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From Solution to Biointerface: Graphene Self-Assemblies of
VaryingLateral Sizes and Surface Properties for Biofilm Control
andOsteodifferentiationZhaojun Jia,† Yuying Shi,† Pan Xiong,†
Wenhao Zhou,† Yan Cheng,*,† Yufeng Zheng,†,‡ Tingfei Xi,†
and Shicheng Wei†,§
†Center for Biomedical Materials and Tissue Engineering, Academy
for Advanced Interdisciplinary Studies, ‡Department of
AdvancedMaterials and Nanotechnology, College of Engineering, and
§Department of Oral and Maxillofacial Surgery, School and Hospital
ofStomatology, Peking University, Beijing 100871, China
*S Supporting Information
ABSTRACT: Bringing multifunctional graphene out of solution
through facile self-assembly to form 2D surface nanostructures,
with control over the lateral size and surfaceproperties, would be
an intriguing accomplishment, especially in biomedical fields
wherebiointerfaces with functional diversity are in high demand.
Guided by this goal, in this work,we built such graphene-based
self-assemblies on orthopedic titanium, attempting toselectively
regulate bacterial activities and osteoblastic functions, which are
both crucial inbone regeneration. Briefly, large-area graphene
oxide (GO) sheets and functionalizedreduced GO (rGO)
micro-/nanosheets were self-assembled spontaneously and
controllablyonto solid Ti, through an evaporation-assisted
electrostatic assembly process and a mussel-inspired one-pot
assembly process, respectively. The resultant layers were
characterized interms of topological structure, chemical
composition, hydrophilicity, and protein adsorptionproperties. The
antibacterial efficacies of the assemblies were examined by
challenging themwith pathogenic Staphylococcus aureus (S. aureus)
bacteria that produce biofilms, wherebyaround 50% antiadhesion
effects and considerable antibiofilm activities were observed
forboth layer types but through dissimilar modes of action. Their
cytocompatibility and osteogenic potential were also
investigated.Interfaced with MC3T3-E1 cells, the functionalized rGO
sheets evoked better cell adhesion and growth than GO sheets,
whereasthe latter elicited higher osteodifferentiation activity
throughout a 28-day in vitro culture. In this work, we showed that
it istechnically possible to construct graphene interface layers of
varying lateral dimensions and surface properties and confirmed
theconcept of using the obtained assemblies to address the two
major challenges facing orthopedic clinics. In addition,
wedetermined fundamental implications for understanding the
surface−biology relationship of graphene biomaterials, in efforts
tobetter design and more safely use them for future
biomedicine.
KEYWORDS: self-assembly, poly(dopamine), graphene nanomaterials,
antibacterial, cytotoxicity, osteogenic
1. INTRODUCTION
The successful “scotch-tape” isolation of graphene, a
honey-comb-structured monatomic nanosheet of sp2-hybridizedcarbon,1
has triggered a wave of development for next-generation 2D planar
nanomaterials. Over the past decade,intensive research has been
rapidly expanding nanoscalegraphene materials from fundamental
physics to a myriad ofwidespread applications, including in
electronics, energy, water,and biomedicine.1−3 In particular,
graphene and graphenederivatives are integrated with biological
systems given thatthey are biocompatibile, multifunctional
(conductive, anti-bacterial, osteogenic, able to deliver drugs,
etc.), inexpensive,and sustainably available.2,4 Of these
materials, graphene oxide(GO), bearing a great deal of functional
groups on its carbonplane, exhibits great promise because of its
good hydrophilicity,chemical reactivity, and solution
processability.5 Herein,graphene-based interfaces with
antibacterial and osteogenicbiofunctionalities are highlighted. On
one hand, to date, intense
studies have employed GO solutions (dispersions) for thepurposes
of bacteria inactivation, giving generally excitingresults.4,6,7 In
contrast, pure-graphene-presenting antimicrobialsurfaces (not
composite coatings, such as GO/hydroxyapatite/Ag) are far
fewer,8−12 thus requiring more focus, especially ontheir
antibacterial actions, which can vary from those observedin
solution.11 On the other hand, GO has displayed
greatosteoconductive and osteoinductive abilities for
regulatingosteoblastic differentiation.13,14 Nevertheless, the
nanotoxicdanger has raised a lively safety discussion. For example,
therough edges of graphene sheets can slice through and
penetratethe membrane and disrupt normal cell functioning.15 It
isexpected that graphene-on-substrate engineering can
restrictgraphene sheet mobility (thus avoiding unwanted
interactions
Received: May 1, 2016Accepted: June 21, 2016Published: June 21,
2016
Research Article
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with cells) and tailor the sheet size and the levels of defects
andsurface functionalization, hence enabling control of
thecytotoxicity below an acceptable level.16 Further, it
allowsconcomitant chemical modification of the graphene to
reinforceits physicochemical properties and perhaps add new
function-alities such as cell- and bacteria-specific
responses.17,18
In orthopedics, Ti-based implants are frequently used forbone
repair and replacement; however, their poor oesteointe-gration and
vulnerability to implant-centered infections remaintwo major
clinical challenges.19,20 Dual-functional Ti surfacesthat depress
bacterial activities while enhancing osteoblasticfunctions are
therefore in dire need of development. To date,considerable
interest has been focused on constructingcomplicated Ti surfaces
with multiple releasable components(normally, combining
bactericides with osteogenic fac-tors19−22). However, these
strategies could potentially sufferone or more limitations in real
applications, such as bacterialresistance, low stability,
decreasing efficiency with time, discretesterilization, high costs,
and tedious and intricate preparationand handling. Thus, it is
better to find a simple, benign,inexpensive, yet unified
biofunctionalization approach that isamenable to practical
implementation. An alternative is totransform dispersed graphene in
situ into biocompatible,multifunctional surface layers that can
selectively influencebiological activities of microbes and living
cells; in turn, theestablished surfaces could serve as an
alternative platform, otherthan solution-based, for better
elucidating the roles of theavailable, diverse
antibacterial/nanotoxicological mechanisms ofgraphene,4 as some of
them (e.g., cell wrapping, membranepenetration, and cellular
intake) can be excluded if the systemdimension (complexity) is
reduced from 3D (solution) to 2D(surface). Such an approach can
also permit an isolatedinvestigation of the wanted physical or
chemical factors(through variable control), as long as the
on-surface grapheneis exquisitely designed.10,12 We believe that
the results will beuseful for both the theoretical understanding
and the practicalutilization of graphene in biomedical fields. This
concept (alsoillustrated in Figure 1a), to the best of our
knowledge, has notyet been tested. Indeed, it is technically
difficult to implement,especially when the size and surface
properties as well ascellular/bacterial behaviors of graphene all
need to beconsidered.To this end, the promising aqueous
self-assembly technique
is highlighted for its ability to form thin layers on
selectedtargets, with simplicity, universality, and easy
thicknesscontrol.23 Based on different assembly principles, it is
possibleto produce 2D graphene layers of varying lateral
dimensionsand surface properties that critically dictate their
biologicalbehaviors. Herein, two flexible, easy-to-use
self-assemblystrategies, depicted in Figure 1b, were developed to
yield twodifferent biofunctional graphene interfacial layers on
metallicTi: (1) macro-to-microscopic GO bulk sheets, as
constructedthrough electrostatic self-assembly assisted by
poly-(ethyleneimine) (PEI) and promoted by evaporation, and
(2)surface-modified submicroscopic sheets of chemically
reduced/functionalized GO, assembled using the novel
one-potreactivity and versatility of dopamine (DA), as inspired
bymarine mussels.24 Catechol-bearing DA is shown to
formsubstrate-independent, adhesive poly(dopamine) (PDA)
layersthrough mild, pH-triggered oxidative self-polymerization,
andPDA is multifunctional: It acts as a secondary platform to
reactfurther with various functional groups (quinone, thiol,
amine,etc.) through dismutation or Michael addition or Schiff
base
reactions, and it is a reducing/stabilization agent for
buildingorganic/inorganic hybrid materials.25 In particular, the
use ofDA to simultaneously reduce, stabilize, and functionalize
GOhas been well-documented.17,26−28 However, prior workemployed
DA−GO binary systems, with the GO as bulkmaterial to be
reduced/modified. Here, we described a uniqueDA−GO−substrate
ternary system, involving a one-stepreaction that not only reduced
and decorated GO but alsoscreened the sheets and selectively
immobilized them in themicro-/nanoscopic range onto the
substrate.In the study, we set out to (1) establish and
characterize
these surface assemblies with varying lateral dimensions,
defectdensities, and functional states; (2) examine whether
theinterfaces simultaneously benefit the antibacterial
activity,cellular compatibility, and osteogenic properties of
orthopedictitanium; and (3) further compare the two assemblies
withrespect to the critically important correlation between
theirphysicochemical characteristics and biological effects
onbacteria/osteoblasts, to advance the current state of
knowledgeabout graphene sheet surfaces. This work was intended to
helpguide better design and use of future graphene
biomaterials.
