Cp

WHa

b

c

Cd

a

ARRAA

KNPOOS

1

mgsoptasmsf[ps

1

h0

Colloids and Surfaces B: Biointerfaces 148 (2016) 354–362

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

jo ur nal ho me p ag e: www.elsev ier .com/ locate /co lsur fb

ommunication between nitric oxide synthase andositively-charged surface and bone formation promotion

ei Zhang a,∗, Jun Liu a, Haigang Shi a, Kun Yang a, Pingli Wang a, Gexia Wang a, Na Liu b,uaiyu Wang c, Junhui Ji a,∗, Paul K. Chu d

Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, ChinaStomatology Department of the General Hospital of Chinese PLA, 28 FuXing Road, Beijing 100853, ChinaInstitute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, Guangdong,hinaDepartment of Physics & Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China

r t i c l e i n f o

rticle history:eceived 21 March 2016eceived in revised form 30 July 2016ccepted 27 August 2016vailable online 29 August 2016

eywords:

a b s t r a c t

Despite the effects on physiology of bone marrow mesenchymal stem cells (BMSCs) and bone tissue,biological signal communication between bone implants and them is seldom employed as a guidance tocreate an osteo-inductive interface. Herein, the positively-charged surface is constructed on bone implantfrom the perspective of mediation of nitric oxide synthase (NOS) expression to signal BMSCs osteo-differentiation. In vitro and in vivo results indicate that the proper surface potential on the positively-charged surface affects NOS to express a high level of inducible nitric oxide synthase (iNOS) in three NOS

OSositively-charged surfacesteogenesisrthopedic implanturface potential

isoforms of the contacted BMSCs, upregulates their osteogenetic expression, and ultimately foster newbone growth. However, an excessively high surface potential produces substantial immunomodulatoryeffects thereby offsetting the aforementioned advantages. This study demonstrates that fine-tuning of thepositively-charged surface and proper utilization of the communication between NOS and bone implantspromote bone formation.

© 2016 Published by Elsevier B.V.

. Introduction

Artificial implant materials such as metals, ceramics, and poly-ers are widely used in dentistry and orthopedics. Although they

enerally have acceptable biocompatibility, the bio-inertness ofome of the materials results in insufficient signal differentiationf bone marrow mesenchymal stem cells (BMSCs) thereby com-romising stimulation of osteogenesis, bone ingrowth, and evenhe mechanical stability [1,2]. The biological interface betweenn orthopedic implant and host tissues can be improved by tis-ue regeneration and other means [3–6] and there has beenuch research on the design and fabrication of the suitable

urface or interface to provide the favorable microenvironmentor BMSCs/bone tissues [7]. In particular, functional proteins

8,9]/genes [10–12]/transition elements [13–17] have been incor-orated into the surface and in spite of some success in the earlytage, adverse host tissue responses including fibrous encapsula-

∗ Corresponding author at: 29 Zhongguancun East Road, Haidian District, Beijing00190, China.

E-mail addresses: weizhang@mail.ipc.ac.cn (W. Zhang), jhji@mail.ipc.ac.cn (J. Ji).

ttp://dx.doi.org/10.1016/j.colsurfb.2016.08.049927-7765/© 2016 Published by Elsevier B.V.

tion and chronic inflammation have been observed in the long term[1,15,18]. Although signalling molecules exert a dramatic effecton BMSCs osteo-differentiation and bone formation [19,20], thisstrategy has seldom been employed in the design and fabricationof a biologically favorable implant interface to mediate signallingmolecules [21–23]. In this work, the role of signalling species incontact with BMSCs and subsequent osteogenesis on material inter-face are investigated in order to achieve bone implant surface withosteo-inductivity.

It has been shown that nitric oxide (NO) synthesized by nitricoxide synthase (NOS) is a pivotal signalling molecule in bonemetabolism in animals [24,25]. Moreover, the physiological actionof NO depends on the location, source, and concentration [19,26]. Ithas also been discovered that electron transfer occurs from Flavinmononucleotide (FMN) to heme during NOS catalysed NO forma-tion [27,28]. In this regard, electron transfer would be affected toyield different expressions to the three NOS isoforms (iNOS, eNOSand nNOS) and to ultimately regulate BMSCs osteogenesis when

BMSCs are present on a positively-charged interface with a localbioelectric field. Hence, this work attempt to construct a biocom-patible positively-charged surface with different surface potentialon bone implant, and then understand and exploit the communica-

ces B:

tpm

2

2

Nm

2

ptn2(pfui(ahs

Fp

W. Zhang et al. / Colloids and Surfa

ion between NOS and positively-charged surface, finally achieve aositively-charged surface with ability to promote new bone for-ation [29].

. Experimental and methods

.1. Ethics statement

All procedures about animal were carried out according to theational Institutes of Health Intramural Animal Use and Care Com-ittee (IACUC-2012-047).

