new from solution to biointerface: graphene self-assemblies of...

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

www.acsami.org

© 2016 American Chemical Society 17151 DOI: 10.1021/acsami.6b05198ACS Appl. Mater. Interfaces 2016, 8, 17151−17165

www.acsami.orghttp://dx.doi.org/10.1021/acsami.6b05198http://pubsdc3.acs.org/action/showImage?doi=10.1021/acsami.6b05198&iName=master.img-000.jpg&w=130&h=134

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.

ACS Applied Materials & Interfaces Research Article

DOI: 10.1021/acsami.6b05198ACS Appl. Mater. Interfaces 2016, 8, 17151−17165

17152

http://dx.doi.org/10.1021/acsami.6b05198http://pubsdc3.acs.org/action/showImage?doi=10.1021/acsami.6b05198&iName=master.img-001.jpg&w=239&h=283

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.



17153

http://dx.doi.org/10.1021/acsami.6b05198

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.



17154


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.



17155

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://dx.doi.org/10.1021/acsami.6b05198http://pubsdc3.acs.org/action/showImage?doi=10.1021/acsami.6b05198&iName=master.img-003.jpg&w=359&h=394

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).



17157

http://dx.doi.org/10.1021/acsami.6b05198http://pubsdc3.acs.org/action/showImage?doi=10.1021/acsami.6b05198&iName=master.img-005.jpg&w=299&h=132http://pubsdc3.acs.org/action/showImage?doi=10.1021/acsami.6b05198&iName=master.img-006.jpg&w=211&h=269

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.



17158

http://pubs.acs.org/doi/suppl/10.1021/acsami.6b05198/suppl_file/am6b05198_si_001.pdfhttp://dx.doi.org/10.1021/acsami.6b05198http://pubsdc3.acs.org/action/showImage?doi=10.1021/acsami.6b05198&iName=master.img-007.jpg&w=389&h=225

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.



17159

http://pubs.acs.org/doi/suppl/10.1021/acsami.6b05198/suppl_file/am6b05198_si_001.pdfhttp://dx.doi.org/10.1021/acsami.6b05198

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



17160

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://dx.doi.org/10.1021/acsami.6b05198

(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.



17162


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



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

thank Ms. Chao Xu from State Key Laboratory of AdvancedOptical Communication Systems & Networks, Peking Uni-versity, for assistance with AFM measurements.

