Research Article Antibacterial Effects and ... · Titanium (Ti) implants with long-term...

Research ArticleAntibacterial Effects and Biocompatibility of Titania Nanotubeswith Octenidine DihydrochloridePoly(lactic-co-glycolic acid)

Zhiqiang Xu1 Yingzhen Lai2 Dong Wu3 Wenxiu Huang3

Sijia Huang1 Lin Zhou1 and Jiang Chen3

1School of Stomatology Fujian Medical University Fuzhou Fujian 350000 China2Department of Oral Medicine Xiamen Medical College Xiamen Fujian 361000 China3Department of Oral Implantology Affiliated Stomatological Hospital of Fujian Medical University Fuzhou Fujian 350002 China

Correspondence should be addressed to Jiang Chen dentistjiangchensinacom

Received 23 January 2015 Revised 11 March 2015 Accepted 13 March 2015

Academic Editor Zufu Lu

Copyright copy 2015 Zhiqiang Xu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Titanium (Ti) implants with long-term antibacterial ability and good biocompatibility are highly desirable materials that can beused to prevent implant-associated infections In this study titania nanotubes (TNTs) were synthesized on Ti surfaces throughelectrochemical anodization Octenidine dihydrochloride (OCT)poly(lactic-co-glycolic acid) (PLGA) was infiltrated into TNTsusing a simple solvent-casting technique OCTPLGA-TNTs demonstrated sustained drug release andmaintained the characteristichollow structures of TNTs TNTs (200 nm in diameter) alone exhibited slight antibacterial effect and good osteogenic activity butalso evidently impaired adhesion and proliferation of bone marrow mesenchymal stem cells (BMSCs) OCTPLGA-TNTs (100 nmin diameter) supported BMSC adhesion and proliferation and showed good osteogenesis-inducing ability OCTPLGA-TNTs alsoexhibited good long-term antibacterial abilitywithin the observation period of 7 dThe synthesized drug carrier with relatively long-term antibacterial ability and enhanced excellent biocompatibility demonstrated significant potential in bone implant applications

1 Introduction

Titanium (Ti) implants are widely used clinically because oftheir high biocompatibility and good mechanical properties[1] However implant-associated infections remain as one ofthe most serious postoperative complications [2] The highincidence of implant-associated infections can be mainlyattributed to the adhered bacteria that form a biofilm thisbiofilm provides the bacteria with high resistance to hostdefenses and antimicrobial therapies [3 4] Conventionalsystemic drug therapies in bones present limitations suchas low efficacy poor bioavailability and toxicity [5] Thuslocalized delivery of antimicrobial agents with time-effectivehandling of infection while potentially eliminating problemsassociated with systemic administration is highly desirable[6 7] Furthermore Ti bioactivity is not ideal and thus maylead to the formation of a fibrous capsule around the implant[5] Fibrous tissues can prevent the contact between hostimmunity sentinel cells and bacteria [2] Hence implant

coatings with enhanced osteogenic activity and antibacterialproperty must be used to prevent infections and elongate theservice life of Ti implants

With the advent of nanotechnology nanostructuredmaterials play fundamental roles in orthopedic researchbecause bones demonstrate a structural hierarchy at thefirst level in the nanometer regime [8] Titania nanotubes(TNTs) fabricated on the Ti surface through electrochemicalanodization have received considerable attentions in ortho-pedic research because of their high bioactivity to promotebone cell growth and cell differentiation unlike unanodizedTi [9ndash12] Moreover TNTs with controllable dimensionshigh surface-to-volume ratio and hollow structures havebeen demonstrated to be superior platforms for local antibi-otic delivery applications [13ndash17]

TNTs as antibiotic carriers are challenged with twodisadvantages that must be addressed before clinical appli-cations First the drug release in proposed drug-deliverysystems is directed through diffusion of drug molecules

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 836939 11 pageshttpdxdoiorg1011552015836939

2 BioMed Research International

from the nanotube structure [5] The release rate is veryfast and short thus limiting the antimicrobial effects toearly-stage peri-implant infections Although polymers andphospholipids were directly coated on the top of drug-loadedTNTs to extend drug release TNTswere buried in the coatingcap hence the potential benefit of TNTs in promoting bonegrowth was diminished [18ndash20] Poly(lactic-co-glycolic acid)(PLGA) has been loaded into TNTs through solvent-castingtechnique to maintain their characteristic hollow structureand sustain drug release [21] However to the best of ourknowledge the biocompatibility of TNTs loaded with PLGAremains unknown Second bacteria are highly adaptable innature which leads to the evolution of strains resistant to con-ventional antibiotics [22] Agents working through unspecificmodes of action are required to overcome this resistancesuch as octenidine dihydrochloride (OCT) OCT approvedas a medicinal substance in several European countries is anestablished bispyridine antiseptic with a broad activity and iscommonly used as wound antiseptic [23 24] It has attractedincreasing attentions because of broad antibacterial spec-trum including antibiotic-resistant bacteria noncytotoxicityat suitable doses satisfactory stability and smaller possibilityto develop resistant bacteria [25 26]

In this study we investigated the possibility of usingcomposite PLGA-TNTs as a carrier for sustained OCTdelivery Despite active studies on using localized deliveryof antimicrobial agents to prevent implant infections therehave been few reports on using antiseptics [27] and noneon using nanotubes to deliver antiseptics In this paper forthe first time OCTPLGA was loaded into TNTS througha simple dip-coating process Sustained antibacterial abilityand biocompatibility were systematically investigated

2 Materials and Methods

21 Fabrication of TNTs on Ti TNTs were fabricated throughanodization on a Ti sheet Ti samples (Alfa-Aesar Ward HillMA USA 1 times 1 times 0025 cm3 998 purity) were degreasedby sonication in acetone and deionized water for 15minThe samples were then eroded for 10 s in 4wt HFndash5molLHNO

3 followed by rinsing with deionized water and drying

in air A two-electrode electrochemical cell using a platinumsheet as the counter-electrode was used Anodization wasperformed using a mixture of 050wtNH

4F + 10 volH

2O

in glycerol at 60V for 5 h After anodization the samples wererinsed with deionized water dried in air and annealed at450∘C for 2 h

22 OCT Loading Two types of samples were preparedThe first type was prepared by loading the mixture ofOCTPLGA into TNTs PLGA (Sigma-Aldrich St LouisMO USA lactide-to-glycolide ratio 65 35 24000Dandash38000Da) and OCT (TCI Shanghai China) were dissolvedin dichloromethane at 15mgmL and 05120583gmL respectivelyThe polymerdrug mixture was loaded into TNTs using adip-coating process at 40∘C for 2 d and then dried in airThe second type was produced by directly loading OCT intoTNTs OCT was dissolved in low-surface tension solvent(ethanol) and forced into TNTs using a vacuum-assisted

physical adsorptionmethod In brief 50120583L of 40120583gmLOCTsolutionwas pipetted onto the nanotube surfaces and allowedto dry under a vacuum desiccator at 20∘C for 1 hThe loadingprocess was repeated three times

