Bacterial Inhibition and Osteoblast Adhesion on Ti Alloy Surfaces

Subscriber access provided by Kaohsiung Medical University

Biological and Medical Applications of Materials and Interfaces

Bacterial inhibition and osteoblasts adhesion on Ti alloy surfaces modified by poly(PEGMA-r-Phosmer) coating Xinnan Cui, Tatsuya Murakami, Yukihiko Tamura, Kazuhiro Aoki, Yu Hoshino, and Yoshiko Miura ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07757 • Publication Date (Web): 26 Jun 2018 Downloaded from http://pubs.acs.org on June 29, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Bacterial Inhibition and Osteoblasts Adhesion on Ti Alloy Surfaces Modified by Poly(PEGMA-r-Phosmer) Coating

Xinnan Cuia, Tatsuya Murakamib, Yukihiko Tamurac, Kazuhiro Aokid, Yu Hoshinoa, Yoshiko Miuraa* a

Department of Chemical Engineering, Graduate School of Engineering, Kyushu

University 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan b

Center for Nano Materials and Technology, Japan Advanced Institute of Science and

Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan c

Pharmacology, Department of Bio-Matrix, Graduate School of Medical and Dental

Sciences, Tokyo Medical and Dental University, Tokyo 113-8549, Japan d

Department of Basic Oral Health Engineering, Graduate School of Medical and

Dental Sciences, Tokyo Medical and Dental University, Tokyo 113-8549, Japan Keywords: titanium alloy, surface modification, bacterial resistance, preosteoblast response, polymer graft

1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT We have synthesized and immobilized PEGMA500-Phosmer to Ti6Al4V surfaces by a simple procedure to reduce bacteria-associated infection without degrading the cell response. Adhered bacteria coverage was lessened to 1% on polymer-coated surfaces when exposed to Escherichia coli, Staphylococcus epidermidis and Streptococcus mutans. Moreover, PEGMA500-Phosmer and homoPhosmer coatings presented better responses to MC3T3-E1 preosteoblast cells when compared with the results for PEGMA2000-Phosmer coated and raw Ti alloy surfaces. The behavior of balancing bacterial inhibition and cell attraction of the PEGMA500-Phosmer coating was explained by the grafted phosphate groups with an appropriate PEG brush length facilitating greater levels of calcium deposition and further fibronectin adsorption when compared with that of the raw Ti alloy surface.


Page 2 of 25

Page 3 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. Introduction Titanium and its alloy are used widely for the fabrication of orthopedic and dental implants because these materials display favorable mechanical strength, fatigue and corrosion resistance, low density and osteoconductivity. Although titanium and its alloy are biocompatible and non-cytotoxic, there can be serious complications and failure during therapeutic implantation 1. For example, a common problem is bacterial infection, which is directly associated with high rates of patient morbidity and mortality. Bacterial adhesion to surfaces often leads to biofilm formation, which represents the main pathogenic form of chronic infections 2-4. Thus, infection-resistant surfaces used as implant materials are urgently needed to improve the success rate and service life of implants constructed with titanium and its alloy 5. Moreover, to integrate with bone tissue and further enhance the success of implants, the implant material should be compatible with osteoblasts

6-7

. Based on these two requirements,

we have developed a simple material and its associated coating method to generate implant surfaces, tailoring the surfaces by exploiting the differences in the response of bacteria and osteoblasts. Significant effort has been made recently to eradicate bacteria-associated infections that lead to inflammatory responses and ultimately implant failure

8-9

.

Successful efforts include increased wettability 10, grafting with zwitterionic polymers 11

, taking advantage of the biocidal properties of silver nanoparticles

12-13

and

silane-based coating systems 14. The PEGylation on Ti and its alloy surfaces is known to decrease non-specific protein adsorption

15-17

, however, improvement of the

anti-bacterial properties by surface PEGylation has rarely been reported. Tailoring surfaces to inhibit bacteria colonization and enhancing osteoblast adhesion represents an even greater challenge. In this regard, many complex coating strategies for implant surfaces have been reviewed

18

. Basically, the coatings integrate advance materials

graft on the surfaces as base, such as PLGA and chitosan polymers 19, dopamine and chitosan coating 18, poly(methacrylic acid sodium salt) 20, polyHEMA 21, heparin and dopamine 22, dopamine and dextran 23, and further more functionalized factors loaded



Page 4 of 25

onto primers. Factors such as silk sericin 20, BMP2 and VEGF 22-23, RGD peptide 24-25 and hydroxyapatite