2. EXPERIMENTAL SECTION2.1. Materials. GO bulk sheets were
obtained from Angstron
Material LLC (Dayton, OH), as synthesized by Hummers’ method.DA
(hydrochloride) and PEI (MW = 10000) were purchased fromAlfa Aesar
and Aladdin, respectively, and used as received. Suspensionsof GO
(1 mg/mL) were freshly prepared by adding 100 mg of GObulk sheets
into 100 mL of 30% (v/v) EtOH and ultrasonically mixing
Figure 1. (a) Conceptual illustration of constructing
multifunctionalgraphene biointerfaces through straightforward
self-assembly forscientific/practical purposes. (b) Routes for the
two assemblystrategies: (1) evaporation-assisted, colloidal
electrostatic assemblyand (2) mussel-inspired, one-pot covalent
assembly. Note: Thegraphene sheets extend farther than depicted,
and the molecularstructure of PDA is simplified.
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for over 6 h to form brown colloidal dispersions.
Commerciallyavailable pure titanium (cpTi) foils with dimensions of
Φ 10 mm × 1mm were polished using SiC paper up to 2000 grit; rinsed
sequentiallyin acetone, alcohol, and deionized water (DI); and then
dried at 60 °Cbefore use.2.2. Evaporation-Assisted Electrostatic
Assembly of GO Bulk
Sheets. In brief, Ti disks were first immersed into a PEI
solution (5mg/mL, pH 7.4) for 30 min and then withdrawn and
rinsedthoroughly to dislodge excess PEI. Thereafter, a GO
suspension (pH∼3.0) and PEI-treated Ti disks were coincubated
without a cover andleft shaking at 37 °C overnight. This induced
evaporation-assistedelectrostatic assembly and gave the so-called
bulk GO.2.3. One-Pot Assembly of rGO-PDA Micro-Nanosheets. The
one-pot surface modification consisted of three components: GO,
DA,and metal Ti. Typically, GO suspensions were first mixed with a
Trisbuffer solution (10 mM) of DA (2 mg/mL) at adjustable
DA/GOratios of 1:1, 2:1, 5:1, and 10:1. A pH value of 8.5 was
achieved. Next,Ti foils were introduced, and the resulting mixtures
were constantlyshaken at 45 °C for 24 h. After that, samples were
withdrawn,ultrasonically treated to detach unbound products, and
further rinsedand dried under nitrogen. For clarity, samples
prepared at DA/GOratios of x are denoted as rGO-PDAx (x = 1, 2, 5,
and 10).2.4. Surface Characterization. Surface morphology and
topology
were observed by field-emission scanning electron microscopy
(FE-SEM, S4800, Hitachi) and atomic force microscopy (AFM,
DimensionICON, Bruker) in contact mode. X-ray photoelectron
spectroscopy(XPS) was performed on an AXIS Ultra spectrometer
(KratosAnalytical, Manchester, U.K.) with Al Kα excitation
radiation (1486.6eV). Fourier transform infrared (FTIR, Nicolet,
Madison, WI) spectrawere collected in transflection mode in the
range of 700−4000 cm−1.Micro-Raman spectra were recorded on a
confocal Raman microscope(Renishaw 1000) under an excitation laser
of Ar+ at 514 nm.2.5. Contact Angle (CA) Measurements. The surface
hydro-
phobicity was determined using the sessile-drop water method,
underambient conditions, on an SL200B Contact Angle System
(KINO,Norcross, GA) equipped with a high-resolution camera.
Measurementswere taken until droplets were well settled on samples
and repeated intriplicate, at six different positions per substrate
type.2.6. Protein Adsorption. Bovine serum albumin (BSA) was
used
as a model protein. Aliquots of BSA solution (1 mg/mL, pH 7.4)
wereintroduced carefully onto different surfaces and incubated at
37 °C for1 h. Samples were collected and rinsed with
phosphate-buffered saline(PBS), and the adsorbed proteins were
eluted using 2% sodiumdodecyl sulfate (SDS, Sigma) under shaking at
37 °C for 2 h.Subsequently, quantification was chieved with a Micro
BCA ProteinAssay Reagent Kit (Thermo Scientific) by measuring
absorbance at570 nm on a microplate reader (Bio-RAD, Hercules,
CA).Alternatively, specimens adsorbed with fluorescein
isothiocyanate-(FITC-) conjugated BSA under identical conditions
were fixed in 4%paraformaldehyde (PFA, in PBS) for 15 min and
subjected tofluorescence imaging under confocal scanning laser
microscopy(CSLM, Nikon ALR-SI) at an excitation wavelength of 488
nm.2.7. Antimicrobial Activity Assays. 2.7.1. Bacteria Culture
and
Inoculation. S. aureus was used and incubated aseptically in
Luria−Bertani (LB) broth at 37 °C. The overnight-grown bacteria
(adjustedto 106−107 cells/mL) were seeded onto each surface
(sterilized byautoclaving) and then incubated for predetermined
time periods. Allexperiments and measurements were carried out in
triplicate.2.7.2. Microbial Viability Assay. To evaluate the
performance of
various surfaces in suppressing bacterial adhesion, a convenient
WST-8-based microbial viability assay (Dojindo, Kumamoto, Japan)
wascarried out according to the manufacturer’s instructions.
Results wereobtained by colorimetrically measuring the formazan dye
yieldedduring microbial metabolism in the presence of WST-8 and
anelectron mediator. For each sample, controls with materials only
wereset to exclude any background interference.2.7.3.
Characterization of Adherent Bacteria by SEM. For SEM
observations, adhered bacterial cells were fixed with 2.5%
(v/v)glutaraldehyde (GA) and dehydrated in serial ethanol
(30−100%).
After being dried in air, the constructs were sputtered with
thin goldlayers prior to analysis.
2.7.4. Live/Dead Staining and CLSM Investigation. The
sampleswere first rinsed with 0.85% physiological saline twice, and
a LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes,
Invitrogen)was used to stain the specimens, as previously
described.29 Imagestacks were recorded successively by CLSM, and
then 3D visual-izations were reconstructed with aid of a
NIS-Elements AR package,version 3.0.
2.7.5. Biofilm Formation Assay by Crystal Violet Staining.
Aftersamples had been cultured for 5 days, 400 μL of PBS was added
twiceto gently remove the loose bacteria. The biofilms on the back
sides ofthe samples were cleaned carefully with a swab immersed in
70% (v/v)EtOH. To visualize biofilm development, the samples were
fixed with4% PFA, stained with 0.1% (w/v) crystal violet for 15
min, and washedthree times with PBS to remove excess stains. After
pictures weretaken, the dyes were eluted with 95% (v/v) EtOH, and
absorbance at570 nm was determined.
2.7.6. Production of Reactive Oxygen Species (ROS). Theformation
of ROS was measured using a sensitive 2′,7′-dichloro-fluorescin
diacetate (DCFH-DA) fluorescent stain method, inaccordance with the
manufacturer’s protocol (Jiancheng Biotech,Nanjing, China).
Briefly, dilute DCFH-DA (nonfluorescent) wascoincubated at 37 °C
with bacteria (on specimens that had beenprecultured for 24 h) for
2 h, and it could be transformed into 2′,7′-dichlorofluorescin
(DCF, fluorescent) in the presence of ROS. CLSMfluorescence images
were collected at 488 (Ex) and 535 (Em) nm intriplicate, and for
each sample, five random data points were selected.ImageJ freeware
was used for data analysis.
2.7.7. Bacterial Impacts on Materials. Ti foils with
grapheneassemblies were cultivated with S. aureus for 48 h.