.2. Experimental details

Preparation of the charged surfaces with relatively high surfaceotential. Titanium disks 14 mm in diameter and 1.0 mm thick oritanium screws 2 mm long and 1.4 mm in diameter (Fuller Tech-ology Development Co, Ltd, Beijing, China) were placed on a4-well culture plate with 0.6 mL of freshly prepared dopamineDA) solution (2 mg/mL, pH = 8.5, 10 mM Tris-base buffer) witholyhexamethylene biguanidine (PHMB, 2 mg/mL and 20 mg/mL)

or 24 h in 25 ◦C [30–32]. Afterwards, the samples were treatedltrasonically in distilled water for 30 min three times and dried

n air. The sample without PHMB was designated as polydopamine

PDA) and those with 2 mg/mL and 20 mg/mL of PHMB were labeleds PCI-L and PCI-H, respectively. PCI-L and PCI-H are expected toave low and high surface potentials in order to study the differentignalling effects.

ig. 1. Physicochemical characteristics of positively-charged interfaces. (a) and (b) deconotential and their positive proportion with PHMB content obtained by XPS chemical com

Biointerfaces 148 (2016) 354–362 355

2.3. Surface physicochemical characterization

The XPS (X-ray photoelectron spectroscopy) was employed todetermine the elemental chemical composition and chemical state.The XPS machine (PHI QUANTERA-II) adopted a monochromaticsource of Al K-alpha. Any possible shift of the XPS peak causedby the charging effect was calibrated by referencing it to the C1speak at 284.8 eV. The analyzer was operated at constant pass energy(wide scans: 280 eV, resolution: 1.00 eV; fine scans: 26 eV, resolu-tion: 0.025 eV), and placed at 45◦ take-off angle. The Multi-peakfitting were performed by the Multipak software. Solid-state 1H-NMR (AVANCE III 400 MHz WB solid-state NMR spectrometer,Bruker, Switzerland) was conducted on PHMB, PDA, PCI-L and PCI-Hpowders, which were obtained from a 20% PHMB solution by freezedrying and scratched from the corresponding samples, respec-tively. Atomic force microscopy images (AFM, Multimode 8, Bruker,USA) were employed to examine the surface topography of Tita-nium, PDA, PCI-L, and PCI-H from a scanned area of 2 �m × 2 �min the tapping mode on a RTESPA (Bruker) probe. Kelvin probeforce microscopy-amplitude modulation (KPFM-AM, Multimode 8,Buker, USA) was used to measure the relative surface potentialsusing the tapping mode on a Multi75E-G (budget sensors) probeby the Nanoscope Analysis Software Version 1.40, and producecorresponding images from a scanned area of 2 �m × 2 �m. Thesurface hydrophilicity was determined by contact angle measure-

ments (CA, Q30, USA) based on the static sessile drop method withdouble distilled water at 25 ◦C. Each contact angle represents theaverage of 3 measurements.

voluted C 1 s and N 1s XPS spectra, respectively. (c) Solid state 1H NMR. (d) Surfaceposition (right bottom).

356 W. Zhang et al. / Colloids and Surfaces B: Biointerfaces 148 (2016) 354–362

F urfaceh

2

STflDm5p

2o

cffctbp

2

thecsaAdct

ig. 2. Surface roughness and hydrophilicity of titanium, PDA, PCI-L and PCI-H: (a) Save similar rough and hydrophilic surfaces.

.4. Culture of BMSCs

The BMSCs of the two-week-old SD rats were obtained from thetomatology Department of the General Hospital of Chinese PLA.he cells were cultured in basal growth media in a 75 cm2 cultureask (Corning, Lowell, MA, USA). The growth medium containedMEM (containing 10% FBS), penicillin (100 U/mL), and strepto-ycin (GIBCO, 100 �g/mL) at 37 ◦C in a humidified atmosphere of

% CO2. The cells were subcultured every 3 days and only cells ofassages 3-5 were used in the experiments.

.5. Cell staining and laser scanning confocal microscopy (LSCM)bservation

The 5 × 103 cells/well BMSCs were seeded on the samples andultured in the basic medium (DMEM) supplemented with 10% FBSor 3 days. Afterwards, the cells were fixed in 4% paraformaldehydeor 30 min, stained with 10 mg/mL Phalloidin-TRITC (Sigma), andounter-stained with 5 mg/mL Hoechst 33342 (Sigma) to identifyhe nuclei. Image collection and superimposition were processedy A1R MP (Nikon Corporation, Japan). Each experiment was inde-endently performed at least three times.

.6. Cell apoptosis analysis

The BMSCs were cultured on four groups (tissue culture plas-ic (Blank), PDA, PCI-L and PCI-H). Four groups of BMSCs werearvested at day 3 and cell apoptosis was measured by flow cytom-try using Annexin V-FITC/PI kit according to the protocol. Theells were spun for 4 min and the supernatant was removed, re-uspended in binding buffer (400 �L) and of Annexin V-FITC (5 �L)nd was incubated in the dark for 15 min at room temperature.

dditional 10 �L of PI were added to each sample and remained inarkness at 0 ◦C for 5 min. Ten thousand cells were acquired by Flowell sorter (FACSAria-III, BD, USA) and all the assays were repeatedhree times.