26

are used to promote cell adhesion, while antibiotics

21, 27-28

and

antimicrobial peptides 25 enhance antibacterial activity. In addition, the topography of surfaces has also been designed to modulate different bacteria and cell adhesion responses, including varying surface roughness 29, fabricating a ZnO/TiO2 nanophase 30

and ZnO/polydopamine/arginine-glycine-aspartic acid-cysteine nanorod arrays on

Ti implants 31. Although these methods have showed varying degrees of effectiveness, inevitably, they require complex fabrication steps that limit their widespread adoption for surface modification of titanium implant materials. Since calcium phosphate-based coatings effectively simulate an osteoblast response to enhance bone regeneration 32-33, the strong binding of phosphate to TiO2 34 has been considered to play an important role in surface modifications. Phosphate adsorption onto titania by a variety of direct surface analysis methods has been described

35-36

. The adsorption mechanism of phosphonic acids to a metal oxide

surface as bidentate and tridentate phosphonate species involves condensation and coordination of phosphonate coupling molecules with the hydroxyl groups on the surface 37-38. Thus, inspired by the phosphate-based modification on titanium and its alloy, we synthesized a polymer composed of the phosphate segment as the binding site and PEG brushes, which are well-known to function as an anti-biofouling agent

39

.

PEGMA (Mn 500 Da) was used because of the anti-bacterial property of PEG and to avoid hindrance to cell attachment. Because of the polymerization, part of the phosphate groups were able to bind to the surfaces and the residual free phosphate groups attracted Ca2+ ions present in the culture medium to form a Ca-P deposition, which is biocompatible with cells 40. Designed with a simple structure and applied to the

Ti

alloy

surface

by

a

practical

method,

this

polymer

presented

dual-functionalization thereby solving the inherent problem associated with bacterial inhibition and simultaneously facilitating cell adhesion. This promising material and its coating strategy should enhance the success rate and service life of orthopedic and dental implants.


Page 5 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In this report, we described the polymerization of the PEGMA500-Phosmer and the immobilization procedure. This polymer was composed of PEG brushes as anti-bacterial functionality and Phosmer to promote cell adhesion without changing the surface topography. Surface modification with PEGMA500-Phosmer was confirmed by surface elemental analysis. Coverage of the polymer coating was measured by coating the fluorescein labeled polymer on the surface. The anti-bacterial property of the polymer coating was evaluated in bacterial inhibition experiments using Escherichia coli, Staphylococcus epidermidis and Streptococcus mutans. The osteoblastic cell line MC3T3-E1 adhered onto the polymer-immobilized and raw Ti alloy surfaces to prove the compatibility of the polymer to cell adhesion. Calcium deposition and fibronectin (Fn) adsorption were investigated to explore the mechanism of the dual-functional performance of the polymer coating.

2. Materials and methods

2.1. Materials

Ti alloy (Ti6Al4V) was purchased from Tokyo Titanium Co., Ltd., Japan. Poly(ethylene glycol) methyl ether methacrylate (PEGMA, Mn 500 Da, 2000 Da) (Sigma-Aldrich, St Louis, MO, USA), ethylene glycol methacrylate phosphate (Phosmer; DAP Co., Ltd, Kyoto, Japan) and 4,4’-azobis(4-cyanovaleric acid) (V-501; Wako Pure Chemical Industries Ltd., Osaka, Japan) were used for polymerization. Escherichia coli (E. coli) ORN 178, Staphylococcus epidermidis (S. epidermidis) JCM 2414 and Streptococcus mutans (S. mutans) ATCC 25175 were purchased from the Japan Collection of Microorganisms, RIKEN BRC, which participates in the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan. MC3T3-E1 cells (derived from mouse) were provided by the RIKEN BioResource Center. The fluorescent monomer 4-acrylamido fluorescein (AFA) was synthesized according to a reported protocol 41. PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.4) was used as the buffer.



Page 6 of 25

Acti-stain™ 488 fluorescent phalloidin (Cytoskeleton, Denver, CO, USA) was used to visualize the cytoskeletal actin of the cells. The anti-fibronectin antibody (ab2413) and the goat anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077) (Abcam, Cambridge, UK) as the secondary antibody were used in the fibronectin immunocytochemical analysis of the surfaces following cell adhesion. An artificial saliva solution was used as the calcium deposition solution that included CaCl2 (1.5 mM), KH2PO4 (0.9 mM), KCl (130 mM), NaN3 (1.0 mM), HEPES (20 mM) and adjusted to pH 7.0 using KOH (1 M). Minimum essential medium α (MEM α, Gibco, USA) supplemented with 10% FBS and 2% penicillin-streptomycin solution was used as the culture medium for MC3T3-E1 osteoblast cells. Fibronectin (Fn; from human plasma, Sigma-Aldrich) was used for protein adsorption experiments.