■ REFERENCES(1) Geim, A. K.; Novoselov, K. S. The Rise of Graphene. Nat. Mater.2007, 6, 183−191.(2) Wang, Z. F.; Zeng, H. D.; Sun, L. Y. Graphene Quantum Dots:Versatile Photoluminescence for Energy, Biomedical, and Environ-mental Applications. J. Mater. Chem. C 2015, 3, 1157−1165.(3) Krishna, K. V.; Menard-Moyon, C.; Verma, S.; Bianco, A.Graphene-Based Nanomaterials for Nanobiotechnology and Biomed-ical Applications. Nanomedicine 2013, 8, 1669−1688.(4) Zou, X. F.; Zhang, L.; Wang, Z. J.; Luo, Y. Mechanisms of theAntimicrobial Activities of Graphene Materials. J. Am. Chem. Soc. 2016,138, 2064−2077.(5) Zhu, Y. W.; Murali, S.; Cai, W. W.; Li, X. S.; Suk, J. W.; Potts, J.R.; Ruoff, R. S. Graphene and Graphene Oxide: Synthesis, Properties,and Applications. Adv. Mater. 2010, 22, 3906−3924.(6) He, J.; Zhu, X.; Qi, Z.; Wang, C.; Mao, X.; Zhu, C.; He, Z.; Li,M.; Tang, Z. Killing Dental Pathogens Using Antibacterial GrapheneOxide. ACS Appl. Mater. Interfaces 2015, 7, 5605−5611.(7) Liu, S. B.; Zeng, T. H.; Hofmann, M.; Burcombe, E.; Wei, J.;Jiang, R. R.; Kong, J.; Chen, Y. Antibacterial Activity of Graphite,Graphite Oxide, Graphene Oxide, and Reduced Graphene Oxide:Membrane and Oxidative Stress. ACS Nano 2011, 5, 6971−6980.(8) Akhavan, O.; Ghaderi, E. Toxicity of Graphene and GrapheneOxide Nanowalls Against Bacteria. ACS Nano 2010, 4, 5731−5736.(9) Li, J.; Wang, G.; Zhu, H.; Zhang, M.; Zheng, X.; Di, Z.; Liu, X.;Wang, X. Antibacterial Activity of Large-Area Monolayer GrapheneFilm Manipulated by Charge Transfer. Sci. Rep. 2014, 4, 4359.(10) Mangadlao, J. D.; Santos, C. M.; Felipe, M. J. L.; de Leon, A. C.C.; Rodrigues, D. F.; Advincula, R. C. On the Antibacterial Mechanismof Graphene Oxide (GO) Langmuir-Blodgett Films. Chem. Commun.2015, 51, 2886−2889.(11) Perreault, F.; de Faria, A. F.; Nejati, S.; Elimelech, M.Antimicrobial Properties of Graphene Oxide Nanosheets: Why SizeMatters. ACS Nano 2015, 9, 7226−7236.(12) Pham, V. T. H.; Truong, V. K.; Quinn, M. D. J.; Notley, S. M.;Guo, Y. C.; Baulin, V. A.; Al Kobaisi, M.; Crawford, R. J.; Ivanova, E. P.Graphene Induces Formation of Pores That Kill Spherical and Rod-Shaped Bacteria. ACS Nano 2015, 9, 8458−8467.(13) Kim, T. H.; Shah, S.; Yang, L. T.; Yin, P. T.; Hossain, M. K.;Conley, B.; Choi, J. W.; Lee, K. B. Controlling Differentiation ofAdipose-Derived Stem Cells Using Combinatorial Graphene Hybrid-Pattern Arrays. ACS Nano 2015, 9, 3780−3790.(14) Jung, H. S.; Lee, T.; Kwon, I. K.; Kim, H. S.; Hahn, S. K.; Lee, C.S. Surface Modification of Multipass Caliber-Rolled Ti Alloy withDexamethasone-Loaded Graphene for Dental Applications. ACS Appl.Mater. Interfaces 2015, 7, 9598−9607.(15) Sanchez, V. C.; Jachak, A.; Hurt, R. H.; Kane, A. B. BiologicalInteractions of Graphene-Family Nanomaterials: An InterdisciplinaryReview. Chem. Res. Toxicol. 2012, 25, 15−34.(16) Bussy, C.; Jasim, D.; Lozano, N.; Terry, D.; Kostarelos, K. TheCurrent Graphene Safety Landscape - A Literature Mining Exercise.Nanoscale 2015, 7, 6432−6435.(17) Liu, H. Y.; Xi, P. X.; Xie, G. Q.; Shi, Y. J.; Hou, F. P.; Huang, L.;Chen, F. J.; Zeng, Z. Z.; Shao, C. W.; Wang, J. SimultaneousReduction and Surface Functionalization of Graphene Oxide forHydroxyapatite Mineralization. J. Phys. Chem. C 2012, 116, 3334−3341.(18) Cui, W.; Li, M. Z.; Liu, J. Y.; Wang, B.; Zhang, C.; Jiang, L.;Cheng, Q. F. A Strong Integrated Strength and Toughness ArtificialNacre Based on Dopamine Cross-Linked Graphene Oxide. ACS Nano2014, 8, 9511−9517.(19) Hu, X. F.; Neoh, K. G.; Shi, Z. L.; Kang, E. T.; Poh, C.; Wang,W. An In Vitro Assessment of Titanium Functionalized withPolysaccharides Conjugated with Vascular Endothelial Growth Factor