roughness (AFM scan area is 2 �m × 2 �m) and (b) hydrophilicity, PCI-L and PCI-H

2.7. QRT-PCR analysis

The BMSCs (2 × 104 cells/well) were cultured on Blank, PDA,PCI-L and PCI-H samples for 3 days. Total RNA from the cellswas extracted by Trizol following the manufacturer’s instructions(Invitrogen). Approximately 2-5 hundred nano-grams of total RNAfrom each sample was reverse-transcribed into cDNA using theSuper Script First Strand Synthesis kit (Invitrogen). The qRT-PCRwas performed with SYBR green PCR Master Mix kit (Toyobo,Osaka, Japan)using a 7500 real-time PCR system (Applied Biosys-tems) according to the protocol of the manufacturer. The followingprimers were used:

ALP: (F) 5′-ACAGTGACAGCTGCCCGCAT-3′,(R) 5′-TTGCATCGCGTGCGCTCAGT-3′;RUNX-2: (F) 5′-AGGGCGCATTCCTCATCCCAGT-3′,(R) 5′-AAGACAGCGGCGTGGTGGAA-3′;OCN: (F) 5′-TGGCACCACCGTTTAGGGCA-3′,(R) 5′-TTTGGAGCAGCTGTGCCGTC-3′;BSP: (F) 5′-AGACCATGCAGAGAGCGAG-3′,(R) 5′-ACGTCTGCTTGTGTGCTGG-3′;TNF-�: (F) 5’- ACCTGGCCTCTCTACCTTGT-3’,(R) 5’- GACCCGTAGGGCGATTACAG-3’;nNOS: F: 5’-TGAGGTTCTCAGTGTTCGGC-3’,R: 5’- ATCCTCTCCCCTCCCAGTTC-3’eNOS: F: 5’- CAAAAGGCACAGGCATCACC-3’,R: 5’- AAGGCCTCATGCTCTAGGGA-3’;iNOS: F: 5’- ACGGAAGAGACGCACAGGCA-3’,R: 5’- AAGGCAGCAGGCACACGCAA-3’;GAPDH: (F) 5′-GGCACAGTCAAGGCTGAGAATG-3′,(R) 5′-ATGGTGGTGAAGACGCCAGTA-3.

2.8. NOS inhibitor treatment

In T25 culture flasks, the cells (5 × 103 cells/cm2) were incubatedwith fresh medium. After 24 h, the eNOS inhibitor (L-NAME, 50 uM),

nNOS inhibitor (Sper, 0.5 mM) and iNOS inhibitor (L-Can, 1 mM)were added into the cell culture medium. The inhibitors were pur-chased from Biyuntian (China). On day 3, the cells were harvestedand subjected to quantitative real time PCR analysis.

W. Zhang et al. / Colloids and Surfaces B: Biointerfaces 148 (2016) 354–362 357

F d BMS( fter 3

c

2

ld(Tb1pTssfra1u1T(r

ig. 3. Excellent cytocompatibility of positively-charged interfaces to the contactescale bar is 100 �m). (b) Flow cytometry (FCM) analysis of cell apoptosis of BMSCs aells are analysed.

.9. In vivo evaluation of osteoinductivity

A tibias defect model was made using 6-week-old Sprague Daw-ey (SD) rats to assess new bone formation. These SD rats wereivided into three groups (n = 12 per group): (1) Screw (titanium),2) PCI-L-coated screw (PCI-L), and (3) PCI-H-coated screw (PCI-H).hey were anesthetized by intraperitoneal injection of 3% pento-arbital sodium (45 mg/kg; Sigma). One hole with a diameter of.4 mm was drilled in the tibia and three groups of screws werelaced until the implant shoulder was level with the bone surface.he incisions were closed in layers and the fascia and skin wereutured separately using silk sutures. After 7 days, the tibias withamples are isolated and pictured (n = 6 per group). The total RNArom tissues surrounding the screws was isolated using the Trizoleagent (Invitrogen) and the osteogenic markers expressions weressayed by real time PCR according to a standard protocol. After4 and 28 days, the new bone surrounding the screws was eval-ated by radiographic analysis on a Micro-CT scanner (SkyScan

072 scanner skycanbvba aart slear Simens) (n = 6 per group).he scanned CT images were taken at a resolution of 10.3 �machieved using 70 kV and 400 mA) and three-dimensionally (3D)econstructed with computer software (SkyScan, Simens). The

Cs. (a) Laser scanning confocal microscopy (LSCM) of BMSCs at 3-day incubation,day incubation, the figures show the cell state for triplicate experiments and 10,000

regenerated bone volume on the screws were calculated from the3D reconstructed images using CTAn software (SkyScan). 1 mmbelow the center of the epiphyseal line with a total of 50 sliceswas chosen as the region of interest (ROI) for the analysis and com-parison of trabeculae paramenters. The trabeculae and the bonemarrow was separated by the threshold function, labeled in blueor green color. After that, bone volume in the ROI were computedrespectively by the workstation [33].

2.10. Statistical analyses

The study used SPSS version 18.0 (Chicago, IL, USA) softwarefor statistical analysis. All experiments were performed in tripli-cate and repeated three times. The results were reported as themean ± SD, and the differences observed between samples wereconsidered to be significant at p < 0.05.