2.2. Polymerization of poly(PEGMA-r-Phosmer), homoPEGMA and homoPhosmer

Monomers, 0.5 mmol PEGMA (Mn 500 Da) and 0.5 mmol Phosmer for PEGMA500-Phosmer, 0.25 mmol PEGMA (Mn 2000 Da) and 0.25 mmol Phosmer for PEGMA2000-Phosmer, 1.0 mmol PEGMA for homoPEGMA or 1.0 mmol Phosmer for homoPhosmer were dissolved in 10 mL of water and bubbled under a nitrogen atmosphere for 30 min. Polymerization began with the decomposition of the initiator V-501 (0.0128 mmol) dissolved in a modicum of methanol and then reacted for 11 h at 70 °C. Scheme of PEGMA500-Phosmer polymerization was shown in Figure 1B. To label the polymer with fluorescence, 0.1% AFA was added to the monomers before polymerization. The physical characterization of polymers and the scheme of AFA labeled PEGMA500-Phosmer polymerization were shown in Table S1, Figure S1 and Scheme S1. Physical characterization of polymers was analyzed by 1H NMR (400 Hz, D2O) (JEOL Ltd., Tokyo, Japan), weight-average molecular weights (Mw) and polydispersities

(Mw/Mn)

of

polymers

were

measured

by

size

exclusion

chromatography (SEC) (Showa Denko, Tokyo, Japan) (Shodex OHpak SB-G guard column, Shodex OH park LB-806 HQ column and 20 µL of polymer solution (10 mg/1 mL) in a 10 mM NaNO3 solution).


Page 7 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.3. Polymer immobilization

Ti alloy plates were cut into 0.5 × 0.5 cm squares (thickness 1 mm) for subsequent use. Processed plates were ultrasonically cleaned for 30 min in each of the following solutions: detergent, isopropanol, acetone and deionized water. All alloy plates were blow-dried and submerged in 1 N H2SO4 at 75 °C for 2 h to remove the metal oxidized layer. After acid etching, those plates were ultrasonically cleaned with deionized water (5 min, refresh the water thrice) and dried with a stream of N2. UV/O3 treatment was carried out on both sides (each 30 min) just before use in polymer immobilization reactions. Treated Ti alloy plates were immersed in a 10 mg/mL PEGMA500-Phosmer solution at 75 °C for 1 min, 10 min, 30 min, 1 h, 3 h, 5 h, 8 h and 24 h. Scheme of polymer immobilization was shown in Figure 1B. To prove the necessity of phosphate groups for surface modification, alloy plates were coated with a homoPEGMA solution (10 mg/mL) at 75 °C for 30 min, 1 h, 3 h, 5 h and 24 h. To investigate the minimum concentration for immobilization on the alloy surfaces, various concentrations of PEGMA500-Phosmer (10, 1, 0.1, 0.01 and 0.001 mg/mL) were used to modify the surfaces at 75 °C for 24 h. The immobilization of AFA labeled PEGMA500-Phosmer, PEGMA2000-Phosmer and homoPhosmer on surfaces was carried out with the same pre-treatment and immersed in a polymer solution at 75 °C for 8 h. Modified surfaces were elementary analyzed using C(1s), O(1s), P(2p) and Ti(2p) X-ray photoelectron spectroscopy (XPS, AXIS-ultra; Shimadzu/Kratos, Kyoto, Japan). The peaks were analyzed with PeakFit V4.12 software (Systat Software, Inc., San Jose, CA, USA). To evaluate the wettability after polymer immobilization, the contact angle was measured on raw and modified Ti alloy surfaces using DropMaster300 (Kyowa Interface Science Co., Ltd., Saitama, Japan). One microliter of water was placed onto the surfaces and data was obtained after 15 s contact time.

2.4. Bacterial adhesion on Ti alloy plates in vitro



E. coli cultured in LB broth (Lennox) medium (Sigma-Aldrich), S. epidermidis cultured in nutrient medium (polypeptone 5 g/L, beef extract 3 g/L and NaCl 5 g/L) and S. mutans cultured in broth medium (BD 237500, Sigma-Aldrich) were constantly shaken at 100 rpm (Shaker NTS-2100, Tokyo Rikakikai Co., Ltd, Tokyo, Japan) in a 37 °C water bath until the cell density was approximately 108 CFU/mL. All the Ti alloy plates were rinsed with 70% ethanol and deionized water to eliminate potential microbial contamination before placed in 24-well culture plates. 0.5 mL of the E. coli, S. epidermidis and S. mutans PBS suspensions were added to each well, and then incubated at 37 °C for 2.5 h, 6 h and 24 h to imitate the bacterial adhesion process. One milliliter of the appropriate medium was added to 6 h and 24 h cultures to ensure the bacterial viability. After cultivation, bacterial cells were fixed for scanning electron microscopy (SEM; SU8000, Hitachi High-Technologies Co., Tokyo, Japan) analysis to evaluate the adhesion on surfaces. The Ti alloy plates were washed with PBS gently to remove non-adherent cells. Bacteria adhered on surfaces were fixed with 2.5% glutaraldehyde at 37 °C for 24 h. After rinsing with PBS twice, the samples were dehydrated in an ethanol series (10, 30, 50, 60, 70 and 80%, each 5 min, and then 90 and 100%, each 10 min) and dried at 37 °C in an incubator overnight.