for Enhanced Osseointegration and Inhibition of Bacterial Adhesion.Biomaterials 2010, 31, 8854−8863.(20) Chua, P. H.; Neoh, K. G.; Kang, E. T.; Wang, W. SurfaceFunctionalization of Titanium with Hyaluronic Acid/ChitosanPolyelectrolyte Multilayers and RGD for Promoting OsteoblastFunctions and Inhibiting Bacterial Adhesion. Biomaterials 2008, 29,1412−1421.(21) Lee, D. W.; Yun, Y. P.; Park, K.; Kim, S. E. Gentamicin andBone Morphogenic Protein-2 (BMP-2)-Delivering Heparinized-Titanium Implant with Enhanced Antibacterial Activity andOsteointegration. Bone 2012, 50, 974−982.(22) Xie, C. M.; Lu, X.; Wang, K. F.; Meng, F. Z.; Jiang, O.; Zhang,H. P.; Zhi, W.; Fang, L. M. Silver Nanoparticles and Growth FactorsIncorporated Hydroxyapatite Coatings on Metallic Implant Surfacesfor Enhancement of Osteoinductivity and Antibacterial Properties.ACS Appl. Mater. Interfaces 2014, 6, 8580−8589.(23) Shao, J. J.; Lv, W.; Yang, Q. H. Self-Assembly of GrapheneOxide at Interfaces. Adv. Mater. 2014, 26, 5586−5612.(24) Kang, S. M.; Hwang, N. S.; Yeom, J.; Park, S. Y.; Messersmith, P.B.; Choi, I. S.; Langer, R.; Anderson, D. G.; Lee, H. One-StepMultipurpose Surface Functionalization by Adhesive Catecholamine.Adv. Funct. Mater. 2012, 22, 2949−2955.(25) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B.Mussel-Inspired Surface Chemistry for Multifunctional Coatings.Science 2007, 318, 426−430.(26) Xu, L. Q.; Yang, W. J.; Neoh, K. G.; Kang, E. T.; Fu, G. D.Dopamine-Induced Reduction and Functionalization of GrapheneOxide Nanosheets. Macromolecules 2010, 43, 8336−8339.(27) Guo, L. Q.; Liu, Q.; Li, G. L.; Shi, J. B.; Liu, J. Y.; Wang, T.;Jiang, G. B. A Mussel-Inspired Polydopamine Coating as a VersatilePlatform for the In Situ Synthesis of Graphene-Based Nano-composites. Nanoscale 2012, 4, 5864−5867.(28) Cheng, C.; Li, S.; Zhao, J.; Li, X. X.; Liu, Z. Y.; Ma, L.; Zhang,X.; Sun, S. D.; Zhao, C. S. Biomimetic Assembly of Polydopamine-Layer on Graphene: Mechanisms, Versatile 2D and 3D Architecturesand Pollutant Disposal. Chem. Eng. J. 2013, 228, 468−481.(29) Jia, Z. J.; Xiu, P.; Li, M.; Xu, X. C.; Shi, Y. Y.; Cheng, Y.; Wei, S.C.; Zheng, Y. F.; Xi, T. F.; Cai, H.; Liu, Z. J. Bioinspired AnchoringAgNPs onto Micro-Nanoporous TiO2 Orthopedic Coatings: Trap-Killing of Bacteria, Surface-Regulated Osteoblast Functions and HostResponses. Biomaterials 2016, 75, 203−222.(30) Konkena, B.; Vasudevan, S. Understanding Aqueous Dispersi-bility of Graphene Oxide and Reduced Graphene Oxide throughpK(a) Measurements. J. Phys. Chem. Lett. 2012, 3, 867−872.(31) Kang, S. M.; Park, S.; Kim, D.; Park, S. Y.; Ruoff, R. S.; Lee, H.Simultaneous Reduction and Surface Functionalization of GrapheneOxide by Mussel-Inspired Chemistry. Adv. Funct. Mater. 2011, 21,108−112.(32) Anderson, T. H.; Yu, J.; Estrada, A.; Hammer, M. U.; Waite, J.H.; Israelachvili, J. N. The Contribution of DOPA to Substrate-PeptideAdhesion and Internal Cohesion of Mussel-Inspired Synthetic PeptideFilms. Adv. Funct. Mater. 2010, 20, 4196−4205.(33) Ku, S. H.; Ryu, J.; Hong, S. K.; Lee, H.; Park, C. B. GeneralFunctionalization Route for Cell Adhesion on Non-Wetting Surfaces.Biomaterials 2010, 31, 2535−2541.(34) Wang, X. L.; Bai, H.; Shi, G. Q. Size Fractionation of GrapheneOxide Sheets by pH-Assisted Selective Sedimentation. J. Am. Chem.Soc. 2011, 133, 6338−6342.(35) Chen, J.; Li, Y. R.; Huang, L.; Jia, N.; Li, C.; Shi, G. Q. SizeFractionation of Graphene Oxide Sheets via Filtration through Track-Etched Membranes. Adv. Mater. 2015, 27, 3654−3660.(36) Neoh, K. G.; Hu, X. F.; Zheng, D.; Kang, E. T. BalancingOsteoblast Functions and Bacterial Adhesion on FunctionalizedTitanium Surfaces. Biomaterials 2012, 33, 2813−2822.(37) Park, J.; Kim, B.; Han, J.; Oh, J.; Park, S.; Ryu, S.; Jung, S.; Shin,J. Y.; Lee, B. S.; Hong, B. H.; Choi, D.; Kim, B. S. Graphene OxideFlakes as a Cellular Adhesive: Prevention of Reactive Oxygen SpeciesMediated Death of Implanted Cells for Cardiac Repair. ACS Nano2015, 9, 4987−4999.