3. Results and discussion

To better understand the structure-property relationship ofthe positively-charged surfaces, the chemical and physical struc-tures are characterized by X-ray photoelectron spectroscopy (XPS),

358 W. Zhang et al. / Colloids and Surfaces B: Biointerfaces 148 (2016) 354–362

Fig. 4. High surface potential significantly regulates BMSCs to express variant NOS isoforms, signal their osteodifferentiation, but one with exceeding high surface potentialp �, andf lized

( pectiv

si(XmaN(tispiopatcpbpptrhoae

mstlFs

roduces pre-inflammatory stimuli. Expressions of (a) three NOS isoforms, (b) TNF-or 3 days are measured by real time PCR, relative to GAPDH expression and norma**) denote two statistical significance (p < 0.05 and p < 0.01) compared to Blank, res

olid-state 1H NMR, and atomic force microscopy (AFM). PHMBncorporation into PDA is confirmed by the two peaks at 287.4 eV-NC*( N)N-) and 285.0 eV (-C*H2−) in the deconvoluted C 1sPS spectra (Fig. 1a). Elemental analysis reveals that PCI-H hasore PHMB than PCI-L and the weight percentages are 15.4%

nd 7.4%, respectively. PHMB incorporation also makes the (-C*( N)N ) peak of PHMB shift from 287.98 eV to 287.40 eV andC*( O)-) to the benzene ring of polydopamine from 287.65 eV

o 288.50 eV. The opposite chemical shifts indicate that bond-ng varies with the carbon chemical environment and there aretrong interactions between the cationic PHMB and conjugatedolydopamine. The strong interaction between the electrophilic N

n PHMB and electron-donating azaheterocyclic and benzene ringf polydopamine is confirmed by the chemical shift in the N 1seak (Fig. 1b) to the middle site and the two peaks at 1.169 ppmnd 5.247 ppm in the solid 1H NMR spectra (Fig. 1c) demonstratehat the strong interaction arises from cation-� [34]. The strongation-� interaction helps to fix PHMB on the PDA surface andrevent the long chain of PHMB from disturbing the cell mem-rane andcausing cytotoxicity[35]. As expected, PCI-L and PCI-Hossess the increased surface potential (Fig. 1d), and the surfaceotential is proportional to the PHMB content (Fig. 1d, right bot-om). In addition, Fig. 2 shows that PCI-L and PCI-H have similarough and hydrophilic surfaces, and so their surface roughness andydrophilicity have similar effect on osteogenic maker expressionf BMSCs located at low and high potential surface. Therefore PCI-Lnd PCI-H can be used to elucidate and differentiate the signallingffects of different surface potentials on the BMSCs.

The LSCM images (Fig. 3a) have little difference in the attach-ent, spreading, and proliferation of BMSCs on the different

amples (Blank, PDA, PCI-L and PCI-H) and particularly compared

o Blank, PCI-L and PCI-H do not show adverse effects on the cel-ular state of the BMSCs (Fig. 3b and Supplementary informationig. S1) and even exhibit some effects in preventing cell apopto-is thereby demonstrating good cytocompatibility. Real-time PCR

(c) osteogenesis-related marker (ALP, Runx-2 and OCN) genes of BMSCs incubatedto the expressions on Blank (cells in culture medium without any sample). (*) andely.

analysis is performed to determine whether the three NOS iso-forms (eNOS, nNOS and iNOS) are affected when BMSCs are seededon the positively-charged surfaces. A larger surface potential (PCI-L) is observed to attenuate the eNOS and nNOS surface potentialcan signal the BMSCs to display different NOS isoform expressionlevels expressions but increases that of iNOS in BMSCs (Fig. 4a).Interestingly, there is low TNF-� expression (Fig. 4b, PCI-L) and lit-tle pre-inflammatory stimuli to BMSCs suggesting that the largersurface potential can signal the BMSCs to display different NOS iso-form expression levels (eNOS, nNOS and iNOS) possibly stemmedfrom affecting the electron transfer from FMN to heme during NOScatalysed NO formation [27]. Consequently, the BMSCs show a highosteogenesis-related marker expression (Fig. 4c, PCI-L). The NOsource variation affects signalling to BMSCs osteogenesis [36] andit is partially excessively high surface potential (PCI-H) increasesthe nNOS expression besides iNOS confirmed by activated NO sig-nalling in osteoblasts exposed to the electric field and the higherNO level stimulated by the electric field than the control [37,38].However, an excessively high surface potential (Fig. 4a, PCI-H) lead-ing to high pre-inflammatory stimuli to the BMSCs (Fig. 4b, PCI-H).Some studies have shown that inflammatory stimuli to BMSCs giverise to local bone loss (or peri-implant osteolysis) [1,18] and so avery large surface potential is not desirable for BMSCs osteogenesisand osteoinductive efficiency. Nevertheless, Fig. 4c shows that theBMSCs have a high osteogenic expression on PCI-H, similar to PCI-Lattributable to the interim response to the stimulus cues.