Thereafter, sampleswere collected, rinsed, and ultrasonically
vibrated to dissociate adheredbacteria.29 The resulting surfaces
were dried and characterized bySEM.
2.8. Cytocompatibility and Osteogenic Activity Assays.2.8.1.
Cell Culture and Seeding. The bone-forming MC3T3-E1cells were
cultivated in α-MEM supplemented with 10% fetal bovineserum (FBS)
and 1% penicillin/streptomycin (pen-strep) in ahumidified incubator
with 5% CO2 at 37 °C. Subconfluent cellswere harvested and seeded
on autoclaved samples at a density of 5 ×104 cells/mL in 48-well
tissue culture plates (TCPS; negative controlalso). The medium was
refreshed every 2−3 days.
2.8.2. Cell Adhesion. Cells were allowed to attach to surfaces
for 4and 8 h postseeding. To visualize cell attachment,
polychromeimmunofluorescence staining was performed in sequence
usingspecific stains of focal adhesions, tubulin cytoskeleton, and
nuclei,namely, Anti-FAK antibody kit (1:100, Abcam),
Tubulin-Tracker RedProbe (1:250, Beyotime), and
4′,6-diamidino-2-phenylindole (DAPI,1:1000; Sigma), respectively,
in accordance with the manufacturers’protocols. Prior to staining,
samples were fixed with 4% PFA for 15min and then subjected to 5
min of permeabilization using 0.1%Triton X-100 and 1 h of blocking
in 1% BSA. For each of three parallelsamples, images were recorded
over five areas by CLSM inmultichannel mode, and afterward,
quantification was performedusing ImageJ software.
2.8.3. Cell Proliferation and Morphology. Cell proliferation
wasquantified using Cell Counting Kits (CCK-8, Dojindo), as
detailedelsewhere,29 based on the measurement of mitochondrial
activity. Inaddition, the cell morphology was determined by CLSM
study of thestained actin cytoskeleton. For staining, the samples
were rinsed threetimes with PBS and fixed in 4% PFA. Fixed cells
were permeabilizedand counterstained with FITC-phalloidin (1:200,
40 min; Sigma) andDAPI (1:1000, 5 min).
2.8.4. Cell Apoptosis. To determine the degree of cell
apoptosis, theextracellular release of lactate dehydrogenase (LDH)
was measured at3 days with an LDH kit (Abcam, Cambridge, MA),
following themanufacturer’ protocol. LDH release at the single-cell
level (i.e.,cellular LDH activity) was determined by normalizing
the total LDHactivity to the number of cells.
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2.8.5. Intracellular ROS Levels. The levels of intracellular ROS
forMC3T3-E1 cells were assayed using a DCFH-DA-based
fluorescencetest similar to that described in section 2.7.6 for
bacteria.2.8.6. Cellular Live/Dead Staining. At 7 days, the
survival abilities
of cells on different samples were assessed by staining living
cells with2 μM Calcein AM and the dead cells with 4 μM PI
(Live/Dead CellStains, Dojindo, Kumamoto, Japan), after which
confocal fluorescenceimages were recorded.2.8.7. Alkaline
Phosphatase (ALP) Activity. The ALP activity was
assayed by measuring the transformation of substrate
p-nitro-phenylphosphate (pNPP; Jiancheng Biotech, Nanjing, China)
into p-nitrophenol (pNP) after culture for 7 days. In short, cells
were washedwith PBS and lysed with 1% Triton X-100 for 40 min, and
then the celllysis was mixed to react with buffered pNPP solution
at 37 °C.Absorbance at 520 nm was read after colorimetric reaction.
The ALPactivity was normalized against total cellular proteins, as
determined byBCA reaction (see section 2.6), and expressed in U per
gram ofprotein.2.8.8. Extracellular Matrix (ECM) Collagen and
Calcium Assays.
Histochemical dyes Sirius Red (SR, 0.1%; Sigma) and Alizarin Red
S(ARS, 2%; Sigma), which bind specifically to ECM collagen
andcalcium salts, were employed following a 21-day and 28-day
cultures,respectively. For staining, cells were fixed in 4% PFA and
rinsed withPBS, and 500 μL of SR or ARS dye solution was added and
incubatedfor 18 h or 15 min, respectively; after thorough washing,
specimenswere dried and photographed. Quantitatively, dyes were
extractedusing 50% 0.2 M NaOH/methanol (for SR) and 10%
cetylpyridiniumchloride (for ARS), and the samples were then read
on a microplate at570 or 562 nm.2.9. Statistical Analysis. All data
in this paper were analyzed using
SPSS 19.0 software. Statistical significance was determined
using one-way analysis of variance (ANOVA) or Student’s t test and
defined as a
p value of less than 0.05. Data values are expressed as mean ±
standarddeviation.
3. RESULTS AND DISCUSSION
3.1. Material Preparation and Characterization. In thisstudy, we
established two graphene-based layer types withvarying sizes and
chemical states in situ on the surface of solidTi. First, GO bulk
sheets were constructed readily byevaporation-assisted
electrostatic assembly (Figure 1b; route1). Actually, this strategy
was motivated by combining theexisting electrostatic assembly and
evaporation-induced inter-facial assembly of GO.23 The aqueous GO
sheets weremoderately charged (negative, because of ionized
carboxylgroups30), but they were metastable and tended to
precipitateonto Ti foils that were precharged with PEI, a
well-knowncationic polyelectrolyte that is rich in amino groups. As
a result,some GO sheets bound to the PEI-modified Ti, initiating
theformation of a GO prelayer with incomplete
coverage.Subsequently, the slowly evaporation of EtOH/water
inducedan intrinsically self-concentrating process of additional
GOsheets at the liquid/prelayer interface, eventually leading
tointerlinked macroscopic GO films on the substrate.23 Themajor
chemical interactions can include electrostatic inter-actions
between PEI and GO and π−π stacking and hydrogen-bonding
interactions among different GO sheets.As an alternative,
chemically modified rGO sheets were
prepared according to the principle of
catecholamine-assisted,facile, spontaneous coassembly (Figure 1b;
route 2).24 Toinitiate this process, mixed solutions of DA and GO
were
Figure 2. Chemical compositions of the graphene assemblies: (a)
Raman spectra of (1) bulk GO and (2−5) rGO-PDAx for x = 1, 2, 5,
and 10,respectively; (b) FTIR spectra; and (c,d) C 1s XPS spectra
of (c) bulk GO and (d) rGO-PDA.
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placed in one pot with Ti foils at an alkaline pH of 8.5 and
atemperature of 45 °C. Note, here, that the DA chemicallymodified
and reduced GO into rGO and, in turn, the resultantrGO acted as a
template for DA to autooxidize and self-polymerize, during which
the brown color of the mixturedarkened over time.17,28,31 In
detail, the reaction might haveproceeded as a three-step process
(Figure S1a):18,28 (1)chemisorption (π−π stacking, aryl−aryl
coupling) or covalentgrafting of DA molecules onto GO and
conversion of GO intorGO (partly reduced) by their catechol groups;
(2) the pH-induced, graphene-templated formation of PDA precursors
andcross-linking of adjacent graphene sheets; (3) further growthand
aggregation of PDA clusters. Simultaneously, the settlingand
immobilization of PDA-functionalized rGO (rGO-PDA)onto Ti metal
took place, probably in three steps as well(Figure S1b): First, PDA
nanolayers formed easily on the Tibecause of the strong bidentate
coordination of DA towardTiO2 species, which are rich on passivated
Ti.
