer/graphene systems.
Materials 2017, 10, 748 29 of 33

due to functionalized graphene addition, and how this translates into cell proliferation and
cytocompatibility compared to simple PLA polymer/graphene systems.
Since PLA polymers can be processed with ease in many different ways such as solution, spinning,
extrusion, and injection, they offer unique possibilities to produce graphene–PLA nanocomposites with
higher-order 3D architectures such as sponge-like macroporous scaffolds, nanofiber mats or hollow
micrometer-sized spheres, which are ideal platforms for surgical reconstruction and drug delivery [97].
Although graphene and GO are able to accelerate the growth, differentiation, and proliferation
of stem cells, and therefore hold great promise in tissue engineering, regenerative medicine, and
other biomedical fields, still many safety questions exist (i.e., cellular uptake mechanisms) hence
incorporation of graphene and/or GO in 3D PLA architectures can minimize and even eliminate
these concerns.
The future seems to be the combination of PLA stereocomplexes with graphene and GO rather
than PLA as PLA stereocomplexes are not only thermomechanically superior but also more flexible in
terms of interfacial engineering, opening up many promising additional applications in delivery of
drugs, genes, proteins, growth factors, and other biomolecules for cancer and other disease therapies.
As seen, advances in PLA/graphene material systems are growing rapidly and this trend, the author
believes, will continue and even speed up in the years to come.

Conflicts of Interest: The author declares no conflict of interest.

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