2.5. In vitro cell adhesion on Ti alloy plates

MC3T3-E1 cells were cultured in prepared medium at 37 °C under 5% CO2 in a humidified atmosphere. PEGMA500-Phosmer, PEGMA2000-Phosmer, homoPhosmer coated and raw Ti alloy plates were placed in 48-well plates. Cells (2 × 104 cell/well) were seeded into each well and incubated at 37 °C under 5% CO2 in a humidified atmosphere for 1, 2 and 3 days. The adhered cells were fixed with 4% formaldehyde for 10 min, permeabilized with 0.2% Triton X-100 for 10 min and then incubated in 2% BSA in PBS for 30 min to block non-specific protein-protein interactions. For actin staining, the cells were incubated with fluorescent phalloidin for 1 h. For Fn


Page 8 of 25

Page 9 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


immunostaining, the cells were incubated with the primary antibody (ab2413, diluted 1:200) for 1 h and then with the secondary antibody (ab150077, diluted 1:1000) for 45 min. 4',6-diamidino-2-phenylindole (DAPI) was used to stain the cell nuclei. All cell morphologies and the Fn distribution were observed by confocal laser scanning microscopy (CLSM; Olympus FluoView FV1200, Tokyo, Japan).

2.6. Calcium deposition and Fn adsorption on Ti alloy plates

An artificial saliva solution was used as the calcium deposition solution. Each sample was vertically soaked in the artificial saliva solution. The solution was refreshed every day and incubated at 37 °C for long term. After 1 d, 2 d, 1 week and 2 weeks the plates were rinsed with copious amounts of water and dried with N2. The deposited calcium on the surfaces was confirmed by XPS Ca(2p). Specimens after 2 d were used for further protein adsorption experiments. 0.5 mL of 10 µg/mL Fn (dissolved in PBS buffer) was added to each plate. After incubating at 37 °C for 1 h, the plates were rinsed with PBS twice, water thrice and dried with N2. The absorbed protein on the surfaces was confirmed by XPS N(1s).

3. Results and discussion

3.1. Surface analysis of polymer immobilized Ti alloy plates

XPS analysis was carried out on raw and modified Ti alloy surfaces to evaluate the polymer immobilization. After immobilization, the existence of phosphorus on coated surfaces was confirmed by observing a peak at ~133.5 eV with no obvious peak observed on the bare surface (Figure 1A). The P(2p) spectrum clearly showed the presence of phosphorus atoms on PEGMA500-Phosmer modified Ti alloy surfaces. The P(2p) spectra of other modified surfaces and XPS spectra with wide range were shown in Figure S2 and S7. HomoPEGMA was synthesized and immobilized on the alloy surfaces under the same conditions to confirm the necessity of phosphate groups



acting as bonding sites. Since the Ti alloy was not composed of C=O, the concentration ratio of C=O to Ti was directly proportional to the polymer immobilization on the surfaces. An example of peak fitting of C(1s) spectrum was shown in Figure S3. As a result, the PEGMA500-Phosmer reached saturation gradually after 3 h. In contrast, the C=O unit proportion of Ti alloy plates immersed in homoPEGMA was much lower than that on PEGMA500-Phosmer coated surfaces and showed no change over time, which showed a similar behavior to that of Ti alloys submerged in water (Figure 2A). The results of surface elemental analysis confirmed the immobilization of PEGMA500-Phosmer on Ti alloy surfaces because of the high affinity of this compound toward metal oxide surfaces. The AFA labeled PEGMA500-Phosmer coating was presented in Figure 2B and showed that the polymer coating on the surface in a uniform film with full coverage. After strong acid etching and ozone treatment, the phosphate groups in the polymers acted as anchors to couple the polymers with hydroxyl groups on Ti alloy surfaces in aqueous environments. The condensation

36, 42

and further spatial repartition

37

of the phosphate groups in the

polymers afforded formation of the P-O-metal bonds whose stability has been shown in numerous metal phosphate and phosphonate compounds 43.

Figure 1. (A) XPS P(2p) spectra of PEGMA500-Phosmer modified and raw Ti alloy surfaces. (B) Scheme of PEGMA500-Phosmer polymerization and immobilization on 10 ACS Paragon Plus Environment

Page 10 of 25

Page 11 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the Ti alloy surface.