32 Second, therGO-PDA sheets could be readily integrated with
the PDA-modified Ti, possibly through covalent cross-links
(mediated byfree DA molecules) and aryl−aryl coupling.18
Additionally, atan earlier stage, the PDA-precursor-modified
graphene sheets
might also associate with the already-modified Ti through
π−πstacking and covalent linkages.28 Third, as PDA grew furtherand
new sheets were deposited, stronger interactions werelikely
established. Nonetheless, the exact reactions might be farmore
complex, so future work on this issue is needed.Predictably, the
DA/GO ratio is a critical factor needing
consideration to obtain ideal layers. To this end, we
preparedrGO-PDAx samples (x = 1, 2, 5, 10) and subjected
them,together with bulk GO, to Raman spectroscopy, a
powerfulanalytical tool for probing the crystal structure,
disorder, andlattice defects of carbonaceous nanomaterials. The
results arepresented in Figure 2a. For GO materials, the Raman
spectrumfeatures the broadening of two associated bands: the G band
atca. 1580 cm−1 and the D band centered at ca. 1350 cm−1.8
Whereas the G peak is general in all sp2 graphitized
structures,the D-mode peak is exclusively assigned to the defect
level andcrystallinity.8 Obviously, after modification, the
intensity of theD peaks (relative to the G peaks) decreased
markedly,indicating that the carbon defects were restored and
thatrGO was obtained. In addition, bulk GO showed the strongestG
band, sequentially followed by rGO-PDA2, rGO-PDA1, andrGO-PDA5,10
(almost lost G bands), indicating that the DA/
Figure 3. Morphologies and microstructures of the assemblies:
(a,b) bulk GO and (c,d) rGO-PDA. Panels b and d show
contrast-enhanced imagesto aid visualization of the ridges/edges in
panels a and c, respectively. (e,f) Corresponding distributions of
(e) ridges and (f) edges. Note that sheetsin tens of nanometers did
exist for rGO-PDA, but their sizes were difficult to measure and
present in panel f.
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GO ratio should be neither too large nor small.
Figuratively,this resembles the process of building with bricks and
glues: Ifthe ratio is large, limited GO (“brick”) is available to
be reducedand immobilized, whereas if the ratio is small, less
PDA(“glue”) is available to stick rGO to Ti. In particular, at a
ratioof 1:1, GO and DA reacted thoroughly to form stable
3D“hydrogels” other than to decorate the substrate (Figure
S2).Similar phenomena were noted before for DA-GO two-component
systems.28 Given these facts, we suggest a feasibleDA/GO ratio of
2, which was fixed in all subsequentexperiments (unless otherwise
stated, rGO-PDA now refersto samples at x = 2). This ratio can
allow both sufficientreduction of GO and effective fixation of rGO
onto titanium.The resultant layers are expected to manifest strong
interfacialbonding, through the occurrence of PDA−rGO (e.g.,
covalentcross-linking,18 π−π stacking28) and TiO2−PDA
(covalentbonds32) interactions (Figure S1). Further, they can
impartspecific biological properties to the modified rGO, as
detailedlater, through the use of the biological adhesive
“musselpower”.33
Despite the flourishing development of graphene, the
simple,economical, and scalable size fractionation of its sheets
hasremained challenging thus far.34 The sonication of GO iscommon
in research works. However, the precursors obtainedare inevitably
split into pieces with wide size distributions.Herein, the lateral
dimensions of graphene were easily refinedthrough the PDA-mediated
one-step aqueous selection.Notably, neither centrifugation nor
filtration was performedto decrease these sizes.34,35 Instead, PDA
reacted with GO andsegregated the products naturally into two
portions: The partwith macro-/microscopic dimensions formed
precipitatesspontaneously, whereas the submicrometer part
preferentiallyassembled onto the substrate. For the underlying
kinetics, wepostulated the “nano-effects” of submicrometer sheets
mighthave provided them with energetically favorable
affinity(reactivity) toward bulk objects. As evidence for this
possibility,a fraction of these sheets (asterisks, Figure S3) were
observedto bind identically to the surface of precipitates,
essentiallyanother type of “substrate”.An overall understanding of
the surface characteristics of
biomaterials is crucial given that osteoblastic cells, as well
asbacteria, respond to characteristics such as surface
chemistry,
topography, and hydrophilicity, which ultimately affects
thesuccess of the implants. The surface physicochemical
propertiesof two assemblies were investigated synchronously.
Thechemical characteristics were revealed by means of
FTIRspectroscopy and XPS. In the FTIR spectra of GO (Figure 2b),the
peaks at 1046, 1216, 1592, and 1728 cm−1 correspond tothe sp2
stretching vibrations of CO, COH, CC/CC,and CO groups,
respectively.17,35 For rGO-PDA2, theoxygen-associated peaks
weakened or disappeared, indicatinga significant degree of
restoration within the grapheneframework or at the edges. A peak
emerged at 1250 cm−1,assigned to the phenolic COH vibrations of
PDA. Never-theless, amino signals were absent, implying that the
polymerlayer was thin. For the control rGO-PDA10 (with thicker
PDA),an additional peak at 1516 cm−1 ascribed to N−H vibrationswas
noted.17 The degree of reduction was revealed by an XPSstudy of the
C 1s core-level band, which can be generally fittedto three
components, corresponding to carbon atoms in severalchemical
environments:34,35 in-plane sp2 carbon (CC/CC) at 284.8 eV,
CO-bound carbon at 286.9 eV, and carbonylcarbon (CO) at 288.5 eV.
For bulk GO (Figure 2c), the CO and CO contents were 42.0% and
8.1%, respectively,signifying an appreciable degree of oxidation.
For rGO-PDA(Figure 2d), the fractions of CO and CO were only
17.6%and 4.0%, respectively.The surface microstructure and topology
were examined by
SEM and AFM. As depicted in Figure 3a,b, bulk GO sheetswere
interconnected in continuous large pieces, similarly tofilms, and
were characterized by a myriad of ridged protrusions,known as
asperities. In addition, the ridges were micro- tomacroscale
(∼1−100 μm; Figure 3e). No apparent edge orcorner was observed. The
AFM measurements in Figure 4a−csuggest a ridge width of several
micrometers, a height of dozensof nanometers, and an average
roughness (Ra) of 28.4 nm. Bycomparison, the mussel-inspired
assembly approach gave rise toa very different graphene counterpart
in rGO-PDA. Theinterface was constituted by sheet aggregates rather
than films(Figure 3c,d). These micronanosheets ranged from
-
roughness of 4.6 nm according to AFM (Figure 4d−f). Of note,the
rGO-PDA exhibited many irregular jagged edges, withnanoneedles
protruding upward at the edge margins. Theseneedles were atomically
sharp, likely generated by long hours offierce sonication that
conferred an extremely high free energyto the edges and made them
curl spontaneously at the ends.3.2. Wettability Properties.
Additionally, the sensitivity of
contact angle (CA) measurements was utilized to assess
thewettability of the resultant surfaces (Figure 5a). High CA
valuesdescribe hydrophobicity, and low angles indicate
hydrophilicity.Initially, the titanium was relatively hydrophobic
(60.4°). DA-GO codeposition improved the surface wetting by
14.2°,probably because of the hydrophilic nature of PDA. The
GOcoverage dramatically decreased the water CAs by 20°, as aresult
of the presence of hydrophilic carboxylic ends on GO.Taken
together, the data in sections 3.1 and 3.2 indicate thesuccessful
interfacial assembly of graphene with varyingstructural and
compositional properties.3.3. Protein Adsorption. The adsorption of
BSA on
different biomaterial surfaces was examined because
theattachment of cells is, to some extent, governed by
theinteractions of membrane integrin with preadsorbed
serumproteins.36 To our delight, both bulk GO and
rGO-PDApreferentially facilitated the absorption of BSA compared
tocpTi, as evidenced by BCA measurements and
fluorescencevisualization (Figure 5b). Factors that strongly
influenceprotein adsorption briefly include surface chemistry,
wettability,and topography (roughness). Here, both assemblies were
morehydrophilic than cpTi, thus favoring the retention of
theprotein. Moreover, bulk GO had a rougher surface and perhapsa
greater capacity for holding protein; it can also adsorbproteins
through hydrophobic interactions, electrostatic forces,and hydrogen
bonding.37 Regarding rGO-PDA, PDA is potentfor covalently grafting
BSA through o-benzoquinone−aminecoupling.29
3.4. Antimicrobial Effects of the Assemblies.3.4.1.
Anti-Adhesion and Anti-Biofilm Activities. Biofilms,sessile
communities of microbial cells, clinically colonize onorthopedic
implants and lead to the outbreak of catastrophicinfections, such
as osteomyelitis. The buildup involves severalsteps, starting with
bacterial attachment on the surface,subsequent cell aggregation and
accumulation into micro-colonies, followed by biofilm maturation,
and ending with thedetachment of cells from the biofilms into
planktonic states toinitiate a new cycle of biofilm formation
elsewhere.38 Oncebiofilms are established, they encase and defend
bacteria fromhost immune responses or antibiotic attacks. It is
generally
accepted that inhibiting microbial anchoring is better
thantreating already-colonized biofilms.36 Hence, the impacts
ofbulk GO and rGO-PDA layers on the antiadhesion andantibiofilm
abilities of Ti disks were assessed against S. aureus, afrequent
pathogenic strain that is reported to be able to infect95% of
subcutaneous implants with only a 102-CFU inoculumin vivo.39
The antiadhesion ability of the obtained surfaces
wasinvestigated after 24 h in culture, by measuring
themitochondrial activity of surface-retained bacteria. As shownin
Figure 6d, both forms of graphene assemblies impressivelyreduced
the adherent bacterial cells relative to cpTi, by 45.0%
Figure 5. Surface properties of the assemblies: (a)
hydrophilicity and (b) protein adsorption. In panel b, the left is
BCA measurements, and the rightis fluorescence adsorption.