23 SpecimenCharacterization Scanning electronmicroscopy(SEM) (JSM-7500F JEOL Tokyo Japan) was used tocharacterize the morphology of the prepared TNTs andOCTPLGA-TNTs The infiltration of PLGA polymer intoTNTs was also assessed by SEM according to the reportof Jia and kerr [21] Scotch tape was used to separate theOCTPLGA-TNTs from Ti foil The tape with OCTPLGA-TNTs then was soaked in 5 vol HF solution for 15min untilall TNTs were etched offThe remaining PLGAwas examinedthrough SEM

24 Contact Angle Determination A 1 120583L drop of distilledwater was delivered on the clean specimen surface with asyringe at a room temperature and in the open atmosphereof the lab Contact angles were measured on the obtainedphotographs (Phoenix 300 SEO Seoul Korea) The meanvalue was calculated from five separate measurements

25 Protein Adsorption Assay A 1mL droplet of DMEM-LGcontaining 10 FCS was pipetted onto each specimen Afterincubation at 37∘C for 2 h the proteins adsorbed onto thesamples were detached using 1 sodium dodecyl sulfate Theprotein concentrations were determined using a MicroBCAprotein assay kit (Pierce Rockford IL USA)

26 Release Profile of OCT OCT has been shown to bestable in chloroform at 40∘C [25] So in vitro OCT releasekinetics of the samples were measured using ultravioletvisible spectroscopy (UV-1750 Shimadzu Kyoto Japan) byrecording the absorption peak at 280 nm which is thecharacteristic excitation wavelength of OCT Two specimensTNTs loaded with OCT and TNTs loaded with OCTPLGAwere immersed in 1-mL of PBS in a glass vial while rotating(60 revolutions per minute) at 37∘C After 1 2 4 and 6 h aswell as after 1 2 3 6 9 12 15 and 18 d 500mL of the solutionwas sampled and fresh PBS was replenished Three sampleswere tested in each time interval and themean valuewas usedin data analysis

27 Bacteria Cultures Staphylococcus aureus (S aureus)(ATCC25923 American Type Culture Collection ManassasVA USA) was cultivated in the brain-heart infusion brothmedium at 37∘C for 12 h and then adjusted to a concentrationof 106 CFUmL The specimens were placed on 24-well cul-ture plates and separately incubated in 1mL of the bacteria-containing medium

271 SEM Observation After being incubated in 1mL ofthe bacteria-containing medium for 6 h the samples wererinsed with PBS fixed with 3 glutaraldehyde dehydrated ingraded ethanol series freeze-dried sputter-coated with thinplatinum layers and observed by SEM

BioMed Research International 3

Table 1 Primers used for qRT-PCR

Gene Forward primer sequence (51015840-31015840) Reverse primer sequence (51015840-31015840)RUNX2 CCTCTGACTTCTGCCTCTGG GATGAAATGCCTGGGAACTGALP GCCTGGACCTCATCAGCATT AGGGAAGGGTCAGTCAGGTTOCN CAAGTCCCACACAGCAACTC CCAGGTCAGAGAGGCAGAATCOL-I ATCTCCTGGTGCTGATGGAC GCCTCTTTCTCCTCTCTGACCGAPDH GGCACAGTCAAGGCTGAGAATG ATGGTGGTGAAGACGCCAGTA

272 Antibacterial Assay In vitro antibacterial activity wasassessed by the plate-counting method After culturing for 14 and 7 days the sample was rinsed in PBS and ultrasonicallyagitated to detach the bacteria from the sample The bacteriasuspensions were recultivated on agar plates for colonycounting The antibacterial rates were calculated using thefollowing formula antibacterial rate () = (CFU of control minusCFU of experimental groups)CFU of control times 100 whereTi served as the control while TNTs and TNTsPLGA consti-tuted the experimental groups

28 Cell Cultures Sprague-Dawley rat bone marrow mes-enchymal stem cells (BMSCs) were purchased from CyagenBiosciences (Guangzhou China) The cells were cultured inDMEM-LG containing 10 FCS at 37∘C and the mediumwas changed every 3 d Cells were used between passage 4and passage 6 in the following experimentsThe samples wereplaced in 24-well plates and the BMSCs were seeded at adensity of 4 times 104well for the cell adhesion assay and 2 times104well for the other assays

281 Cell Morphology After culturing for 2 d the sampleswere rinsed with PBS fixed with 25 glutaraldehyde dehy-drated in graded ethanol series freeze-dried sputter-coatedwith thin platinum layers and observed by SEM

282 Adhesion and Proliferation For the cell adhesion assaythe adherent cells were fixed and stained with 46-diamidino-2-phenylindole (DAPI Sigma-Aldrich) after culturing for 051 and 2 h Images were captured from five random fields bya fluorescence microscope and the cell number in each fieldwas determined To assess cell proliferation the cell numberswere assessed using Cell Counting Kit-8 (CCK-8 BeyotimeShanghai China) assay after seeding for 1 3 and 7 d

283 Gene Expressions The expression levels of osteogen-esis-related genes including runt-related transcription factor2 (RUNX2 a key transcript factor for osteogenic differ-entiation) alkaline phosphatase (ALP an early marker forosteogenic differentiation) osteocalcin (OCN a late markerfor osteogenic differentiation) and type 1 collagen (COL-1 a main collagen found in bones) were measured usingquantitative reverse transcription polymerase chain reaction(qRT-PCR) Total RNA was extracted using Trizol (Invitro-gen Carlsbad CA USA) after culturing for 2 weeks TotalRNA was then reverse-transcribed with a cDNA ReverseTranscription Kit (TaKaRa Shiga Japan) and qRT-PCRanalysis was performed on an ABI Prism 7500 real-time PCR

cycler (Applied Biosystems Carlsbad CA USA) using SYBRPremix Ex Taq II (TaKaRa) The primers for the target genesare listed in Table 1 The expression levels of the target geneswere normalized to that of the housekeeping gene GAPDH

29 Statistical Analysis All data were expressed as the meanplusmn standard deviation (SD) One-way ANOVA and Student-Newman-Keuls post hoc test were used to determine the levelof significance119875 lt 005was considered to be significant and119875 lt 001 was considered to be highly significant

3 Results

31 Specimen Characterization The SEM images of TNTswith and without OCTPLGA loading are shown in Figure 1TNTs (200 nm in diameter) were neatly and uniformlyarranged over the anodized Ti surface in Figure 1(a) Thediameter decreased to 100 nm after OCTPLGA loadingand the intertubular areas were filled with PLGA as shownin Figure 1(b) The side view of TNTs and the image ofthe remaining PLGA after TNTs removal are shown inFigures 1(c) and 1(d) respectively The remaining PLGA wasuniformly arranged in most of the areas and its length wassimilar to the length of TNTs Thus PLGA demonstrated anexcellent infiltration depth into TNTs

32 Static Contact Angles Drop images were captured by avideo camera in the direction perpendicular to the surface(Figure 2) For hydrophilic material the contact angle islower than 90 degrees and the smaller the contact angle thegreater the hydrophilicity Multiple comparisons (Figure 2)revealed that TNTs showed the greatest surface hydrophilic-ity whereas Ti exhibited the greatest surface hydrophobicity