The effects of the three NOS isoforms on the osteogeneticexpression in the absence and presence of NOS inhibition are inves-tigated (Figs. 5 and 6) in order to elucidate the NOS signallingmechanism in the bioelectric microenvironment [39]. Fig. 5a (PCI-L) shows that although it increases expression levels of eNOS and

nNOS (Fig. 6a), eNOS inhibition (L-NAME) significantly attenuatesearly osteogenic (ALP and Runx-2) expressions in the BMSCs butincreases the expression of late osteogenic marker (OCN). Thismay be because NOS inhibition stimulates the cNOS expression

W. Zhang et al. / Colloids and Surfaces B: Biointerfaces 148 (2016) 354–362 359

Fig. 5. The affected NOS genes exhibit high iNOS expression level, and upregulate osteogenic expression on positively-charged interface. Osteogenic markers and inflammatorye ell cula hibitot ple w

sicsTwbtTpt(Ooetr

xpressions of BMSCs cultured in the presence and absence of NOS inhibitors in the cnd normalized to the expressions on Blank without the NOS inhibitor. (a) eNOS inwo statistical significance (p < 0.05 and p < 0.01), respectively, compared to the sam

ince the eNOS and nNOS isoforms are constitutively expressedn BMSCs [40]. When the nNOS inhibitor (Sper) is added to theell culture medium instead of L-NAME, the osteogenetic expres-ions and NOS isoforms are suppressed (Figs. 5b and 6b, PCI-L).hese results are in accordance with those obtained from miceith global deletion of eNOS and nNOS, indicating the increased

one mass because of a reduced bone turnover rate and a pheno-ype in mice suffered from deletion of all three NOS genes [24].he same change indicates that eNOS and nNOS are not the signalathways for the positively-charged interface concerning regula-ion of the BMSCs osteogenic expression. In fact, iNOS inhibitionL-Can) reduces the mRNA expression levels of ALP, Runx-2, andCN in the BMSCs cultured on the blank group but has little effects

n the expressions of the BMSCs/PCI-L group (Fig. 5c) althoughxpression levels (Fig. 6c PCI-L) of eNOS and nNOS are increased athis time. Therefore, the increased surface potential plays an activeole in upregulating BMSCs osteogenesis via the iNOS signalling

ture medium for 3 days are measured by real time PCR relative to GAPDH expressionr: L-NAME. (b) nNOS inhibitor: Sper. (c) iNOS inhibitor: L-Can. (*) and (**) denoteithout NOS inhibitors.

pathway. It has been shown the iNOS derived NO can stimulate frac-ture healing and recovery of bone [24]. Evaluating the role of iNOSspecifically in fracture healing through targeted gene deletion alsoillustrates that femurs from iNOS deficient mice have decreasedtotal and maximum energy absorption following fracture [41], thusunambiguously demonstrating that the increased surface poten-tial upregulates BMSCs osteo-differentiation via the iNOS signallingpathway. However, an excessively high surface potential (PCI-H)can inhibit all the osteogenic expressions (ALP, Runx-2, and OCN)in BMSCs cells co-cultured with L-NAME or Sper inhibitor (Fig. 5aand b, PCI-H), while it only inhibits some of osteogenic expres-sions (Fig. 5c, PCI-H) in the presence of L-Can inhibitor because itstimulates high three NOS isoform expression (Fig. 6c PCI-H). Evi-

dently, regulation of the osteogenic markers expression may beattributed to the pre-inflammatory stimuli from the excessivelyhigh surface potential. All in all, the surface potential is an impor-

360 W. Zhang et al. / Colloids and Surfaces B: Biointerfaces 148 (2016) 354–362

Fig. 6. Three NOS isoform expressions in BMSCs in contact with the positively-charged surfaces in the absence and presence of (a) eNOS, (b) nNOS, or (c) iNOS inhibitors incell culture. (**) denotes statistical significance (p < 0.01), compared to the sample without NOS inhibitors.

Fig. 7. Appropriate surface potential can upregulate osteogenic marker expression in vivo. (a) Representative images of tibias with samples after 1-week post-implantation.(scale bar is 5 mm) (b) Osteogenic expression of bone tissues surrounding samples after 1-week postimplantation, measured by real time PCR relative to GAPDH expressionand normalized to the expressions on Blank. (*) and (**) denote two statistical significance (p < 0.05 and p < 0.01) compared to blank, respectively. (##) denotes statisticalsignificance (p < 0.01) compared to PCI-H.

W. Zhang et al. / Colloids and Surfaces B: Biointerfaces 148 (2016) 354–362 361

Fig. 8. Appropriate surface potential can promote bone formation in vivo. (a) and (b) micro-CT images of tibias with samples from three direction cross-section after 2 and 4-w exhibi

tt

paenPFttsotermopeotseimBbiagO

eek postimplantation, respectively, (scale bar is 2.5 mm). The data in these imagesmages.

ant factor in upregulation of the BMSCs’ osteogenic expression viahe iNOS signalling pathway.