Figure 2. (A) Relative immobilization amounts of PEGMA500-Phosmer and homoPEGMA coated Ti alloy surfaces as a function of time. Plates were immersed in water as the control (n = 3 for each condition). (B) CLSM images of AFA labeled PEGMA500-Phomer coated (left) and unlabeled PEGMA500-Phomer coated (right) surfaces.

Furthermore, Figure S4 summarized the mass concentration of phosphorous on PEGMA500-Phosmer immobilized surfaces as the immersion time in the polymer solution changing. The immobilization proceeded fast and reached saturation after 3 h when the concentration was 10 mg/mL and preserved at 75 °C. The immobilization process reached saturation with a 1 mg/mL PEGMA500-Phosmer solution at 75 °C for 24 h (Figure S5). The surface wettability was measured by contact angle analysis. After pre-treatment, the contact angle was 48.4 ± 0.9° and altered to 40.9 ± 1.1° after polymer immobilization (Figure S6). This observed change in the contact angle revealed that the alloy surfaces were originally hydrophilic, which was potentially capable of working as implant materials, and the hydrophilicity was increased by PEGMA500-Phosmer coating.

3.2. Bacterial adhesion on modified and raw Ti alloy plates 11 ACS Paragon Plus Environment


Statistical analysis of the bacterial adhesion area on surfaces was summarized in Figure 3 and SEM images showing adhesion after 2.5 h incubation of bacteria were shown in Figure 4. Additional SEM images of other adhesion incubation periods were shown in Figure S8. The number of E. coli adhering to raw Ti alloy surfaces increased over time, whereas negligible amounts of E. coli adhered to the modified surfaces. For S. epidermidis, after 6 h incubation the bacterial adherence area was nearly 12%, which was double the level observed after incubation for 2.5 h. In contrast, S. epidermidis adherence to modified surfaces was reduced significantly, with the area of coverage at different incubation times lower than 1%. S. mutans showed high biocompatibility to the raw Ti alloy inducing a high tainted area on the surface even after a short (2.5 h) incubation period contrasting to the dramatically reduced adhesion on the modified surfaces. Compared with PEGMA500-Phosmer coated surface, where bacteria were hardly observed (adhered coverage close to 0%), the performance of PEGMA2000-Phosmer was less satisfying that still 10% (compared with raw surfaces) bacteria adhered on the surfaces. About 50% bacterial adhesion (compared with result on raw surfaces) was reduced on homoPhosmer coated surfaces and no reduction showed on homoPEGMA coated surfaces (results shown in Figure S9). In summary, bacterial adhesion to the surface was reduced drastically with only ~1% coverage on PEGMA500-Phosmer modified surfaces.


Page 12 of 25

Page 13 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. The bacterial adhesion area on raw and PEGMA500-Phosmer immobilized Ti alloy surfaces after exposure to E. coli, S. epidermidis and S. mutans (108 CFU/mL) suspensions for 2.5, 6 and 24 h. The results on modified surfaces were highlighted with slashes (n = 3, 25 images were taken on five separate areas of each sample; all the images were analyzed by ImageJ). Statistical comparison between the raw and PEGMA500-Phosmer coated conditions was tested. *Significant difference (t-test and Tukey’s test, P < 0.05); n.s., no significance.

Figure 4. Representative SEM images of raw (above) and PEGMA500-Phosmer coated (blow) Ti alloy surfaces after exposure with S. epidermidis, S. mutans and E. coli suspensions for 2.5 h. Scale bar, 10 µm. 13 ACS Paragon Plus Environment


Page 14 of 25

The primary attachment of bacteria between interfaces was generally mediated by non-specific interactions that are reversible and controllable

44-45

. Thus, to render

the surfaces with anti-bacteria behavior, PEG brushes were covalently combined and grafted to the interfaces. An efficient sterically hindering cloud formed by the high flexibility of the PEG chains could diminish bacteria interactions 44. According to our previous results 46, PEG brushes grafted to surfaces with high density were capable of inhibiting bacterial attachment even with short brushes (PEGMA, Mn 500 Da). The free phosphate groups of the polymer increased the electronegativity, which was repulsive against the negative surface potential of bacteria cell membrane. The combination of the moderating physical force offered by PEG brushes and repulsive electrostatic feature provided by Phosmer on the polymer-modified surfaces offered an effective approach to reduce bacterial adhesion levels and eliminate the formation of biofilm. Thus, this synergy of PEGMA500-Phosmer showed better inhibitory property than the single composition like homoPEGMA and homoPhosmer. In our previous study, we have we concluded that the PEGMA ratio of 40%~60% in copolymers could inhibit the bacterial adhesion effectively

46

. Although it has been

reported that protein adsorption could be reduced by PEGylation on Ti alloy surfaces, bacterial inhibition has rarely been mentioned because single PEGylation may make surfaces unfavorable for cell adhesion. Nonetheless, PEGylation carried out in our PEGMA500-Phosmer

coating

showed

efficient

bacterial

resistance

toward

gram-negative (E. coli) and gram-positive (S. epidermidis) strains, and especially reduced S. mutans levels that provided the possibility of using this coating in oral hygiene and dentistry fields.