Figure 6. Antibacterial activities of different samples against
S. aureus:(a) Typical bacterial morphology, (b) live (green)/dead
(red) status,(c) biofilm formation (visualized by crystal violet),
(d) relativeadherence of bacteria, (e) quantification of biofilm
contents in panel c.Note: In panel b, the overlaying of red and
green can appear green-yellow. In panel c, patches of aggregate
biofilms are indicated bycircles; the arrows point to bulk GO
sheets (black, reflective) thatinterfered with biofilm observation
(they were discriminated frombiofilms by size and reflector
effects).
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for rGO-PDA and by 55.1% for bulk GO. In addition, SEM
wasperformed to obtain evidence of membrane disfigurations(Figure
6a). In this case, bacteria adhered to and proliferatedreadily on
cpTi and formed aggregates of cells whose wallsremained intact.
Upon encountering rGO-PDA, however, thebacterial membrane typically
became deflated. In particular,bulk GO resulted in generally
wrinkled and damagedmorphologies.To visualize the viable state of
the bacteria, in situ live/dead
fluorescence imaging was performed, as depicted in Figure
6b.Many bacterial colonies were easily observed on cpTi, whichwere
active and ready to form 3D mature biofilms. In contrast,much fewer
microbes were anchored on the rGO-PDA. Inaddition, the majority of
cells were individuals, and some wereeven killed (red
fluorescence). Bulk GO retained many fewerbacteria than cpTi but
slightly more than rGO-PDA. Never-theless, the bacterial death was
greatest on bulk GO.The morphologies and structures of graphene
materials are
responsible, more or less, for their antibacterial
abilities.However, they can be changed by the physiological
milieuand/or by interactions with bacteria. Hence, the
structuralstability of the materials was investigated preliminarily
in vitro.As shown in Figure S6, exposure of the samples to
bacterialsuspensions for relatively long times did not result in
obviousde-adhesion of layers, nor did it significantly alter
themorphologies/structures of the two assemblies, indicatingtheir
long-term usefulness.Furthermore, the antibiofilm activities were
surveyed by
crystal violet staining after a prolonged culture of 5 days. As
isevident in Figure 6c, large-area, dense biofilm patches
wereformed macroscopically on cpTi. In sharp contrast, all
surfacestreated with graphene remained relatively clear, with only
a fewsmall, disperse zones of violet stains. Quantitatively, the
S.aureus biofilm content was reduced by 30.5% and 40.7% byrGO-PDA
and bulk GO, respectively (Figure 6e). Takentogether, the results
indicate that Ti implants with grapheneself-assemblies alone, in
the absence of any other bactericides,were potent for suppressing
initial bacteria anchoring and
delaying subsequent biofilm formation. In clinics, this
couldgreatly enhance the chance for the remaining bacteria to
beeradicated by host immunity or that for early infections, if
any,to be diagnosed and treated in a timely manner, therebyavoiding
serious infections.
3.4.2. Antibacterial Mechanisms. Graphene is a
well-knownantimicrobial agent that is able to inactivate or kill
microbes byinteracting with membranal and intracellular
components(lipids, proteins, DNA/RNA, etc.) through π−π
stacking,hydrogen bonds, and electrostatic adsorption.4 These
phys-icochemical interactions have derived a group of
antibacterialmechanisms (Figure 7a), briefly including
nanoknives/nano-needles through the physical action of sharp
edges/corners,oxidative stress through ROS production or charge
transfer,membrane wrapping/trapping originating from the
flexibility oflarge graphene sheets, extraction of membrane lipids
derivedfrom overwhelming hydrophobic attraction between sp2
carbons and lipid molecules (“nanoscale dewetting”),
andspontaneous insertion that penetrates lipid bilayers.4,12
Moregenerally, these actions fall into two categories:
membranedamage and oxidative stress (Figure 7b). The former is more
orless associated with physical or mechanical modes of
interactionwith the cell membrane, whereas the latter is
typicallyaccompanied by a series of metabolic events. However,
mostof the described actions rely heavily on dispersed
graphenesheets (graphene solutions were used) that are able to
assumedynamic exposure, fully penetrate the membrane, and
interactactively with components inside bacteria. By contrast,
theantimicrobial behaviors of on-surface graphene have rarely
beenstudied (see the summary in Table 1). In these cases,
threemajor mechanisms seem applicable, namely,
nanoknives/nanoneedles, ROS production, and charge transfer.
Usingthese mechanisms, we attempt to establish putative
mechanisticlinks for the graphene interfaces in this
work.Principally, early research endeavors have ascribed nano-
graphene-induced bacterial death to “nanoknives” effects,
whichhighlight the role of nanosheets’ sharp edges that act
likecutters to laterally incise bacterial membranes, causing
Figure 7. (a,b) Summaries of graphene-related antimicrobial
mechanisms paying special attention to their applicability for
solutions and surfaces. (c)Plausible antibacterial mechanisms of
graphene for (c-1) rGO-PDA and (c-2) bulk GO. (d) Principle of
infection control by tuning the lateral sizeand surface properties
of graphene self-assemblies.
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intracellular substances to leak and, consequently, cells to
die.8
Howvever, micro-/nanosheets such as rGO-PDA are
assembledquasiparallel rather than perpendicular to the substrate,
makinga near-orthogonal cut impossible. Recently, a study by Pham
etal. expanded the “cutting” theory, by pointing out that
graphenesheet surfaces, actually, induce the formation of pores
withinthe bacterial membrane instead of simply cutting through
it,which altered the osmotic pressure and then inducedmembrane
collapse (here, called the nanoneedle effect).12
From a similar viewpoint, we speculate that the spherical
S.aureus, once settled on the jagged rGO-PDA sheets
(havingdefected, atomically sharpened edges, with needlelike
nano-structures; Figure 4f), can encounter a constant,
ultrastrongflow of mechanical pressure, imposed by the
peripheralnanoneedles of various sheets, that is able to pierce
themembrane to form localized nanopores. These local,
multipointpores, if sufficient, can presumably initiate “crack”
propagationand trigger the efflux of cytoplasmic components such
asproteins and DNA (Figure 7a). The needles could furtherretard
bacteria from growing on them, thus delaying the onsetof biofilms.
Note also that the described effect is likely randomand cumulative
(not instantaneous) and should, therefore, bemore inhibitory than
lethal (Figure 6b).Comparatively, the scenario for bulk GO sheets,
large in area
and entirely immobilized, interacting with bacteria is
ratherdifferent; it would be difficult for such sheets to trap or
wrapthe bacteria as dispersed large films do, and edge effects
similarto those of nanosheets would also not be possible.10
Indeed,something like a ridge, if tiny and sharp enough, could
alsoimpose mechanical pressure on bacteria. However, for GOsheets
prepared by our method, the ridges’ lateral dimensionsspanned
across orders of magnitude, from ∼1 to 100 μm, ascale much greater
than that of S. aureus cells (typically, 500nm−1 μm). Considering
the large ridge width (mostly severalmicrometers) and rough surface
landscape, they would betterbe described as blunt than sharp.