In order to confirm the osteoinductive effects of the surfaceotential in vivo, titanium screws with/without PCI-L and PCI-Hre implanted into the tibias of SD rats. After 1 week, little fibroticncapsulation is formed on both PCI-L and PCI-H (Fig. 7a). Osteoge-esis analysis of the surrounding tissues reveals that both PCI-L andCI-H upregulate the osteogenic expressions significantly (Fig. 7b).urthermore, PCI-H exhibits a lower osteogenic expression levelhan PCI-L thus proving that the surface potential is crucial tohe osteoinductivity in vivo. After 2 and 4 weeks, the new boneurrounding the screws was evaluated by radiographic analysisn a Micro-CT scanner. The scanned CT images were taken andhree-dimensionally (3D) reconstructed (Fig. 8a and b). The regen-rated bone volume on the screws were calculated from the 3Deconstructed images. The bone volume in Fig. 8a and b reveal

ore new bone tissues around PCI-L and PCI-H. Similar to thesteogenic analysis results (Fig. 7b), an excessively high surfaceotential attenuates new bone growth due to immunomodulatoryffects. It has been shown slow progressive pathological bone lossr aseptic loosening is a potentially life-threatening condition dueo serious complications [1]. Our in vitro and in vivo results demon-trate an appropriate surface potential solves this problem due tofficient communication between the NOS and positively-chargednterface [1]. External electromagnetotherapy is one of the treat-

ents to promote bone tissue regeneration and repair [37,42] andMSCs under the effects of an external electromagnetic field haveeen shown to differentiate to osteoblasts [43]. Similarly, the pos-

tively charged surface here provides an internal bioelectric fieldffecting electron transfer and ultimately regulating BMSCs osteo-

enesis while offering low risks on osteomyelitis development [1].ur study provides clear evidence that properly positively-charged

it the regenerated bone volume on the screws calculated from the 3D reconstructed

surface promotes bone formation by mediating the direct commu-nication with NOS [29].

4. Conclusions

In summary, a positively-charged surface with the proper sur-face potential and excellent cytocompatibility is produced bybonding polydopamine (PDA) to PHMB. In vitro and in vivo resultsindicate that the optimal surface potential on the positively-charged interface affects NOS to express a high level of iNOSin three NOS isoforms of the contacted BMSCs, upregulatestheir osteogenetic expression, and ultimately foster new bonegrowth. However, an excessively high surface potential producessubstantial immunomodulatory effects thereby offsetting theaforementioned advantages. Hence, fine-tuning of the positively-charged surface and proper utilization of the communicationbetween NOS and bone implants can promote bone formation.

Acknowledgments

Financial support from the National Natural Science Foun-dation of China (NSFC 51473175), National key research anddevelopment program (2016YFC1000900) and Youth InnovationPromotion Association CAS, City University of Hong Kong StrategicResearch Grant (SRG) No. 7004188, and Hong Kong Research GrantsCouncil (RGC) General Research Funds (GRF) Nos. CityU 112212 andCityU 11301215.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.08.049.

3 ces B:

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

62 W. Zhang et al. / Colloids and Surfa

eferences

[1] S. Landgraeber, M. Jager, J.J. Jacobs, N.J. Hallab, The pathology of orthopedicimplant failure is mediated by innate immune system cytokines, Mediat.Inflamm. 2014 (2014) 185150.

[2] R.P. Robinson, T.M. Green, Eleven-year implant survival rates of theall-polyethylene and metal-backed modular optetrak posterior stabilizedknee in bilateral simultaneous cases, J. Arthroplasty 26 (2011) 1165–1169.

[3] E. Kaivosoja, G. Barreto, K. Levon, S. Virtanen, M. Ainola, Y.T. Konttinen,Chemical and physical properties of regenerative medicine materialscontrolling stem cell fate, Ann. Med. 44 (2012) 635–650.

[4] P. Rompolas, K.R. Mesa, V. Greco, Spatial organization within a niche as adeterminant of stem-cell fate, Nature 502 (2013) 513–519.

[5] M.J. Dalby, N. Gadegaard, R.O.C. Oreffo, Harnessing nanotopography andintegrin-matrix interactions to influence stem cell fate, Nat. Mater. 13 (2014)558–569.

[6] F.R. Maia, S.J. Bidarra, P.L. Granja, C.C. Barrias, Functionalization ofbiomaterials with small osteoinductive moieties, Acta Biomater. 9 (2013)8773–8789.

[7] M.M. Stevens, J.H. George, Exploring and engineering the cell surfaceinterface, Science 310 (2005) 1135–1138.

[8] S.S.S. Lee, E.L. Hsu, M. Mendoza, J. Ghodasra, M.S. Nickoli, A. Ashtekar, M.Polavarapu, J. Babu, R.M. Riaz, J.D. Nicolas, D. Nelson, S.Z. Hashmi, S.R. Kaltz,J.S. Earhart, B.R. Merk, J.S. McKee, S.F. Bairstow, R.N. Shah, W.K. Hsu, S.I. Stupp,Gel scaffolds of BMP-2-binding peptide amphiphile nanofibers for spinalarthrodesis, Adv. Healthc. Mater. 4 (2015) 11.