3.3. Cell adhesion on modified and raw Ti alloy plates

Statistical analysis of cell adhesion amount per surface area after 1, 2 and 3 d were summarized in Figure 5. The amount of cell adhesion increased as the culture time extended. The PEGMA500-Phosmer and homoPhosmer coated surfaces enhanced


Page 15 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the levels of cell adhesion when compared with the results on raw surfaces. In particular, cell adhesion to the homoPhosmer coated surface after 3 d was double the amount of cells when compared with the number on raw surfaces. In contrast, PEGMA2000-Phosmer coated surfaces showed no increase when compared with that of the raw surfaces. Representative CLSM images of MC3T3-E1 cell morphology and Fn distribution on surfaces are shown in Figure 6. After the first 24 h (Figure 6A), cell adhesion on PEGMA500-Phosmer and homoPhosmer coated surfaces showed more filopodia, whereas cells on PEGMA2000-Phosmer and raw surfaces appeared spherical without filopodia extensions. After 2 d (Figure 6B), on the homoPhosmer coated surface cells were observed to have more lamellipodia around the surface and the cells were elongated with a fusiform fibroblastic shape. Similarly, the cells on PEGMA500-Phosmer coated surface extended better than on the PEGMA2000-Phosmer coated and raw surfaces. The cells were fully extended after 3 d (Figure 6C), displaying spreading with polytonal shapes with numerous filopodia and lamellipodia, and cell-to-cell contacts were observed. After initial contact to substrates, cells began to expend metabolic energy and subsequent spreading involved active mechanisms such as actin polymerization

47-49

. Following initial contact on phosphate-enriched

surfaces, actin filaments of cells enriched from spreading formed long bundles to configure a front-to-tail polarity, which was not as apparent on the raw surfaces (Figure 6). This result suggested that grafting of the phosphate groups to the surfaces improved cell adherence, cellular extension and intracellular actin cytoskeleton formation. The observed cell adhesion amount was consistent with the Fn distribution on the surfaces, as visualized by immunocytostaining. More Fn was associated with the PEGMA500-Phosmer and homoPhosmer coated surfaces around cells when compared with that of the on raw and PEGMA2000-Phosmer coated surfaces. Interestingly, even though PEG brushes were grafted onto the surface by PEGMA500-Phosmer coating, this PEGylation didn’t prevent cell attachment contrary to the PEGMA2000-Phosmer. As documented, the length of the PEG brushes and the brush density influenced the anti-bacterial property. The approach taken to modify the surface might also alter the surface topography and further influenced cell activity. In



Page 16 of 25

this study, PEG brushes of PEGMA2000-Phosmer grafted to the surfaces reduced Fn adsorption and cell adhesion, which was similar to previous reports

50-51

. Thus, we

concluded that cell adhesion and bacterial inhibition can be achieved by grafting the phosphate groups onto the Ti alloy surfaces with combining with adequate length of PEG brushes.

Figure 5. The amount of adhered cells on PEGMA500-Phosmer, PEGMA2000-Phosmer, homoPhosmer coated and raw Ti alloy surfaces after incubation for 1, 2 and 3 d with MC3T3-E1 cell (2 × 104 cells/mL, 0.5 mL) suspensions. The cells were counted based on pictures taken by CLSM (n = 4, 20 images taken from five separate areas of each sample). Statistical comparison between the raw and PEGMA500-Phosmer coated conditions was tested. *Significant difference (t-test and Tukey’s test, P < 0.05); n.s., no significance.


Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Representative CLSM images of MC3T3 cell morphology and Fn distribution on PEGMA500-Phosmer, PEGMA2000-Phosmer, homoPhosmer coated and 17 ACS Paragon Plus Environment


Page 18 of 25

raw Ti alloy surfaces after incubation for 1 d (A), 2 d (B) and 3 d (C). Scale bar, 30 µm. Images above presented F-actin stained by fluorescent phalloidin and nuclei counterstained

with

DAPI.

Images

blow

showed

the

Fn

examined

by

immunocytochemistry and nuclei counterstained with DAPI.

3.4. Calcium deposition and Fn adsorption on phosphate-enriched surfaces

Because Fn visualized on the surfaces was in accordance with the cell adhesion results, we hypothesized that the free phosphate groups grafted on the surfaces promoted Fn adsorption, which induced further cell adhesion. Since the pI of Fn is 5.39, it can be postulated that divalent cations such as Ca2+ play a critical role in mediating cell attachment.