33 Protein Adsorption Assay The amounts of adsorbedproteins from 10 FCS after 2 h of incubation are pre-sented in Figure 3 to elucidate subsequent cellular responsesOCTPLGA-TNTs with higher hydrophilicity absorbedmoreproteins than control Ti However paradoxically TNTs withthe highest hydrophilicity had the fewest protein aggregatesIt is because the protein aggregates (asymp30-nm-size regime)initially attach only to the available surfaces that are the topportion of the nanotubewalls [11 12] and these aggregates aretoo small to anchor on TNTs with a diameter of 200 nmTheprotein aggregates could easily attach to OCTPLGA-TNTsprobably because the larger intertubular areas which werefilled with PLGA providedmore effective nucleation sites forprotein adsorption

Huang
高亮


(a) (b)

(c) (d)

Figure 1 SEM images (a) TNTs with a diameter of 200 nm (b) OCTPLGA-TNTs with a diameter of 100 nm (c) side view of TNTs and (d)remaining PLGA

Ti OCTPLGA-TNTs TNTs0

20

40

60

Wat

er co

ntac

t ang

le (d

eg)

Ti OCT TNTsPLGA-TNTs

lowastlowast

lowastlowast

Figure 2 Photographs of water droplets on different samples and multiple-comparison results of contact angles on different samples (meanplusmn SD119873 = 5 lowast119875 lt 005 lowastlowast119875 lt 001)



10

20

30

40lowastlowastlowastlowast

lowast

Adso

rbed

pro

tein

(120583g

mL)

Figure 3 Protein adsorption to the samples after 2 h of immersionin DMEM-LG containing 10 FCS (mean plusmn SD119873 = 3 lowast119875 lt 005lowastlowast119875 lt 001)

34 OCT Release Figure 4 shows the OCT release time pro-files from TNTs and OCTPLGA-TNTs in PBS For the OCTdirectly released from the nanotubes the release kineticscould be described in two phases a high percentage (80) ofthe drug released in the first 6 h (initial burst release) and thena slow release for the following 2 d By contrast in the case ofPLGA-TNTs the drug release pattern was directed throughthe transport of drug through the polymermatrix and the rateof polymer degradation [19 21] so the burst release decreasedfrom 80 to 48 (versus TNTs) and the extended overallrelease increased from 2 d to 15 d

35 Antimicrobial Activity Figures 5(a) 5(b) and 5(c) showthe qualitative SEM assessments of bacteria incubated withsamples It could be observed that many multiple bacterialcolonies formed colony masses on the surfaces of Ti andTNTs In strong contrast very few single bacterial colonieswere detected on OCTPLGA-TNTS

Antibacterial activity was evaluated for 7 d as shownin Figure 5 On day 1 OCTPLGA-TNTs loaded with OCTgenerated a high antibacterial rate of 100 Although a slightdecrease in the antibacterial rates was observed as timeincreased a high antibacterial rate of 972 was maintaineduntil day 7 This finding suggests an effective and long-termantibacterial activity against S aureus TNTs alone exhibitedslight antibacterial rate of about 20 which was relativelyconstant with time

36 Cell Morphology The BMSCs displayed dramaticallydifferent shapes related to the topography of the substrateas shown in Figure 6 Most of the BMSCs on Ti appearedround and spread poorly with no cellular extensions andfilopodia propagation indicative of undifferentiated BMSCs(Figures 6(a) 6(b) and 6(c)) By contrast the cells onOCTPLGA-TNTs (Figures 6(d) 6(e) and 6(f)) and TNTs(Figures 6(g) 6(h) and 6(i)) had to extend across the tubesto locate a protein-deposited surface on intertubular areasfor initial contact thereby becoming more extended with

a large number of prominent filopodia and unidirectionallamellipodia extensions compared with Ti and pure TNTswith a larger diameter presented stronger induction

37 Adhesion and Proliferation The highest numbers of ini-tial adherent cells on OCTPLGA-TNTs and lowest numbersof initial adherent cells on TNTs are shown in Figure 7Upon contact of an implant surface with blood the proteinsavailable in the serum adsorb to the surface within an initialincubation time and mediate the subsequent cellular perfor-mance [11 28] Cell attachment is believed to be significantlygreater on material surfaces with more protein adhesions[29ndash33] Thus OCTPLGA-TNTs with more protein adhe-sions induced more cell attachment at an early time than TiandTNTswith the fewest protein aggregates induced the leastattachment

Cell proliferation was measured by the CCK-8 assay(Figure 8) On day 1 the cell numbers on OCTPLGA-TNTswere slightly smaller than those onTiThis slight proliferationsuppressive effect was possibly related to the differentiationtendency of BMSCs because of the reciprocal relationshipbetween cell proliferation and differentiation [34 35] Theinhibition of BMSC proliferation was not serious or long Bydays 3 and 7 the cell growth on 100 nm OCTPLGA-TNTscaught up because of the large surface area available for cellcolonization This finding indicates that OCTPLGA-TNTsdid not impair cell viability and could support cell prolifera-tionHowever cell proliferation onTNTswas obviously lowerthan that on the Ti control and OCTPLGA-TNTs and thistrend becamemore evident with timeThe very low adhesionat the early stage can potentially lead to cell quiescence oreven apoptosis by anoikis a type of programmed cell deaththrough ldquohomelessnessrdquo [36]

38 Gene Expressions The expression levels of osteogenesis-related genes including RUNX2 ALP OCN and COL-1 wereassessed by qRT-PCRThe results are shown in Figure 9 Thethree topographies explored in this study induced differentgene expression levels Previous study reported that elongatedBMSCs are prone to undergo osteogenesis [37 38] so theeffects of nanotubes on inducing fast and good distribution ofBMSCs favored the osteogenic ability OCTPLGA-TNTs andTNTs significantly promoted the expression of osteogenesis-related genes and demonstrated excellent osteogenic activitywith the latter exhibiting a higher promotion

4 Discussions

With the steadily increasing demand for implants a properapproach that can endow biomaterials with long-termantibacterial ability and biointegration has been actively pur-sued [28] In the present study OCTPLGA was loaded intoTNTs using a simple solvent-casting technique The designsustained OCT release and maintained the characteristichollow structure of TNTs OCTPLGA-TNTs showed goodantibacterial characteristics and biointegration and are thusimportant to prevent implant-associated infections

PLGA whichwas approved by theUS FDA as therapeuticmaterial was selected as the OCT carrier in this study


0 40 80 120 160 200 240 280 320 360

16

24

32

Time (h)

Dru

g re

leas

e am

ount

(ppm

)

OCT-TNTsOCTPLGA-TNTs

(a)

0 2 4 6

80

4840

60

80

Dru

g re

leas

e (

)

Time (h)


(b)

Figure 4 In vitro release profiles in PBS (a) accumulative amount of OCT release from the samples and (b) accumulative percentage of OCTrelease from the samples

Ti

(a)

OCTPLGA-TNTs

(b)

TNTs

(c)

0 1 2 3 4 5 6

972

7 8

20

40

60

80

100

120

Ant

ibac

teria

l rat

e (

)