Therefore, the chance that thebacteria perceive the mechanical
feature and are further incisedor punctured is fairly small,
excluding so-called “physicaldamage” as a major bactericidal route
here (if any, it couldaccount for a small part). Given its superior
bacteria-killingcapacity (Figure 6b), other modes of action should
exist forbulk GO. The first possibility is an oxidative
mechanisminvolving ROS overproduction. Excess ROS is known to
oxidizefatty acids and yield lipid peroxides that affect
respiratory chainreactions, thereby disrupting membrane integrity
and resultingin the loss of viability.7,40 ROS-mediated oxidative
stress wascorroborated through use of the DCFH-DA fluorescence
probe(Figure S4). The stronger the fluorescence, the larger
theamount of ROS. Clearly, interfacing bacteria with bothgraphene
assemblies, particularly with bulk GO, triggeredelevated ROS
levels. Another rationalization is the charge-transfer mechanism
that interrupts the membrane respiratorychain and also gives rise
to oxidative stress.9 The respiratorychain of bacteria is known to
rely on electron transport toproduce energy for bacterial survival
and maintenance. Inprinciple, for a bacteria/graphene/metal system,
a circuit can beestablished through the “membrane@graphene@TiO2”
junc-tions15 that allows for electrons to be transferred from
themicrobial membrane to the graphene film and then to
theunderlying metal (Figure 7a). This circuit acts as a dynamicpump
that extracts membranal electrons rapidly and steadilyuntil the
initial negative membrane potential is reversed,culminating in
disrupted cellular components, destroyedT
able
1.Con
structionof
Graph
ene-Based
Antibacterial
Interfaces
andTheirToxicityMechanism
s
substrate
physiochem
icalcharacteristicsof
graphene
methods
ofconstructio
n
bacterial
strain(s)
testeda
major
antibacterialfindings
plausibleantibacterialmechanism
sref
stainlesssteel
graphene
(GO,rGO)nanowalls
electrophoretic
depositio
n(EPD
)S.aureus,
E.coli
rGO
nanowallsweremoretoxicthan
GO
mem
branedamageby
sharpedges,
charge
transfer
bydirect
contact
Akhavan
and
Ghaderi8
(2010)
Cu,
Ge,SiO
2large-area
monolayer
film
CVD
direct
grow
th(for
CuandGe),
CVD
+PM
MA-assistedwet
transfer
(for
SiO
2)
S.aureus,
E.coli
filmson
CuandGewereantibacterial,
whereas
that
onSiO
2was
not
charge
transfer
Liet
al.9(2014)
poly(ethyleneterephthalate)
(PET
)flat,large-areaGO
Langmuir−Blodgett(LB)technique
E.coli
layer-dependentactio
n;edge
effectswere
notintegralforGO
tobe
antim
icrobial
chem
icalfactorsassociated
with
basal
plane
Mangadlao
etal.10
(2015)
filtermem
braneof
mixed
cellulose
ester
subm
icrometer
GO
sheetsof
area
0.01
and0.65
μm2
vacuum
filtration
E.coli
antim
icrobialactivity
increasedas
sheetsize
decreased
oxidativemechanism
sassociated
with
sheetdefect
density
Perreaultetal.11
(2015)
alum
inafiltermem
brane
graphene
nanosheetsof
varyingedge
lengthsandangles
oforientation
vacuum
filtration
S.aureus,
P.aeruginosa
edge
density
was
crucial
form
ationof
poresthat
caused
mem
braneosmoticimbalance
Pham
etal.12
(2015)
titanium
ridged,
large-area
GO
sheets(films)
evaporation-assisted
electrostatic
assembly
S.aureus
strong
bacteria-killing,zone-dependent
mem
branedistortio
n,good
antibiofilm
ability
oxidativestress
mediatedby
ROS
and/or
charge
transfer
currentstudy
titanium
jagged,surface-m
odified,
micro-/nanosheetsof
rGO
mussel-inspiredone-potassembly
S.aureus
antifouling,lim
itedbacteria-killing,good
antibiofilm
ability
mem
branepressure
bynanoneedles,
surfacephysicochemicalproperties
currentstudy
aE.
coli=Escherichiacoli;
S.aureus
=Staphylococcus
aureus;P.
aeruginosa
=Pseudomonas
aeruginosa.
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membrane integrity, and eventual microbial death. Actually,
thisaction can be extrapolated from an interesting,
zone-dependentantibacterial phenomenon (Figure S5): The degree
ofmembrane distortion for bacteria in planar zones was muchgreater
than that near the ridges, which was validated byrepetitive
experiments and analysis. The reasoning is as follows:The
graphene−substrate contact (prerequisite for a circuit) forthe
planar zones should be better than for the ridged surfaceswhere
metal−film gaps are possible; as a result, the formershould have
easier, faster electron transfer and, thus, imposemore severe
membrane disruption.Therefore, we suggest that membrane stress by
nanoneedles
and oxidative stress through charge transfer or ROS werepossibly
dominant mechanisms enabling the antibacterialactivities of rGO-PDA
and bulk GO, respectively. Nevertheless,for graphene-related
antimicrobials, the destructive effects canbe multiple. For
example, Liu et al. systematically investigatedthe bacterial
toxicity of four graphene types (graphite, graphiteoxide, GO, and
rGO) and found that both membrane andoxidation stress made
contributions.7 In another study, rGOand GO nanowalls were used to
kill S. aureus, and the formerwas revealed to be more toxic, due to
both better chargetransfer and more sharpened edges.8 In this
regard, it is morereasonable that the aforementioned mechanisms are
shared byboth types of graphene assemblies, but in varying degrees,
asschematically represented in Figure 7c. Further, we deduce
thatoxidative stress is a more active toxic mechanism for
graphenematerials, at least for bulk graphene at the interface.
Becausethese antimicrobial activities exhibit excellent
biologicalpersistence, they can inactivate bacterial replication
and retardbiofilm formation in the long run. In addition,
as-treateddevices should have long shelf lives, and their efficacy
is unlikelyto be impaired by routine sterilization (here, all
specimens wereautoclaved in a standard manner, yet with good
antibacterialeffects), which is common in clinical practice.
Further, becauseof the robust mechanical nature of graphene18 and
the as-mentioned substrate−sheet interactions (section 3.1),
relativelygood structural/chemical stability of the material in
physio-logical milieus is possible (Figure S6). Therefore,
on-the-surfacegraphene might offer a viable alternative to
antibiotic- or silver-releasing surfaces whose effects deplete over
time or arecomprised by harsh sterilization.A major aspect
distinguishing graphene-based interfaces from
graphene solutions is that, in addition to an intrinsic
bacteria-killing ability (discussed above), their antibacterial
activity canalso stem from the surface properties that repel
bacterialadhesion, alter the adhesion phase, and restrict cell
growth(Figure 7d).41 For example, surface wettability and
topography(roughness) are critical considerations in controlling
micro-organism retention. For wetting, principally bacteria
withhydrophobic natures prefer hydrophobic surfaces, and
viceversa.42 S. aureus cells are hydrophobic (CA ≈ 72°43),
soexposing them to hydrophilic surfaces decreases the
amountsadhered (Figure 6d). Concerning topological
configuration,generally, a lower roughness mitigates fouling, as it
providesfewer anchor sites for germ retention and lowers the
membraneshear forces.41 Notably, for rGO-PDA, the needlelike
nano-topography at the edge margins can impart, aside from
pore-forming death, additional fungal-repealing capabilities to
thesurfaces. On one hand, we believe that bacteria tended
tocircumvent the harmful needles by instinct, so most of themwere
likely to be held planktonic. On the other, a
nanofeaturedtopography can play an integral role in reducing
initial bacterial
attachment. For example, Liu et al. confirmed that
nanoscaleroughness (∼14 nm) alone hampered the surface adhesion
andgrowth of both Escherichia coli (E. coli) and S. aureus for up
to 2days.44 However, the roughness should not be too
large;otherwise, more bacteria can be retained, as is the case for
bulkGO (Figure 6b). Additionally, the effects of other
surfaceproperties such as surface chemistry and electrostatic
chargeshave also been noted.41 The functional groups in the
PDAmatrix include quinone, carboxy, amino, imine, and
phenolgroups.45 In particular, the amine groups are anticipated
toendow the substrate with positive charges that disruptmembrane
charges (from −20 to −200 mV9) and to reactwith the functional
groups (e.g., thiols) from bacteriamembrane components
(peptidoglycan, porins, lipopolysac-charides, phospholipid,
etc.).46 This can lead to alteredmetabolic activity and cellular
integrity, thereby influencingbacterial adherence and even
survival. These surface-specificeffects should be viewed as a
whole, together with the intrinsickilling ability of graphene
itself, to rationalize the observedvariations. Although a better
elaboration of the role of each partis still needed, we can roughly
conclude that the intrinsic killingability and surface
physicochemical properties were the keycontributors for bulk GO and
rGO-PDA, respectively, to beantibacterial and to resist biofilm
formation (also included inTable 1).