Thus,

we

investigated calcium

deposition

on

PEGMA500-Phosmer coated and raw Ti alloy surfaces (Figure 7A), and Fn adsorption on the deposited surfaces (Figure 7B). The results showed that calcium deposition on the polymer-coated surfaces reached saturation faster than on the raw Ti surface. More Ca2+ ions attracted greater adsorption of Fn adsorption on the surfaces.

Figure

7.

(A) Atomic concentration of Ca(2p) measured by XPS on

PEGMA500-Phosmer coated and raw surfaces after immersion in an artificial saliva solution. (B) Atomic concentration of N(1s) measured by XPS after fibronectin adsorption on calcium deposited (2 d) surfaces. 18 ACS Paragon Plus Environment

Page 19 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ca2+ ions with positive charge favored the Fn or other cell-adhesive proteins from medium actively, thereby leading to cell adhesion and spreading 52. In this study, surfaces pre-coated with a number of free phosphate groups attracted more Ca2+ ions deposition electrostatically. As the deposition of calcium proceeds, phosphate groups that co-existed with Ca2+ ions former promoted the adsorption of Fn in the routine culture medium. By coordinating those cell attachment-promoting proteins to the integrins at the cell membrane, osteoblasts adhered and spread onto the surfaces 53-55. The effect of Ca-P deposition has also been proven to improve biocompatibility and osteoconductive properties to cells and bone using plasma-sprayed hydroxyapatite layer and biomimetic and electrolytic octacalcium phosphate coatings

40, 57

56

onto the

implants. However, different from the pre-calcification methods on the surfaces, our study avoided the complex process and bacterial colonization during the initial culture stage. Without forming the hydroxyapatite or structured crystal on the surfaces, Ca-P deposition in nanoscale was powerful to the promotion of protein adsorption and subsequent cell adhesion 58.

4. Conclusions

In this study, we showed that a simple surface modification method on Ti6Al4V implant material rendered the surface with anti-bacterial property and promoted binding activity of the MC3T3-E1 osteoblastic cell line. We synthesized the PEGMA500-Phosmer material and immobilized the polymer successfully to the Ti alloy surfaces. Immobilization of PEGMA500-Phosmer was confirmed by XPS and the importance of the phosphate groups in providing highly active bonding sites was shown via comparison with a homoPEGMA coating on the Ti surface. Bacterial adhesion to the surface was reduced to ~1% coverage on PEGMA500-Phosmer modified surfaces when exposed to E. coli, S. epidermidis and S. mutans suspensions for even 24 h because of the PEGylation coordinated with negative-charged phosphate groups. Importantly, after the PEGMA500-Phosmer coating, the surfaces



presented good compatibility to MC3T3-El cells. The grafted phosphate groups with appropriate length of PEG brushes attracted more Ca2+ ions and fibronectin in the culture medium, thereby facilitating greater cell adhesion on the surfaces. These results have presented a practical material and coating method for implants, which may enhance early cell adhesion and bacterial inhibition simultaneously. This material offers potential application prospects in dental and orthopedic implantations.

Supporting Information Supplementary Table S1, Figure S1 and Scheme S1: Physical characterization of polymers and scheme of polymerization; Figure S2-S7: Surface analysis and characterization of polymer immobilized Ti alloy surfaces; Figure S8 and S9: Representative SEM images of polymer-coated and raw surfaces after bacterial adhesion.

Acknowledgments We are grateful to Dr. Midori Watanabe of Kyushu University and Mr. Ichiro Kimura of Japan Advanced Institute of Science and Technology for the measurements. This study was supported by the Grant-in-Aid for Scientific Research B (JP15H03818) and Grant-in-Aid for Scientific Research on Innovative Areas (JP18H04420). This work was performed under the Cooperative Research Program of "Network Joint Research Center for Materials and Devices."

Conflict of interest The authors declare no conflict of interest.

References

(1) Devorah, S.-A.; Amir, L.; Liran, L. Failure Causes, Timing, and Cluster Behavior: An 8-Year Study of Dental Implants. Implant Dentistry 2008, 17 (2), 200-207, DOI: 10.1097/ID.0b013e3181777906. (2) Ploux, L.; Ponche, A.; Anselme, K. Bacteria/Material Interfaces: Role of the Material and