17164

http://dx.doi.org/10.1021/acsami.6b05198http://pubs.acs.org/action/showLinks?system=10.1021%2Ftx200339h&pmid=21954945&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlGjt73Nhttp://pubs.acs.org/action/showLinks?pmid=24074389&crossref=10.2217%2Fnnm.13.140&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFensr3Lhttp://pubs.acs.org/action/showLinks?pmid=25960040&crossref=10.1002%2Fadma.201501271&coi=1%3ACAS%3A528%3ADC%252BC2MXotVCmu7k%253Dhttp://pubs.acs.org/action/showLinks?crossref=10.1039%2FC4TC02536A&coi=1%3ACAS%3A528%3ADC%252BC2cXitFOrt7zNhttp://pubs.acs.org/action/showLinks?system=10.1021%2Fja200218y&pmid=21449601&coi=1%3ACAS%3A528%3ADC%252BC3MXktVCntro%253Dhttp://pubs.acs.org/action/showLinks?system=10.1021%2Fja200218y&pmid=21449601&coi=1%3ACAS%3A528%3ADC%252BC3MXktVCntro%253Dhttp://pubs.acs.org/action/showLinks?pmid=17947576&crossref=10.1126%2Fscience.1147241&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFOjtb%252FOhttp://pubs.acs.org/action/showLinks?pmid=17330084&crossref=10.1038%2Fnmat1849&coi=1%3ACAS%3A528%3ADC%252BD2sXit1Khtrg%253Dhttp://pubs.acs.org/action/showLinks?pmid=20061015&crossref=10.1016%2Fj.biomaterials.2009.12.020&coi=1%3ACAS%3A528%3ADC%252BC3cXptlyntA%253D%253Dhttp://pubs.acs.org/action/showLinks?pmid=23580891&crossref=10.1002%2Fadfm.201200177&coi=1%3ACAS%3A528%3ADC%252BC38Xls1eisbc%253Dhttp://pubs.acs.org/action/showLinks?system=10.1021%2Facsnano.5b02067&pmid=26091689&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVKitr%252FMhttp://pubs.acs.org/action/showLinks?system=10.1021%2Fnn202451x&pmid=21851105&coi=1%3ACAS%3A528%3ADC%252BC3MXhtVyls73Lhttp://pubs.acs.org/action/showLinks?pmid=21603098&crossref=10.1002%2Fadfm.201000932&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFClurvOhttp://pubs.acs.org/action/showLinks?pmid=24852899&crossref=10.1002%2Fadma.201400267&coi=1%3ACAS%3A528%3ADC%252BC2cXosV2rtb0%253Dhttp://pubs.acs.org/action/showLinks?pmid=25582092&crossref=10.1039%2FC4CC07836E&coi=1%3ACAS%3A528%3ADC%252BC2cXitFegtr3Ehttp://pubs.acs.org/action/showLinks?system=10.1021%2Facsami.5b01069&pmid=25705785&coi=1%3ACAS%3A528%3ADC%252BC2MXjtF2kt70%253Dhttp://pubs.acs.org/action/showLinks?system=10.1021%2Fam501428e&pmid=24720634&coi=1%3ACAS%3A528%3ADC%252BC2cXlvVSjsrw%253Dhttp://pubs.acs.org/action/showLinks?system=10.1021%2Fnn507149w&pmid=25919434&coi=1%3ACAS%3A528%3ADC%252BC2MXntlCms70%253Dhttp://pubs.acs.org/action/showLinks?pmid=20706983&crossref=10.1002%2Fadma.201001068&coi=1%3ACAS%3A528%3ADC%252BC3cXhtFKksrfLhttp://pubs.acs.org/