Time (d)OCTPLGA-TNTsTNTs

(d)

Figure 5 SEM images of S aureus incubated for 6 h on the samples (a) control Ti (b) OCTPLGA-TNTs and (c) TNTs (d) shows in vitroantibacterial rate profiles of the samples

because of its excellent biocompatibility and biodegradabilitywithout interrupting osseointegration [39] As expected ourproposed design enabled slow and sustained release for 15 dusing the composite OCTPLGA-TNTs S aureus known forits extensive resistance to antibiotics is the most common

cause of implant infections [40] and was therefore chosen forthe study The antibacterial ability of the OCTPLGA-TNTsdecreased gradually with time and the tendency was consis-tent with the OCT time release profiles A postimplantationperiod of 6 h has been identified as the ldquodecisive periodrdquowhen


Ti

(a) (b) (c)

OCT

PLG

A-TN

Ts

(d) (e) (f)

TNTs

(g) (h) (i)

Figure 6 SEM pictures with three different magnifications of 200x (the first column) 800x (the second column) and 2000x (the thirdcolumn) showing the morphology of BMSCs after 2 d of culture on samples

the implant is particularly susceptible to surface colonization[28] The very few single bacterial colonies in Figure 5(b)(versus many multiple bacterial colonies in Figures 5(a) and5(c)) showed that the burst OCT release of 48 in thefirst 6 h could effectively inhibit bacterial adhesion on theOCTPLGA-TNT samples though the larger OCT release of80 of TNTs may have resulted in less bacteria adhesion onthe TNTs After this stage few drugs are needed to preventfurther infection with the help of the host defense [41]The antibacterial assay showed that the slow and sustainedOCT release from OCTPLGA-TNTS also exhibited a goodlong-term antibacterial ability within the observation periodof 7 d The long-term antibacterial ability with slow andsustained OCT release is meaningful to preventing implantinfection while avoiding potential side effects associatedwith OCT overdose in clinical practice Our results alsoshowed that TNTs without drug loading slightly inhibitedthe antibacterial activity This finding is in accordance withreports that controlled titania nanotube formation through

anodization and heat treatment which decrease the contactangles of water and form crystalline TiO

2 leading to a

decreased bacterial adhesion on them [42]BMSCs are multipotent stem cells that can give rise

to various adult cell types including osteoblasts [43] andmost osteoblastic cells that colonize the implant surface toinduce bone growth originate from BMSCs [44] Hence it iscrucial to evaluate the behavior of BMSCs on OCTPLGA-TNTs before the clinical application Cell assay showed thatOCTPLGA-TNTs could support BMSCs adhesion and pro-liferation This finding indicates that excellent antibacterialability can be obtained from OCT at concentrations lowerthan the values to induce BMSC damage This result is inaccordance with previous reports [25 45] in which OCT ismore toxic to S aureus than tomammalian cells under similartest conditions resulting in a good biocompatibility with con-comitant antibacterial activity Furthermore OCTPLGA-TNTs promoted cell spreading and thus demonstrated excel-lent osteogenic activity Although PLGA can be replaced by


0

100

200

300

400

Cel

l num

ber

scop

e

Ti OCTPLGA-TNTs TNTs

30 60 120

(min)

lowast

lowast

lowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

TiOCTPLGA-TNTsTNTs

Figure 7 Fluorescence images of initial adherent BMSCs stained with DAPI after 1 h and cell numbers measured by counting cells for 05 1and 2 h (mean plusmn SD119873 = 5 lowast119875 lt 005 lowastlowast119875 lt 001)

00

05

10

15

TiOCTPLGA-TNTsTNTs

lowastlowast

lowastlowast

lowastlowast

lowast lowast

1 3 7

(d)

Abso

rban

ce (4

50

nm)

Figure 8 Cell proliferation measured by CCK-8 assay after culturing BMSCs on three different samples for 1 3 and 7 d (mean plusmn SD119873 = 3lowast119875 lt 005 lowastlowast119875 lt 001)



1

2

3

4m

RNA

RU

NX2

GA

PDH lowastlowast

lowastlowast

(a)


1

2

3

4

mRN

A A

LPG

APD

H lowastlowast

lowastlowast

(b)


1

2

3

4

mRN

A O

CNG

APD

H

lowastlowast

lowast

(c)


2

4

6

8

10

mRN

A C

OL-

1G

APD

H

lowastlowast

lowastlowast

(d)

Figure 9 Relative expressions of (a) RUNX2 (b) ALP (c) OCN and (d) COL-1 by BMSCs seeding on different substrates for 2 weeks (meanplusmn SD119873 = 3 lowast119875 lt 005 lowastlowast119875 lt 001)

serum proteins and extracellular matrices after degradationthe initial changes (within the first 24 h) in the cell shapeand cytoskeletal distribution would decide the fate of stemcell differentiation [46] In addition a good cell distributionon the biomaterial surface is important in winning the ldquoracefor the surfacerdquo against bacteria thereby aiding to combatinfection [47] Although TNTs alone showed the best abilityto promote cell spreading and osteogenic differentiation itobviously impaired cell adhesion and proliferation

The technique reported in this study can be readilyextended to other types of local drug-delivery applicationsto produce desirable biological effects The incorporatedamount and release rates can also be readily controlled byvarying the drug concentration and the lactide-to-glycolideratio

5 Conclusions

In summary OCTPLGA was loaded into TNTs withoutcompletely filling the nanotubes using a simple solvent-casting technique to obtain sustained OCT release OCTPLGA-TNTs showed good antibacterial effects which could

prevent postoperation complications and even late cases ofinfection while osteogenic activity was enhanced Moreoverthe fabrication process of OCTPLGA-TNTs is simple eco-nomical and versatile Thus OCTPLGA-TNTs are highlyattractive for biomedical implants because of their controlleddrug release long-term antibacterial efficacy and promotionof osseointegration

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work is supported by the Key Specialist Facility of FujianProvince (20121589) and the Science and Technology Projectof Fujian Province (2013Y0036)The authors gratefully thankthe Fujian Key Laboratory of Polymer Materials for facilitat-ing TNTs preparation They also thank Mr Feng Shi and MrJianhe Liang for technical assistance


References

[1] L Actis L Gaviria T Guda and J L Ong ldquoAntimicrobial sur-faces for craniofacial implants state of the artrdquo Journal of theKorean Association of Oral and Maxillofacial Surgeons vol 39no 2 pp 43ndash54 2013

[2] J Gallo M Holinka and C S Moucha ldquoAntibacterial surfacetreatment for orthopaedic implantsrdquo International Journal ofMolecular Sciences vol 15 no 8 pp 13849ndash13880 2014

[3] D RMonteiro L FGorupA S Takamiya AC Ruvollo-FilhoE R D Camargo and D B Barbosa ldquoThe growing importanceof materials that prevent microbial adhesion antimicrobialeffect ofmedical devices containing silverrdquo International Journalof Antimicrobial Agents vol 34 no 2 pp 103ndash110 2009