3.5. Cytocompatibility of the Assemblies. The abilitiesof cells
to anchor to a substrate and to proliferate and furtherexpress
destined functions critically dictate the long-termsuccess of an
implant and, in an equal sense, the fate of theregenerating tissue.
In particular, in orthopedics, theseinterfacial responses are
associated with biomaterials’ surfaceproperties,47 and they are
often examined by using bone-forming cells (e.g., MC3T3-E1) that
synthesize extracellularmatrix (ECM, primarily collagen) and
control its mineraliza-tion. This ex vivo model makes it possible
to gain a quickimpression of whether a suggested surface
modification isefficacious in improving implant biocompatibility
and en-couraging its integration with bone tissue.First, the
initial cell−material interactions were investigated,
including (1) substrate attachment, (2) cell spreading, and
(3)cytoskeleton development. Here, the cell attachment hinged
onboth surface conditions and cultivation time (Figure 8a,c).
After4 h of culture, the number of osteoblasts (nuclei, blue) on
rGO-PDA was clearly the greatest, and their focal adhesions
(whitearrows) were notable. In addition, a well-organized
cytoskele-ton was visualized by red tubulin tracker, indicating
fairly good,dynamic stretching. Bulk GO also evoked higher cell
affinityand better morphology than cpTi, but they were inferior
tothose of rGO-PDA. This result is attributable to the unique
roleof PDA in mediating strong biological anchorage and
superbstretching during cell adhesion.33,45,46 PDA not only
helpedadsorb more serum proteins (Figure 5b), it might also
haveprovided them with better conformations, thus establishing
afavorable matrix for rapid cell recruitment, stable adherence,and
effective cytoskeleton construction.45 As the time wasincreased to
8 h, the remaining planktonic cells continued toadhere to each
surface. Still, the rGO-PDA groups had the mostcells, richest
tubulin networks, and best spreading. Moreover,the F-actin
development was evaluated after 1 and 3 days(Figure 8b). After 1
day, compared to those on cpTi, theosteoblasts on rGO-PDA exhibited
a higher degree of cellextension and greater stress fiber
formation; for bulk GO,however, some cells were better spread, but
some were worse
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(red arrows). Nevertheless, all groups showed contiguous
andhealthy F-actin networks at 3 days.Next the cells’ metabolic
function during proliferation was
assessed by measuring the mitochondrial activity and expressedas
cell viability relative to TCPS (negative control) (Figure 8d).At
day 2, compared with the cpTi cells (91.1%), the rGO-PDAgroups
(83.9%) exhibited slightly impaired viability, whereasthe bulk GO
cells (62.8%) had the worst cell metabolism.Nonetheless, the
following 2 days witnessed a noticeableincrease for them all, and
at day 6, every surface ended withviability values of around 100%,
indicating their bioacceptablesurface properties for long-term
implantation applications.Bulk GO has greater cytotoxicity than
rGO-PDA. Many
earlier studies have validated the nanotoxicity of
graphenematerials of different sizes in dispersed solutions. For
instance,nanosheet graphene with lateral dimensions of 100 nm−5
μmcan be easily taken up and then accumulate in cells.48 Li et
al.reported edge-first insertion, penetration, and internalization
ofgraphene microsheets (multilayered, 0.5−10 μm) into the
lipidbilayers of three cell types by Brownian motion and
lipidattraction.49 Presumably, macrosheets with extremely
largeareas, say, 20−100 μm (larger than most cells), might also
posehazards, fo example, by means of “wrapping/encapsulation”akin
to that toward bacteria, causing nutrient deprivation.11
Nevertheless, considering the restricted sheet mobility, as
aresult of covalent or electrostatic layer−substrate links,
free-floating sheets are unlikely. Thus, the chance for the
otherwiserisky penetration and cellular uptake of nanoflakes (e.g.,
rGO-PDA) or for the wrapping of host cells by large films (e.g.,
bulkGO) should be critically low. For rGO-PDA, its
bettercytocompatibility was attributed to the surface
modification.According to a recent report, among different
graphenebiomaterials, those with chemical functionalization
generallydisplay improved safety profiles.16 A growing body of
evidence
is showing that PDA on various nanomaterials can attenuatetheir
adverse biological effects.46,50 By contrast, less is knownabout
the impaired cell viability for bulk GO, an interestingmatter as
shall be explored below.For biomaterials physically interacting
with cell membranes,
toxicity cascades will likely be triggered as first the
disruption ofthe membrane integrity, then the leakage of
intracellularenzymes (e.g., LDH), and finally apoptosis or even
cell death.29
For simplicity, LDH was chosen as an indicator for evidence
ofthese damages, given that it leaks immediately into
thesurrounding medium once membrane-associated apoptosisoccurs and
that it is stable in the extracellular environmentfor a long time.
As presented in Figure 9c, bulk GO did induce
the strongest production of LDH, indicative of seriousmembrane
damage, whereas rGO-PDA had a mild impact onmembrane structure. The
trend is just the reverse of thatobserved for bacteria (section
3.4), yet it can also be interpretedin terms of the differences in
size among rGO-PDA, bulk GO,and cells (bacteria). The simple fact
is that osteoblasts (10−50μm) are much larger than S. aureus (
-
mitochondrial activity provided by bulk GO, as seen in
Figure8d.Nevertheless, these negative impacts became less
significant
as cultivation went on, probably attributed to the fact that
oldercells began to get adapted while newer cells contacted less
withsubstrate (e.g., through overlapping growth). Furthermore,
weverified this by cellular live/dead staining at day 7. As shown
inFigure 9e, around 100% confluence was reached for all groups;cell
survival was basically satisfactory with few dead cells
(redfluorescence). It should be admitted that GO materials had
cell-assisting aspects. For example, their polar functional groups
(OH, COOH) were revealed to interact with the polarcomponents of
cells and the culture medium.37 Also, the largesurface area served
as active sites available for cells to spreadand grow.53 Therefore,
once the negative effects weakened inlate stage, improved cell
compatibility and probably strongerosteodifferentiation instead of
cytotoxicity shall be envisioned.Similarly, such staged cell
behaviors were observed withnanosilver coatings.29 These suggest
the term “biocompati-bility” needs revision, or at least, more
dialectical interpretationwhen adopted for risk assessment when
developing novelnanobiomaterials.3.6. Osteogenic Differentiation
Properties. The level of
ALP activity, widely used as an early hallmark of
osteoblastdifferentiation, was examined at day 7. As shown in
Figure 10a-4, the ALP expression for rGO-PDA and, especially, for
bulkGO was notably improved in comparison to that for cpTi.
Theresults were further confirmed by ALP staining (Figure
10a-1−a-3). The modified substrates elicited much darker blue
stains(red arrows). In the long term, the differentiated
osteoblastswill secrete collagen and assemble collagen fibers
extracellularly,
which later become mineralized, and ultimately, a highlycomplex
and hierarchical architecture of bone matrix will beorganized.47 To
this end, matrix collagen secretion by MC3T3-E1 at day 21 was
measured by SR staining and quantified(Figure 10b,d). Compared to
cpTi, higher collagen content anddenser patterns were observed for
titanium decorated withgraphene assemblies, especially for bulk GO.
Furthermore, theARS Ca2+ content at day 28 was measured, which
corroboratedthe collagen assessment (Figure 10c). Notably, larger
calciumnodules (arrows, Figure 10d) were deposited on the
modifiedsurfaces. During substrate-controlled biomineralization,
the rateof compound Ca−P formation is a key index for defining
thebioactivity of corresponding biomaterials. And this in turn
isfacilitated by the available functional groups on materials
thataffect the alternative electrostatic absorption of Ca2+ and
PO4
3−
ions. For rGO-PDA, the catechol/amine groups of PDA, notonly
direct efficient interaction with the graphene sheets butalso serve
as active sites recruiting mineral ions, therebyenhancing apatite
nucleation and growth.17 For bulk GO, thepolar groups (COOH, OH)
can have similar effects. Inaddition, the unique topographical
ridges can be viewed as kindof “graphene patterns” providing
biophysical cues for furtherenhancing the differentiation.