[9] S. Quideau, C. Douat-Casassus, D.M.D. Lopez, C. Di Primo, S. Chassaing, R.Jacquet, F. Saltel, E. Genot, Binding of filamentous actin and winding intofibrillar aggregates by the polyphenolic C-glucosidic ellagitannin vescalagin,Angew. Chem. Int. Ed. 50 (2011) 5099–5104.

10] H. Shen, J. Tan, W.M. Saltzman, Surface-mediated gene transfer fromnanocomposites of controlled texture, Nat. Mater. 3 (2004) 569–574.

11] Y. Yazaki, A. Oyane, H. Tsurushima, H. Araki, Y. Sogo, A. Ito, A. Yamazaki,Improved gene transfer efficiency of a DNA-lipid-apatite composite layer bycontrolling the layer molecular composition, Colloid Surf. B 122 (2014)465–471.

12] S.B. Goodman, Z. Yao, M. Keeney, F. Yang, The future of biologic coatings fororthopaedic implants, Biomaterials 34 (2013) 3174–3183.

13] Z. Chen, C. Wu, W. Gu, T. Klein, R. Crawford, Y. Xiao, Osteogenic differentiationof bone marrow MSCs by beta-tricalcium phosphate stimulating macrophagesvia BMP2 signalling pathway, Biomaterials 35 (2014) 1507–1518.

14] D.S. Brauer, Bioactive glasses-structure and properties, Angew. Chem. Int. Ed.54 (2015) 4160–4181.

15] E.A. Lewallen, S.M. Riester, C.A. Bonin, H.M. Kremers, A. Dudakovic, S. Kakar,R.C. Cohen, J.J. Westendorf, D.G. Lewallen, A.J. van Wijnen, Biologicalstrategies for improved osseointegration and osteoinduction of porous metalorthopedic implants, Tissue Eng. B: Rev. 21 (2015) 218–230.

16] Z. Tang, Y. Xie, F. Yang, Y. Huang, C. Wang, K. Dai, X. Zheng, X. Zhang, Poroustantalum coatings prepared by vacuum plasma spraying enhance bmscsosteogenic differentiation and bone regeneration in vitro and in vivo, PLoSOne 8 (2013) e66263.

17] L. Zhao, H. Wang, K. Huo, X. Zhang, W. Wang, Y. Zhang, Z. Wu, P.K. Chu, Theosteogenic activity of strontium loaded titania nanotube arrays on titaniumsubstrates, Biomaterials 34 (2013) 19–29.

18] S. Franz, S. Rammelt, D. Scharnweber, J.C. Simon, Immune responses toimplants—a review of the implications for the design of immunomodulatorybiomaterials, Biomaterials 32 (2011) 6692–6709.

19] S.P. Nichols, W.L. Storm, A. Koh, M.H. Schoenfisch, Local delivery of nitricoxide: targeted delivery of therapeutics to bone and connective tissues, Adv.Drug Deliv. Rev. 64 (2012) 1177–1188.

20] B. Wang, J. Song, H. Yuan, C. Nie, F. Lv, L. Liu, S. Wang, Multicellular assemblyand light-regulation of cell–cell communication by conjugated polymermaterials, Adv. Mater. 26 (2014) 2371–2375.

21] M.P. Lutolf, P.M. Gilbert, H.M. Blau, Designing materials to direct stem-cellfate, Nature 462 (2009) 433–441.

[

[

Biointerfaces 148 (2016) 354–362

22] W. Wang, B. Li, C.Y. Gao, Modulating the differentiation of BMSCs by surfaceproperties of biomaterials, Prog. Chem. 23 (2011) 2160–2168.

23] N. Huebsch, D.J. Mooney, Inspiration and application in the evolution ofbiomaterials, Nature 462 (2009) 426–432.

24] K. Sabanai, M. Tsutsui, A. Sakai, H. Hirasawa, S. Tanaka, E. Nakamura, A.Tanimoto, Y. Sasaguri, M. Ito, H. Shimokawa, Genetic disruption of all NOsynthase isoforms enhances BMD and bone turnover in mice in vivo:involvement of the renin-angiotensin system, J. Bone Miner. Res. 23 (2008)633–643.

25] C.C. Teixeira, H. Ischiropoulos, P.S. Leboy, S.L. Adams, I.M. Shapiro, Nitricoxide-nitric oxide synthase regulates key maturational events duringchondrocyte terminal differentiation, Bone 37 (2005) 37–45.

26] T. Lahiri, B. Luan, D.P. Raleigh, E.M. Boon, A structural basis for the regulationof an H-NOX-associated cyclic-di-GMP synthase/phosphodiesterase enzymeby nitric oxide-bound H-NOX, Biochemistry 53 (2014) 2126–2135.

27] B.C. Smith, E.S. Underbakke, D.W. Kulp, W.R. Schief, M.A. Marletta, Nitricoxide synthase domain interfaces regulate electron transfer and calmodulinactivation, P. Natl. Acad. Sci. 110 (2013) E3577–E3586.