[4] T-F C Mah and G A OrsquoToole ldquoMechanisms of biofilm resis-tance to antimicrobial agentsrdquo Trends in Microbiology vol 9no 1 pp 34ndash39 2001

[5] K Gulati M S Aw D Findlay and D Losic ldquoLocal drugdelivery to the bone by drug-releasing implants perspectives ofnano-engineered titania nanotube arraysrdquoTherapeutic Deliveryvol 3 no 7 pp 857ndash873 2012

[6] M Zilberman and J J Elsner ldquoAntibiotic-eluting medicaldevices for various applicationsrdquo Journal of Controlled Releasevol 130 no 3 pp 202ndash215 2008

[7] G Gao D Lange K Hilpert et al ldquoThe biocompatibility andbiofilm resistance of implant coatings based on hydrophilicpolymer brushes conjugated with antimicrobial peptidesrdquo Bio-materials vol 32 no 16 pp 3899ndash3909 2011

[8] K S Brammer C J Frandsen and S Jin ldquoTiO2nanotubes for

bone regenerationrdquo Trends in Biotechnology vol 30 no 6 pp315ndash322 2012

[9] J Park S Bauer K von der Mark and P Schmuki ldquoNanosizeand vitality TiO

2nanotube diameter directs cell faterdquo Nano

Letters vol 7 no 6 pp 1686ndash1691 2007[10] J Park S Bauer K A Schlegel F W Neukam K D von Mark

and P Schmuki ldquoTiO2nanotube surfaces 15 nmmdashan optimal

length scale of surface topography for cell adhesion and differ-entiationrdquo Small vol 5 no 6 pp 666ndash671 2009

[11] S Oh K S Brammer Y S J Li et al ldquoStem cell fate dictatedsolely by altered nanotube dimensionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 7 pp 2130ndash2135 2009

[12] K S Brammer S Oh C J Cobb LM Bjursten H V D Heydeand S Jin ldquoImproved bone-forming functionality on diameter-controlled TiO

2nanotube surfacerdquo Acta Biomaterialia vol 5

no 8 pp 3215ndash3223 2009[13] K C Popat M Eltgroth T J LaTempa C A Grimes and T

A Desai ldquoDecreased Staphylococcus epidermis adhesion andincreased osteoblast functionality on antibiotic-loaded titaniananotubesrdquo Biomaterials vol 28 no 32 pp 4880ndash4888 2007

[14] G E Aninwene II C Yao and T J Webster ldquoEnhancedosteoblast adhesion to drug-coated anodized nanotubular tita-nium surfacesrdquo International Journal of Nanomedicine vol 3no 2 pp 257ndash264 2008

[15] H Z Zhang Y Sun A Tian et al ldquoImproved antibacterialactivity and biocompatibility on vancomycin-loaded TiO

2nan-

otubes in vivo and in vitro studiesrdquo International Journal ofNanomedicine vol 8 pp 4379ndash4389 2013

[16] P Chennell E Feschet-Chassot T Devers K O Awitor SDescamps and V Sautou ldquoIn vitro evaluation of TiO

2nan-

otubes as cefuroxime carriers on orthopaedic implants for the

prevention of periprosthetic joint infectionsrdquo InternationalJournal of Pharmaceutics vol 455 no 1-2 pp 298ndash305 2013

[17] W-T Lin H-L Tan Z-L Duan et al ldquoInhibited bacte-rial biofilm formation and improved osteogenic activity ongentamicin-loaded titania nanotubes with various diametersrdquoInternational Journal of Nanomedicine vol 9 no 1 pp 1215ndash1230 2014

[18] M S Aw ldquoControlling drug release from titania nanotubearrays using polymer nanocarriers and biopolymer coatingrdquoJournal of Biomaterials and Nanobiotechnology vol 2 no 5 pp477ndash484 2011

[19] K Gulati S Ramakrishnan M S Aw G J Atkins D MFindlay andD Losic ldquoBiocompatible polymer coating of titaniananotube arrays for improved drug elution and osteoblastadhesionrdquo Acta Biomaterialia vol 8 no 1 pp 449ndash456 2012

[20] M Kazemzadeh-Narbat B F L Lai C Ding J N Kizhakke-dathu R E W Hancock and R Wang ldquoMultilayered coatingon titanium for controlled release of antimicrobial peptides forthe prevention of implant-associated infectionsrdquo Biomaterialsvol 34 no 24 pp 5969ndash5977 2013

[21] H Jia and L L Kerr ldquoSustained ibuprofen release using com-posite poly(lactic-co-glycolic acid)titanium dioxide nanotubesfrom Ti implant surfacerdquo Journal of Pharmaceutical Sciencesvol 102 no 7 pp 2341ndash2348 2013

[22] R Johnson ldquoAntibiotics resistance is restoredrdquo Nature Chem-istry vol 6 no 9 pp 754ndash755 2014

[23] N-O Hubner J Siebert and A Kramer ldquoOctenidine dihy-drochloride a modern antiseptic for skin mucous membranesand woundsrdquo Skin Pharmacology and Physiology vol 23 no 5pp 244ndash258 2010

[24] T Koburger N-O HubnerM Braun J Siebert andA KramerldquoStandardized comparison of antiseptic efficacy of triclosanPVP-iodine octenidine dihydrochloride polyhexanide andchlorhexidine digluconaterdquo Journal of Antimicrobial Chemo-therapy vol 65 no 8 pp 1712ndash1719 2010

[25] G Baier A Cavallaro K Friedemann et al ldquoEnzymatic degra-dation of poly(l-lactide) nanoparticles followed by the releaseof octenidine and their bactericidal effectsrdquo NanomedicineNanotechnology Biology andMedicine vol 10 no 1 pp 131ndash1392014

[26] S A Baskaran A Upadhyay I Upadhyaya V Bhattaram andK Venkitanarayanan ldquoEfficacy of octenidine hydrochloride forreducing Escherichia coliO157 H7 Salmonella spp and Listeriamonocytogenes on cattle hidesrdquo Applied and EnvironmentalMicrobiology vol 78 no 12 pp 4538ndash4541 2012

[27] S Weckbach A Moricke H Braunwarth P Goroncy-BermesM Bischoff and F Gebhard ldquoOctenidine in combination withpolymethylmethacrylate a new option for preventing infec-tionrdquoArchives of Orthopaedic and Trauma Surgery vol 132 no1 pp 15ndash20 2012

[28] K G Neoh X Hu D Zheng and E T Kang ldquoBalancingosteoblast functions and bacterial adhesion on functionalizedtitanium surfacesrdquo Biomaterials vol 33 no 10 pp 2813ndash28222012

[29] F Grinnell and M K Feld ldquoFibronectin adsorption on hydro-philic and hydrophobic surfaces detected by antibody bind-ing and analyzed during cell adhesion in serum-containingmediumrdquoThe Journal of Biological Chemistry vol 257 no 9 pp4888ndash4893 1982

[30] K Webb V Hlady and P A Tresco ldquoRelative importanceof surface wettability and charged functional groups on NIH


3T3 fibroblast attachment spreading and cytoskeletal organi-zationrdquo Journal of Biomedical Materials Research vol 41 pp422ndash430 1998