Micropatterned geometries (5−50 μm) of graphene were shown to be
effective at steering stemcell fate.13 Furthermore, the
amphiphilicity, aromatic scaffoldnature, and electrical
conductivity (electrical stimuli) ofgraphene probably provided
concerted efforts in bothcases.13,54 All together, both kinds of
sheets created anosteogenesis-enhancing microenvironment that is
anticipatedto benefit implant−tissue integration.
Figure 10. Osteodifferentiation properties of the graphene
interfaces: (a) (a-1−a-3) ALP staining and (a-4) ALP activity,
(b,c) quantification of (b)collagen and (c) calcium contents (*p
< 0.05), and (d) optical images of collagen and calcium stains.
Arrows indicate calcium nodules.
ACS Applied Materials & Interfaces Research Article
DOI: 10.1021/acsami.6b05198ACS Appl. Mater. Interfaces 2016, 8,
17151−17165
17162
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3.7. Applications and Perspectives. Undoubtedly, bothlarge and
small graphenes have respective merits, with whichbroader
biomedical opportunities can readily be found. Forinstance,
large-area GO is an ideal matrix for building versatilepatterns, 3D
networks, and scaffolds for stem cell cultures andbone tissue
engineering.13 Also, the enlarged planar surface areaprovides
abundant sites for efficient loading of aromatic drugmolecules such
as dexamethasone (osteogenic) and tetracycline(antibacterial)
through π−π stacking for local drug delivery.14,55rGO-PDA
micro-/nanosheets, having more electrochemicallyactive edging
sites, hold promise in the modification ofelectrodes/devices for
high-performance detection or biosens-ing.56 Taking advantage of
the secondary reactive nature ofPDA,24 one can further charge
micro-/nanovehicles withinorganic particulates, bioactive minerals,
and drugs/biomole-cules for advanced multifunctional implants or
other biomedicaldevices in future.15,17,27,57
Fundamentally speaking, the concept of applying grapheneonto a
surface is also appealing because it offers another viableroute
instead of dispersion to investigate the intricate yetcontroversial
issues regarding graphene materials,4 especially forthe accurate
determination of their bactericidal nature. On onehand, the surface
properties of graphene, if well-devised, canallow for a focus on
the effects from sole factor, such as the baseplane, by
lowering/excluding the effects from other factors,such as sheet
size and edge density. On the other, a properchoice of substrate
can make the identification of substrate-sensitive mechanisms, such
as charge transfer, much easier.However, this area, with limited
research attention, is still in itsinfancy (Table 1). Its
development, as we see it, hinges greatlyon progress in
nanotechnology and methodologies that canengender a host of
designer graphene-based interfaces, ideallywith controllable
physicochemical characteristics. As listed inTable 1, the currently
available techniques/methods consist ofchemical vapor deposition
(CVD), vacuum filtration, Lang-muir−Blodgett (LB), and
electrophoretic deposition (EPD),with substrates including
polymers, inorganics, and semi-conductors and metals, yielding
graphenes with dimensionsranging from nanometer to macroscopic
scales. Indeed, eachone is unique in regard to establishing a
respective model forthe demonstration of a specific theory. Yet,
there is significantroom for these theories to be improved,
entailing research onother models by alternative methods. The
self-assembly ofgraphene at interfaces, as exemplified here, is
essentially easy,cheap, and general. By further careful
engineering,23 diverseinterfaces can be obtained, which could
greatly help in allowingfundamental studies of graphene (Figure
1a).To meet these extremely high expectations, ongoing efforts
are warranted. For one example, the molecular
assemblymechanisms, interlayer and layer−substrate interactions,
andlong-term physiological/mechanical stability of the
assembliesshould be further explored. In addition, the
techniques/materials themselves require improvements to increase
thematerials’ interfacial strength (for bulk GO) and to maximizethe
antibacterial/osteodifferentiation efficiencies (for rGO-PDA)
toward clinical applications. By utilizing the chemicalversatility
of GO/PDA, the former is possible through theintroduction of strong
covalent cross-links (Figure S7a),58
whereas the latter can be fulfilled by surface
decoratingnanosilver,27 antibiotics,55 and osteogenic
molecules14,57
(Figure S7b). Furthermore, it remains an important task
toexploit the efficiency and safety in vivo, as well as to deepen
the
understanding of the structure−function correlations ofgraphene
sheet surfaces.
4. CONCLUSIONSIn this work, we demonstrated the possibility of
impartingintrinsically dual-functional (i.e., antibacterial and
osteogenic)properties of graphene to a metallic implant surface, to
solvethe pressing need for enhanced infection control and
tissueintegration in orthopedics. Two simple, straightforward
self-assembly routes were established to form in situ on
titaniumsubstrate-anchored, 2D graphene structures with
compositionaland dimensional variations (i.e., macro-/microscale
bulk GO vssubmicroscale rGO-PDA). The former was based on an
easyevaporation-assisted electrostatic assembly process, whereas
thelatter was rendered by exploiting a facile, mussel-inspired
self-polymerization and the one-pot reactivity of PDA.The resultant
interface layers with differing structures and
chemistries altered the hydrophilicity and protein adsorption
ofthe substrate. Both modifications were confirmed to havepotential
to generate sterile implant surfaces with antiadhesionand
antibiofilm activities, in the absence of extra antimicrobials,yet
without much compromising their biocompatibility. Inaddition, by
using the assemblies, we managed to achieveimpressive osteogenic
functions, which, coupled with the aboveantimicrobial properties
and with the increasing availability anddecreasing cost of
graphene, bodes well for the furtherdevelopment of graphene-enabled
biomedical care andtherapeutic utility. This work should enrich the
library ofgraphene-based nanotechnologies and facilitate the
manage-ment of graphene-concerned health issues.
■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting
Information is available free of charge on theACS Publications
website at DOI: 10.1021/acsami.6b05198.
Schemes of plausible reaction mechanisms, stability
ofpostreaction solutions at various ratios, typical morphol-ogy of
the reacted mixture for rGO-PDA, surface ROSlevels with bacteria,
zone-dependent antibacterial behav-ior of bulk GO sheets,
structural stability of materials inbacterial tests, and proposed
routes for further materialimprovements (PDF)
■ AUTHOR INFORMATIONCorresponding Author*Address: Center for
Biomedical Materials and TissueEngineering, Academy for Advanced
Interdisciplinary Studies,Peking University, No. 5 Yi-He-Yuan Road,
Hai-Dian District,Beijing 100871, China. Tel./Fax: +86-10-62753404.
E-mail:[email protected] authors declare no competing
financial interest.
■ ACKNOWLEDGMENTSThis work was jointly supported by the National
NaturalScience Foundation of China (Nos. 31370954 and 51431002),the
Project of Scientific and Technical Plan of Beijing
(No.Z141100002814008), and the State Key Laboratory
ofBioelectronics Open Research Fund of China (Chien-ShiungWu
Laboratory, No. 08400-413-161-002). Z.J. gratefullyacknowledge
financial support from the China ScholarshipCouncil (CSC, file
number 201506010201). The authors also
ACS Applied Materials & Interfaces Research Article
DOI: 10.1021/acsami.6b05198ACS Appl. Mater. Interfaces 2016, 8,
17151−17165
17163
http://pubs.acs.org/doi/suppl/10.1021/acsami.6b05198/suppl_file/am6b05198_si_001.pdfhttp://pubs.acs.org/doi/suppl/10.1021/acsami.6b05198/suppl_file/am6b05198_si_001.pdfhttp://pubs.acs.orghttp://pubs.acs.org/doi/abs/10.1021/acsami.6b05198http://pubs.acs.org/doi/suppl/10.1021/acsami.6b05198/suppl_file/am6b05198_si_001.pdfmailto:[email protected]://dx.doi.org/10.1021/acsami.6b05198
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thank Ms. Chao Xu from State Key Laboratory of AdvancedOptical
Communication Systems & Networks, Peking Uni-versity, for
assistance with AFM measurements.
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