action/showLinks?pmid=22257725&crossref=10.1016%2Fj.biomaterials.2012.01.018&coi=1%3ACAS%3A528%3ADC%252BC38XhsFKitb0%253Dhttp://pubs.acs.org/action/showLinks?system=10.1021%2Fjacs.5b11411&pmid=26824139&coi=1%3ACAS%3A528%3ADC%252BC28XhslKlsrk%253Dhttp://pubs.acs.org/action/showLinks?crossref=10.1016%2Fj.cej.2013.05.019&coi=1%3ACAS%3A528%3ADC%252BC3sXhtVOrsb3Ehttp://pubs.acs.org/action/showLinks?pmid=22945453&crossref=10.1039%2Fc2nr31547e&coi=1%3ACAS%3A528%3ADC%252BC38XhtlCjsbbEhttp://pubs.acs.org/action/showLinks?system=10.1021%2Fnn503755c&pmid=25106494&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlaisbjMhttp://pubs.acs.org/action/showLinks?system=10.1021%2Facsami.5b03431&pmid=25909563&coi=1%3ACAS%3A528%3ADC%252BC2MXntF2ntbw%253Dhttp://pubs.acs.org/action/showLinks?system=10.1021%2Facsami.5b03431&pmid=25909563&coi=1%3ACAS%3A528%3ADC%252BC2MXntF2ntbw%253Dhttp://pubs.acs.org/action/showLinks?system=10.1021%2Fma101526k&coi=1%3ACAS%3A528%3ADC%252BC3cXht1ahu7zJhttp://pubs.acs.org/action/showLinks?system=10.1021%2Fnn5066028&pmid=25840606&coi=1%3ACAS%3A528%3ADC%252BC2MXlvV2jsL8%253Dhttp://pubs.acs.org/action/showLinks?pmid=24619247&coi=1%3ACAS%3A528%3ADC%252BC2cXht1Wjtb7Mhttp://pubs.acs.org/action/showLinks?system=10.1021%2Facsnano.5b03368&pmid=26166486&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFGitbvKhttp://pubs.acs.org/action/showLinks?system=10.1021%2Fnn101390x&pmid=20925398&coi=1%3ACAS%3A528%3ADC%252BC3cXht1Kht77Khttp://pubs.acs.org/action/showLinks?crossref=10.1002%2Fadfm.201001692&coi=1%3ACAS%3A528%3ADC%252BC3cXhs1Sltr%252FEhttp://pubs.acs.org/action/showLinks?system=10.1021%2Fjz300236w&pmid=26286412&coi=1%3ACAS%3A528%3ADC%252BC38Xjs1Cmtb8%253Dhttp://pubs.acs.org/action/showLinks?pmid=22289658&crossref=10.1016%2Fj.bone.2012.01.007&coi=1%3ACAS%3A528%3ADC%252BC38Xjs1yhsrc%253Dhttp://pubs.acs.org/action/showLinks?system=10.1021%2Fjp2102226&coi=1%3ACAS%3A528%3ADC%252BC38XivVWmsg%253D%253Dhttp://pubs.acs.org/action/showLinks?pmid=26513414&crossref=10.1016%2Fj.biomaterials.2015.10.035http://pubs.acs.org/action/showLinks?pmid=26513414&crossref=10.1016%2Fj.biomaterials.2015.10.035http://pubs.acs.org/action/showLinks?pmid=18190959&crossref=10.1016%2Fj.biomaterials.2007.12.019&coi=1%3ACAS%3A528%3ADC%252BD1cXhtVGjs7Y%253Dhttp://pubs.acs.org/action/showLinks?pmid=25807416&crossref=10.1039%2FC5NR00236B&coi=1%3ACAS%3A528%3ADC%252BC2MXjslCrs78%253Dhttp://pubs.acs.org/action/showLinks?pmid=20800276&crossref=10.1016%2Fj.biomaterials.2010.08.006&coi=1%3ACAS%3A528%3ADC%252BC3cXht1egsL3O