[31] A M G Borges L O Benetoli M A Licınio et al ldquoPolymerfilms with surfaces unmodified and modified by non-thermalplasma as new substrates for cell adhesionrdquo Materials Scienceand Engineering C vol 33 no 3 pp 1315ndash1324 2013

[32] Y Wang C Wen P Hodgson and Y Li ldquoBiocompatibility ofTiO2

nanotubes with different topographiesrdquo Journal ofBiomedical Materials ResearchmdashPart A vol 102 no 3 pp743ndash751 2014

[33] X Liu Q Feng A Bachhuka and K Vasilev ldquoSurface chemicalfunctionalities affect the behavior of human adipose-derivedstem cells in vitrordquo Applied Surface Science vol 270 pp 473ndash479 2013

[34] G S Stein J B Lian and T A Owen ldquoRelationship of cellgrowth to the regulation of tissue-specific gene expression dur-ing osteoblast differentiationrdquoThe FASEB Journal vol 4 no 13pp 3111ndash3123 1990

[35] I Wall N Donos K Carlqvist F Jones and P Brett ldquoModifiedtitanium surfaces promote accelerated osteogenic differentia-tion of mesenchymal stromal cells in vitrordquo Bone vol 45 no1 pp 17ndash26 2009

[36] E A Cavalcanti-Adam D Aydin V C Hirschfeld-Warnekenand J P Spatz ldquoCell adhesion and response to syntheticnanopatterned environments by steering receptor clusteringand spatial locationrdquo HFSP Journal vol 2 no 5 pp 276ndash2852008

[37] R McBeath D M Pirone C M Nelson K Bhadriraju and CS Chen ldquoCell shape cytoskeletal tension and RhoA regulatestem cell lineage commitmentrdquo Developmental Cell vol 6 no4 pp 483ndash495 2004

[38] K A Kilian B Bugarija B T Lahn and M Mrksich ldquoGeo-metric cues for directing the differentiation of mesenchymalstem cellsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 107 no 11 pp 4872ndash4877 2010

[39] S Y Lee J Y Koak S J Heo S K Kim S J Lee and SY Nam ldquoOsseointegration of anodized titanium implantscoated with poly(lactide-co-glycolide)basic fibroblast growthfactor by electrosprayrdquo The International Journal of Oral ampMaxillofacial Implants vol 25 no 2 pp 315ndash320 2010

[40] D Campoccia L Montanaro and C R Arciola ldquoThe signifi-cance of infection related to orthopedic devices and issues ofantibiotic resistancerdquo Biomaterials vol 27 no 11 pp 2331ndash23392006

[41] L Zhao P K Chu Y Zhang and ZWu ldquoAntibacterial coatingson titanium implantsrdquo Journal of BiomedicalMaterials ResearchPart B Applied Biomaterials vol 91 no 1 pp 470ndash480 2009

[42] B Ercan E Taylor E Alpaslan and T J Webster ldquoDiameterof titanium nanotubes influences anti-bacterial efficacyrdquo Nan-otechnology vol 22 no 29 Article ID 295102 2011

[43] C Vater P Kasten and M Stiehler ldquoCulture media for the dif-ferentiation of mesenchymal stromal cellsrdquo Acta Biomaterialiavol 7 no 2 pp 463ndash477 2011

[44] R Tasso F Fais D Reverberi F Tortelli and R CanceddaldquoThe recruitment of two consecutive and different waves ofhost stemprogenitor cells during the development of tissue-engineered bone in amurinemodelrdquo Biomaterials vol 31 no 8pp 2121ndash2129 2010

[45] G Muller and A Kramer ldquoBiocompatibility index of antisepticagents by parallel assessment of antimicrobial activity and cel-lular cytotoxicityrdquo The Journal of Antimicrobial Chemotherapyvol 61 no 6 pp 1281ndash1287 2008

[46] K Kolind KW Leong F Besenbacher andM Foss ldquoGuidanceof stem cell fate on 2D patterned surfacesrdquo Biomaterials vol 33no 28 pp 6626ndash6633 2012

[47] A Gao R Hang X Huang et al ldquoThe effects of titania nan-otubes with embedded silver oxide nanoparticles on bacteriaand osteoblastsrdquo Biomaterials vol 35 no 13 pp 4223ndash42352014

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


from the nanotube structure [5] The release rate is veryfast and short thus limiting the antimicrobial effects toearly-stage peri-implant infections Although polymers andphospholipids were directly coated on the top of drug-loadedTNTs to extend drug release TNTswere buried in the coatingcap hence the potential benefit of TNTs in promoting bonegrowth was diminished [18ndash20] Poly(lactic-co-glycolic acid)(PLGA) has been loaded into TNTs through solvent-castingtechnique to maintain their characteristic hollow structureand sustain drug release [21] However to the best of ourknowledge the biocompatibility of TNTs loaded with PLGAremains unknown Second bacteria are highly adaptable innature which leads to the evolution of strains resistant to con-ventional antibiotics [22] Agents working through unspecificmodes of action are required to overcome this resistancesuch as octenidine dihydrochloride (OCT) OCT approvedas a medicinal substance in several European countries is anestablished bispyridine antiseptic with a broad activity and iscommonly used as wound antiseptic [23 24] It has attractedincreasing attentions because of broad antibacterial spec-trum including antibiotic-resistant bacteria noncytotoxicityat suitable doses satisfactory stability and smaller possibilityto develop resistant bacteria [25 26]

In this study we investigated the possibility of usingcomposite PLGA-TNTs as a carrier for sustained OCTdelivery Despite active studies on using localized deliveryof antimicrobial agents to prevent implant infections therehave been few reports on using antiseptics [27] and noneon using nanotubes to deliver antiseptics In this paper forthe first time OCTPLGA was loaded into TNTS througha simple dip-coating process Sustained antibacterial abilityand biocompatibility were systematically investigated

2 Materials and Methods

21 Fabrication of TNTs on Ti TNTs were fabricated throughanodization on a Ti sheet Ti samples (Alfa-Aesar Ward HillMA USA 1 times 1 times 0025 cm3 998 purity) were degreasedby sonication in acetone and deionized water for 15minThe samples were then eroded for 10 s in 4wt HFndash5molLHNO

3 followed by rinsing with deionized water and drying

in air A two-electrode electrochemical cell using a platinumsheet as the counter-electrode was used Anodization wasperformed using a mixture of 050wtNH

4F + 10 volH

2O

in glycerol at 60V for 5 h After anodization the samples wererinsed with deionized water dried in air and annealed at450∘C for 2 h

22 OCT Loading Two types of samples were preparedThe first type was prepared by loading the mixture ofOCTPLGA into TNTs PLGA (Sigma-Aldrich St LouisMO USA lactide-to-glycolide ratio 65 35 24000Dandash38000Da) and OCT (TCI Shanghai China) were dissolvedin dichloromethane at 15mgmL and 05120583gmL respectivelyThe polymerdrug mixture was loaded into TNTs using adip-coating process at 40∘C for 2 d and then dried in airThe second type was produced by directly loading OCT intoTNTs OCT was dissolved in low-surface tension solvent(ethanol) and forced into TNTs using a vacuum-assisted

physical adsorptionmethod In brief 50120583L of 40120583gmLOCTsolutionwas pipetted onto the nanotube surfaces and allowedto dry under a vacuum desiccator at 20∘C for 1 hThe loadingprocess was repeated three times