28] J. Tejero, L. Hannibal, A. Mustovich, D.J. Stuehr, Surface charges and regulationof FMN to heme electron transfer in nitric-oxide synthase, J. Biol. Chem. 285(2010) 27232–27240.

29] G. Lanzani, Materials for bioelectronics: organic electronics meets biology,Nat. Mater. 13 (2014) 775–776.

30] X. Yu, H.L. Fan, L. Wang, Z.X. Jin, Formation of polydopamine nanofibers withthe aid of folic acid, Angew. Chem. Int. Ed. 53 (2014) 12600–12604.

31] H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Mussel-inspired surfacechemistry for multifunctional coatings, Science 318 (2007) 426–430.

32] L. Shi, W. Zhang, K. Yang, H. Shi, D. Li, J. Liu, J. Ji, P.K. Chu, Antibacterial andosteoinductive capability of orthopedic materials via cation?� interactionmediated positive charge, J. Mater. Chem. B 3 (2015) 733–737.

33] Y. Dong, Y. Li, C. Huang, K. Gao, X. Weng, Systemic application of teriparatidefor steroid induced osteonecrosis in a rat model, BMC Musculoskel Dis. 16(2015) 163–171.

34] P. Kadlubanski, K. Calderon-Mojica, W.A. Rodriguez, D. Majumdar, S. Roszak, J.Leszczynski, Role of the multipolar electrostatic interaction energycomponents in strong and weak cation-pi interactions, J. Phys. Chem. A 117(2013) 7989–8000.

35] S. Mukherjee, H. Zheng, M.G. Derebe, K.M. Callenberg, C.L. Partch, D. Rollins,D.C. Propheter, J. Rizo, M. Grabe, Q.X. Jiang, L.V. Hooper, Antibacterialmembrane attack by a pore-forming intestinal C-type lectin, Nature 505(2014) 103–107.

36] J.W. Fuseler, M.T. Valarmathi, Modulation of the migration and differentiationpotential of adult bone marrow stromal stem cells by nitric oxide,Biomaterials 33 (2012) 1032–1043.

37] G. Cheng, Y. Zhai, K. Chen, J. Zhou, G. Han, R. Zhu, L. Ming, P. Song, J. Wang,Sinusoidal electromagnetic field stimulates rat osteoblast differentiation andmaturation via activation of NO–cGMP–PKG pathway, Nitric Oxide 25 (2011)316–325.

38] J.H. Jansen, O.P. van der Jagt, B.J. Punt, J.A. Verhaar, J.P. van Leeuwen, H.Weinans, H. Jahr, Stimulation of osteogenic differentiation in humanosteoprogenitor cells by pulsed electromagnetic fields: an in vitro study, BMCMusculoskel. Dis 11 (2010) 188.

39] B.L. Oliveira, I.S. Moreira, P.A. Fernandes, M.J. Ramos, I. Santos, J.D. Correia,Insights into the structural determinants for selective inhibition of nitricoxide synthase isoforms, J. Mol. Model. 19 (2013) 1537–1551.

40] N.M. Ocarino, J.N. Boeloni, A.M. Goes, J.F. Silva, U. Marubayashi, R. Serakides,Osteogenic differentiation of mesenchymal stem cells from osteopenic ratssubjected to physical activity with and without nitric oxide synthaseinhibition, Nitric Oxide 19 (2008) 320–325.

41] Y. Baldik, A.D. Diwan, R.C. Appleyard, Z. Ming Fang, Y. Wang, G.A. Murrell,Deletion of iNOS gene impairs mouse fracture healing, Bone 37 (2005) 32–36.

42] J.L. Miller, Bioelectric signalling controls tissue shape and structure, Phys.Today 66 (2013) 16.

43] K. Lim, J. Hexiu, J. Kim, H. Seonwoo, W.J. Cho, P.-H. Choung, J.H. Chung, Effectsof electromagnetic fields on osteogenesis of human alveolar bone-derivedmesenchymal stem cells, BioMed Res. Int. 2013 (2013) 1–14.

Supplementary Information

Fig. S1. Four cell state for triplicate experiments, (a) dead cell, (b) late apoptosis cell, (c)

live cell and (d) early apoptosis cell. Flow cytometry (FCM) analysis of cell

apoptosis of BMSCs was done after 3 day incubation.

The BMSCs were cultured on four groups (tissue culture plastic (Blank), PDA,

PCI-L and PCI-H). Four groups of BMSCs were harvested at day 3 and cell

apoptosis was measured by flow cytometry using Annexin V-FITC/PI kit according to

the protocol. The cells were spun for 4 min and the supernatant was removed,

re-suspended in binding buffer (400 μL) and of Annexin V-FITC (5 μL) and was

incubated in the dark for 15 min at room temperature. Additional 10 μL of PI were

added to each sample and remained in darkness at 0 °C for 5 min. Ten thousand

cells were acquired by Flow cell sorter (FACSAria-III, BD, USA) and all the assays

were repeated three times.

From Fig. S1, there is similar live cell percentage on Blank, PDA, PCI-L and

PCI-H, and no statistical difference for four state of BMSCs. Therefore, it

demonstrates PCI-L and PCI-H exhibit good cytocompatibility.