(38) Arciola, C. R.; Campoccia, D.; Speziale, P.; Montanaro, L.;Costerton, J. W. Biofilm Formation in Staphylococcus ImplantInfections. A Review of Molecular Mechanisms and Implications forBiofilm-Resistant Materials. Biomaterials 2012, 33, 5967−5982.(39) Zimmerli, W.; Waldvogel, F. A.; Vaudaux, P.; Nydegger, U. E.Pathogenesis of Foreign-Body Infection - Description and Character-istics of an Animal-Model. J. Infect. Dis. 1982, 146, 487−497.(40) Gurunathan, S.; Han, J. W.; Dayem, A. A.; Eppakayala, V.; Kim,J.-h. Oxidative Stress-Mediated Antibacterial Activity of GrapheneOxide and Reduced Graphene Oxide in Pseudomonas Aeruginosa. Int.J. Nanomed. 2012, 7, 5901−5914.(41) Meng, J. X.; Zhang, P. C.; Wang, S. T. Recent Progress inBiointerfaces with Controlled Bacterial Adhesion by Using Chemicaland Physical Methods. Chem. - Asian J. 2014, 9, 2004−2016.(42) An, Y. H.; Friedman, R. J. Concise Review of Mechanisms ofBacterial Adhesion to Biomaterial Surfaces. J. Biomed. Mater. Res. 1998,43, 338−348.(43) Gallardo-Moreno, A. M.; Pacha-Olivenza, M. A.; Saldana, L.;Perez-Giraldo, C.; Bruque, J. M.; Vilaboa, N.; Gonzalez-Martin, M. L.In Vitro Biocompatibility and Bacterial Adhesion of Physico-Chemically Modified Ti6Al4V Surface by Means of UV Irradiation.Acta Biomater. 2009, 5, 181−192.(44) Liu, L. T.; Ercan, B.; Sun, L. L.; Ziemer, K. S.; Webster, T. J.Understanding the Role of Polymer Surface Nanoscale Topography onInhibiting Bacteria Adhesion and Growth. ACS Biomater. Sci. Eng.2016, 2, 122−130.(45) Dang, Y.; Xing, C. M.; Quan, M.; Wang, Y. B.; Zhang, S. P.; Shi,S. Q.; Gong, Y. K. Substrate Independent Coating Formation andAnti-Biofouling Performance Improvement of Mussel InspiredPolydopamine. J. Mater. Chem. B 2015, 3, 4181−4190.(46) Madhurakkat Perikamana, S. K.; Lee, J.; Lee, Y. B.; Shin, Y. M.;Lee, E. J.; Mikos, A. G.; Shin, H. Materials from Mussel-InspiredChemistry for Cell and Tissue Engineering Applications. Biomacro-molecules 2015, 16, 2541−2555.(47) Goriainov, V.; Cook, R.; Latham, J. M.; Dunlop, D. G.; Oreffo,R. O. C. Bone and Metal: An Orthopaedic Perspective onOsseointegration of Metals. Acta Biomater. 