23 SpecimenCharacterization Scanning electronmicroscopy(SEM) (JSM-7500F JEOL Tokyo Japan) was used tocharacterize the morphology of the prepared TNTs andOCTPLGA-TNTs The infiltration of PLGA polymer intoTNTs was also assessed by SEM according to the reportof Jia and kerr [21] Scotch tape was used to separate theOCTPLGA-TNTs from Ti foil The tape with OCTPLGA-TNTs then was soaked in 5 vol HF solution for 15min untilall TNTs were etched offThe remaining PLGAwas examinedthrough SEM

24 Contact Angle Determination A 1 120583L drop of distilledwater was delivered on the clean specimen surface with asyringe at a room temperature and in the open atmosphereof the lab Contact angles were measured on the obtainedphotographs (Phoenix 300 SEO Seoul Korea) The meanvalue was calculated from five separate measurements

25 Protein Adsorption Assay A 1mL droplet of DMEM-LGcontaining 10 FCS was pipetted onto each specimen Afterincubation at 37∘C for 2 h the proteins adsorbed onto thesamples were detached using 1 sodium dodecyl sulfate Theprotein concentrations were determined using a MicroBCAprotein assay kit (Pierce Rockford IL USA)

26 Release Profile of OCT OCT has been shown to bestable in chloroform at 40∘C [25] So in vitro OCT releasekinetics of the samples were measured using ultravioletvisible spectroscopy (UV-1750 Shimadzu Kyoto Japan) byrecording the absorption peak at 280 nm which is thecharacteristic excitation wavelength of OCT Two specimensTNTs loaded with OCT and TNTs loaded with OCTPLGAwere immersed in 1-mL of PBS in a glass vial while rotating(60 revolutions per minute) at 37∘C After 1 2 4 and 6 h aswell as after 1 2 3 6 9 12 15 and 18 d 500mL of the solutionwas sampled and fresh PBS was replenished Three sampleswere tested in each time interval and themean valuewas usedin data analysis

27 Bacteria Cultures Staphylococcus aureus (S aureus)(ATCC25923 American Type Culture Collection ManassasVA USA) was cultivated in the brain-heart infusion brothmedium at 37∘C for 12 h and then adjusted to a concentrationof 106 CFUmL The specimens were placed on 24-well cul-ture plates and separately incubated in 1mL of the bacteria-containing medium

271 SEM Observation After being incubated in 1mL ofthe bacteria-containing medium for 6 h the samples wererinsed with PBS fixed with 3 glutaraldehyde dehydrated ingraded ethanol series freeze-dried sputter-coated with thinplatinum layers and observed by SEM











3 Results




Huang
高亮


(a) (b)

(c) (d)



20

40

60

Wat

er co

ntac

t ang

le (d

eg)


lowastlowast

lowastlowast




10

20

30


lowast

Adso

rbed

pro

tein

(120583g

mL)










4 Discussions




0 40 80 120 160 200 240 280 320 360

16

24

32

Time (h)

Dru

g re

leas

e am

ount

(ppm

)


(a)

0 2 4 6

80

4840

60

80

Dru

g re

leas

e (

)

Time (h)


(b)


Ti

(a)

OCTPLGA-TNTs

(b)

TNTs

(c)

0 1 2 3 4 5 6

972

7 8

20

40

60

80

100

120

Ant

ibac

teria

l rat

e (

)


(d)





Ti

(a) (b) (c)

OCT

PLG

A-TN

Ts

(d) (e) (f)

TNTs

(g) (h) (i)




2 leading to a




0

100

200

300

400

Cel

l num

ber

scop

e


30 60 120

(min)

lowast

lowast

lowast

lowastlowast



TiOCTPLGA-TNTsTNTs


00

05

10

15

TiOCTPLGA-TNTsTNTs

lowastlowast

lowastlowast

lowastlowast

lowast lowast

1 3 7

(d)

Abso

rban

ce (4

50

nm)




1

2

3

4m

RNA

RU

NX2

GA

PDH lowastlowast

lowastlowast

(a)


1

2

3

4

mRN

A A

LPG

APD

H lowastlowast

lowastlowast

(b)


1

2

3

4

mRN

A O

CNG

APD

H

lowastlowast

lowast

(c)


2

4

6

8

10

mRN

A C

OL-

1G

APD

H

lowastlowast

lowastlowast

(d)




5 Conclusions





Acknowledgments



References






















2nan-



2nan-












































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials













3 Results




Huang
高亮


(a) (b)

(c) (d)



20

40

60

Wat

er co

ntac

t ang

le (d

eg)


lowastlowast

lowastlowast




10

20

30


lowast

Adso

rbed

pro

tein

(120583g

mL)










4 Discussions




0 40 80 120 160 200 240 280 320 360

16

24

32

Time (h)

Dru

g re

leas

e am

ount

(ppm

)


(a)

0 2 4 6

80

4840

60

80

Dru

g re

leas

e (

)

Time (h)


(b)


Ti

(a)

OCTPLGA-TNTs

(b)

TNTs

(c)

0 1 2 3 4 5 6

972

7 8

20

40

60

80

100

120

Ant

ibac

teria

l rat

e (

)


(d)





Ti

(a) (b) (c)

OCT

PLG

A-TN

Ts

(d) (e) (f)

TNTs

(g) (h) (i)




2 leading to a




0

100

200

300

400

Cel

l num

ber

scop

e


30 60 120

(min)

lowast

lowast

lowast

lowastlowast



TiOCTPLGA-TNTsTNTs


00

05

10

15

TiOCTPLGA-TNTsTNTs

lowastlowast

lowastlowast

lowastlowast

lowast lowast

1 3 7

(d)

Abso

rban

ce (4

50

nm)




1

2

3

4m

RNA

RU

NX2

GA

PDH lowastlowast

lowastlowast

(a)


1

2

3

4

mRN

A A

LPG

APD

H lowastlowast

lowastlowast

(b)


1

2

3

4

mRN

A O

CNG

APD

H

lowastlowast

lowast

(c)


2

4

6

8

10

mRN

A C

OL-

1G

APD

H

lowastlowast

lowastlowast

(d)




5 Conclusions





Acknowledgments



References






















2nan-



2nan-












































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b)

(c) (d)



20

40

60

Wat

er co

ntac

t ang

le (d

eg)


lowastlowast

lowastlowast




10

20

30


lowast

Adso

rbed

pro

tein

(120583g

mL)










4 Discussions




0 40 80 120 160 200 240 280 320 360

16

24

32

Time (h)

Dru

g re

leas

e am

ount

(ppm

)


(a)

0 2 4 6

80

4840

60

80

Dru

g re

leas

e (

)

Time (h)


(b)


Ti

(a)

OCTPLGA-TNTs

(b)

TNTs

(c)

0 1 2 3 4 5 6

972

7 8

20

40

60

80

100

120

Ant

ibac

teria

l rat

e (

)


(d)





Ti

(a) (b) (c)

OCT

PLG

A-TN

Ts

(d) (e) (f)

TNTs

(g) (h) (i)




2 leading to a




0

100

200

300

400

Cel

l num

ber

scop

e


30 60 120

(min)

lowast

lowast

lowast

lowastlowast



TiOCTPLGA-TNTsTNTs


00

05

10

15

TiOCTPLGA-TNTsTNTs

lowastlowast

lowastlowast

lowastlowast

lowast lowast

1 3 7

(d)

Abso

rban

ce (4

50

nm)




1

2

3

4m

RNA

RU

NX2

GA

PDH lowastlowast

lowastlowast

(a)


1

2

3

4

mRN

A A

LPG

APD

H lowastlowast

lowastlowast

(b)


1

2

3

4

mRN

A O

CNG

APD

H

lowastlowast

lowast

(c)


2

4

6

8

10

mRN

A C

OL-

1G

APD

H

lowastlowast

lowastlowast

(d)




5 Conclusions





Acknowledgments



References






















2nan-



2nan-












































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





10

20

30


lowast

Adso

rbed

pro

tein

(120583g

mL)










4 Discussions




0 40 80 120 160 200 240 280 320 360

16

24

32

Time (h)

Dru

g re

leas

e am

ount

(ppm

)


(a)

0 2 4 6

80

4840

60

80

Dru

g re

leas

e (

)

Time (h)


(b)


Ti

(a)

OCTPLGA-TNTs

(b)

TNTs

(c)

0 1 2 3 4 5 6

972

7 8

20

40

60

80

100

120

Ant

ibac

teria

l rat

e (

)


(d)





Ti

(a) (b) (c)

OCT

PLG

A-TN

Ts

(d) (e) (f)

TNTs

(g) (h) (i)




2 leading to a




0

100

200

300

400

Cel

l num

ber

scop

e


30 60 120

(min)

lowast

lowast

lowast

lowastlowast



TiOCTPLGA-TNTsTNTs


00

05

10

15

TiOCTPLGA-TNTsTNTs

lowastlowast

lowastlowast

lowastlowast

lowast lowast

1 3 7

(d)

Abso

rban

ce (4

50

nm)




1

2

3

4m

RNA

RU

NX2

GA

PDH lowastlowast

lowastlowast

(a)


1

2

3

4

mRN

A A

LPG

APD

H lowastlowast

lowastlowast

(b)


1

2

3

4

mRN

A O

CNG

APD

H

lowastlowast

lowast

(c)


2

4

6

8

10

mRN

A C

OL-

1G

APD

H

lowastlowast

lowastlowast

(d)




5 Conclusions





Acknowledgments



References






















2nan-



2nan-












































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0 40 80 120 160 200 240 280 320 360

16

24

32

Time (h)

Dru

g re

leas

e am

ount

(ppm

)


(a)

0 2 4 6

80

4840

60

80

Dru

g re

leas

e (

)

Time (h)


(b)


Ti

(a)

OCTPLGA-TNTs

(b)

TNTs

(c)

0 1 2 3 4 5 6

972

7 8

20

40

60

80

100

120

Ant

ibac

teria

l rat

e (

)


(d)





Ti

(a) (b) (c)

OCT

PLG

A-TN

Ts

(d) (e) (f)

TNTs

(g) (h) (i)




2 leading to a




0

100

200

300

400

Cel

l num

ber

scop

e


30 60 120

(min)

lowast

lowast

lowast

lowastlowast



TiOCTPLGA-TNTsTNTs


00

05

10

15

TiOCTPLGA-TNTsTNTs

lowastlowast

lowastlowast

lowastlowast

lowast lowast

1 3 7

(d)

Abso

rban

ce (4

50

nm)




1

2

3

4m

RNA

RU

NX2

GA

PDH lowastlowast

lowastlowast

(a)


1

2

3

4

mRN

A A

LPG

APD

H lowastlowast

lowastlowast

(b)


1

2

3

4

mRN

A O

CNG

APD

H

lowastlowast

lowast

(c)


2

4

6

8

10

mRN

A C

OL-

1G

APD

H

lowastlowast

lowastlowast

(d)




5 Conclusions





Acknowledgments



References






















2nan-



2nan-












































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Ti

(a) (b) (c)

OCT

PLG

A-TN

Ts

(d) (e) (f)

TNTs

(g) (h) (i)




2 leading to a




0

100

200

300

400

Cel

l num

ber

scop

e


30 60 120

(min)

lowast

lowast

lowast

lowastlowast



TiOCTPLGA-TNTsTNTs


00

05

10

15

TiOCTPLGA-TNTsTNTs

lowastlowast

lowastlowast

lowastlowast

lowast lowast

1 3 7

(d)

Abso

rban

ce (4

50

nm)




1

2

3

4m

RNA

RU

NX2

GA

PDH lowastlowast

lowastlowast

(a)


1

2

3

4

mRN

A A

LPG

APD

H lowastlowast

lowastlowast

(b)


1

2

3

4

mRN

A O

CNG

APD

H

lowastlowast

lowast

(c)


2

4

6

8

10

mRN

A C

OL-

1G

APD

H

lowastlowast

lowastlowast

(d)




5 Conclusions





Acknowledgments



References






















2nan-



2nan-












































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0

100

200

300

400

Cel

l num

ber

scop

e


30 60 120

(min)

lowast

lowast

lowast

lowastlowast



TiOCTPLGA-TNTsTNTs


00

05

10

15

TiOCTPLGA-TNTsTNTs

lowastlowast

lowastlowast

lowastlowast

lowast lowast

1 3 7

(d)

Abso

rban

ce (4

50

nm)




1

2

3

4m

RNA

RU

NX2

GA

PDH lowastlowast

lowastlowast

(a)


1

2

3

4

mRN

A A

LPG

APD

H lowastlowast

lowastlowast

(b)


1

2

3

4

mRN

A O

CNG

APD

H

lowastlowast

lowast

(c)


2

4

6

8

10

mRN

A C

OL-

1G

APD

H

lowastlowast

lowastlowast

(d)




5 Conclusions





Acknowledgments



References






















2nan-



2nan-












































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





1

2

3

4m

RNA

RU

NX2

GA

PDH lowastlowast

lowastlowast

(a)


1

2

3

4

mRN

A A

LPG

APD

H lowastlowast

lowastlowast

(b)


1

2

3

4

mRN

A O

CNG

APD

H

lowastlowast

lowast

(c)


2

4

6

8

10

mRN

A C

OL-

1G

APD

H

lowastlowast

lowastlowast

(d)




5 Conclusions





Acknowledgments



References






















2nan-



2nan-












































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








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CeramicsJournal of







Biomaterials




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CoatingsJournal of

Advances in








MaterialsJournal of


Nano

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CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Research Article Antibacterial Effects and ... · Titanium (Ti) implants with long-term...

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Transcript of Research Article Antibacterial Effects and ... · Titanium (Ti) implants with long-term...