2014, 10, 4043−4057.(48) Horvath, L.; Magrez, A.; Burghard, M.; Kern, K.; Forro, L.;Schwaller, B. Evaluation of the Toxicity of Graphene Derivatives onCells of the Lung Luminal Surface. Carbon 2013, 64, 45−60.(49) Li, Y. F.; Yuan, H. Y.; von dem Bussche, A.; Creighton, M.;Hurt, R. H.; Kane, A. B.; Gao, H. J. Graphene Microsheets Enter Cellsthrough Spontaneous Membrane Penetration at Edge Asperities andCorner Sites. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 12295−12300.(50) Hong, S.; Kim, K. Y.; Wook, H. J.; Park, S. Y.; Lee, K. D.; Lee,D. Y.; Lee, H. Attenuation of the In Vivo Toxicity of Biomaterials byPolydopamine Surface Modification. Nanomedicine 2011, 6, 793−801.(51) Zhang, Y. B.; Ali, S. F.; Dervishi, E.; Xu, Y.; Li, Z. R.; Casciano,D.; Biris, A. S. Cytotoxicity Effects of Graphene and Single-WallCarbon Nanotubes in Neural Phaeochromocytoma-Derived PC12Cells. ACS Nano 2010, 4, 3181−3186.(52) Li, Y.; Liu, Y.; Fu, Y. J.; Wei, T. T.; Le Guyader, L.; Gao, G.; Liu,R. S.; Chang, Y. Z.; Chen, C. Y. The Triggering of Apoptosis inMacrophages by Pristine Graphene through the MAPK and TGF-BetaSignaling Pathways. Biomaterials 2012, 33, 402−411.(53) Pinto, A. M.; Gonca̧lves, I. C.; Magalhaẽs, F. D. Graphene-BasedMaterials Biocompatibility: A Review. Colloids Surf., B 2013, 111,188−202.(54) Wang, C. H.; Guo, Z. S.; Pang, F.; Zhang, L. Y.; Yan, M.; Yan, J.H.; Li, K. W.; Li, X. J.; Li, Y.; Bi, L.; Han, Y. S. Effects of GrapheneModification on the Bioactivation of Polyethylene-Terephthalate-Based Artificial Ligaments. ACS Appl. Mater. Interfaces 2015, 7,15263−15276.(55) Mishra, R. K.; Segal, E.; Lipovsky, A.; Natan, M.; Banin, E.;Gedanken, A. New Life for an Old Antibiotic. ACS Appl. Mater.Interfaces 2015, 7, 7324−7333.(56) Huang, N.; Zhang, S.; Yang, L. Q.; Liu, M. L.; Li, H. T.; Zhang,Y. Y.; Yao, S. Z. Multifunctional Electrochemical Platforms Based onthe Michael Addition/Schiff Base Reaction of Polydopamine Modified

Reduced Graphene Oxide: Construction and Application. ACS Appl.Mater. Interfaces 2015, 7, 17935−17946.(

new from solution to biointerface: graphene self-assemblies of...

Documents