Page 20 of 25

Page 21 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cell Wall Properties. J. Adhes. Sci. Technol. 2010, 24 (13-14), 2165-2201, DOI: 10.1163/016942410x511079. (3) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Bacterial Biofilms: A Common Cause of Persistent Infections. Science 1999, 284 (5418), 1318-1322, DOI: 10.1126/science.284.5418.1318. (4) Costerton, W.; Veeh, R.; Shirtliff, M.; Pasmore, M.; Post, C.; Ehrlich, G. The application of biofilm science to the study and control of chronic bacterial infections. J. Clin. Invest. 2003, 112 (10), 1466-1477, DOI: 10.1172/JCI20365. (5) Darouiche, R. O. Treatment of Infections Associated with Surgical Implants. N. Engl. J. Med. 2004, 350, 1422-1429, DOI: 10.1056/NEJMra035415. (6) D.Boyan, B.; W.Hummert, T.; D.Dean, D.; ZviSchwartz. Role of material surfaces in regulating bone and cartilage cell response. Biomaterials 1996, 17 (2), 137-146, DOI: 10.1016/0142-9612(96)85758-9. (7) Reyes, C. D.; Petrie, T. A.; Burns, K. L.; Schwartz, Z.; Garcia, A. J. Biomolecular surface coating to enhance orthopaedic tissue healing and integration. Biomaterials 2007, 28 (21), 3228-3235, DOI: 10.1016/j.biomaterials.2007.04.003. (8) Banerjee, I.; Pangule, R. C.; Kane, R. S. Antifouling coatings: recent developments in the design of surfaces that prevent fouling by proteins, bacteria, and marine organisms. Adv Mater 2011, 23 (6), 690-718, DOI: 10.1002/adma.201001215. (9) Zhao, L.; Chu, P. K.; Zhang, Y.; Wu, Z. Antibacterial coatings on titanium implants. J. Biomed. Mater. Res., Part B 2009, 91 (1), 470-480, DOI: 10.1002/jbm.b.31463. (10) 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 biomaterialia 2009, 5 (1), 181-192, DOI: 10.1016/j.actbio.2008.07.028. (11) Rodriguez-Palomo, A.; Monopoli, D.; Afonso, H.; Izquierdo-Barba, I.; Vallet-Regí, M. Surface zwitterionization of customized 3D Ti6Al4V scaffolds: a promising alternative to eradicate bone infection. J. Mater. Chem. B 2016, 4 (24), 4356-4365, DOI: 10.1039/c6tb00675b. (12) Zhao, L.; Wang, H.; Huo, K.; Cui, L.; Zhang, W.; Ni, H.; Zhang, Y.; Wu, Z.; Chu, P. K. Antibacterial nano-structured titania coating incorporated with silver nanoparticles. Biomaterials 2011, 32 (24), 5706-5716, DOI: 10.1016/j.biomaterials.2011.04.040. (13) Flores, C. Y.; Diaz, C.; Rubert, A.; Benitez, G. A.; Moreno, M. S.; Fernandez Lorenzo de Mele, M. A.; Salvarezza, R. C.; Schilardi, P. L.; Vericat, C. Spontaneous adsorption of silver nanoparticles on Ti/TiO2 surfaces. Antibacterial effect on Pseudomonas aeruginosa. J Colloid Interface Sci 2010, 350 (2), 402-408, DOI: 10.1016/j.jcis.2010.06.052. (14) Matinlinna, J. P.; Tsoi, J. K.; de Vries, J.; Busscher, H. J. Characterization of novel silane coatings on titanium implant surfaces. Clin Oral Implants Res 2013, 24 (6), 688-697, DOI: 10.1111/j.1600-0501.2012.02504.x. (15) Tosatti, S.; Paul, S. M. D.; Askendal, A.; VandeVondele, S.; Hubbell, J. A.; Tengvall, P.; Textor, M. Peptide functionalized poly(l-lysine)-g-poly(ethylene glycol) on titanium: resistance to protein adsorption in full heparinized human blood plasma. Biomaterials 2003, 24 (27), 4949-4958, DOI: 10.1016/s0142-9612(03)00420-4. (16) Hansson, K. M.; Tosatti, S.; Isaksson, J.; Wettero, J.; Textor, M.; Lindahl, T. L.; Tengvall,



P. Whole blood coagulation on protein adsorption-resistant PEG and peptide functionalised PEG-coated titanium surfaces. Biomaterials 2005, 26 (8), 861-872, DOI: 10.1016/j.biomaterials.2004.03.036. (17) Liu, S.; Chen, L.; Tan, L.; Cao, F.; Bai, L.; Wang, Y. A high efficiency approach for a titanium surface antifouling modification: PEG-o-quinone linked with titanium via electron transfer process. J. Mater. Chem. B 2014, 2 (39), 6758-6766, DOI: 10.1039/c4tb01014k. (18) Neoh, K. G.; Hu, X.; Zheng, D.; Kang, E. T. Balancing osteoblast functions and bacterial adhesion on functionalized titanium surfaces. Biomaterials 2012, 33 (10), 2813-2822, DOI: 10.1016/j.biomaterials.2012.01.018. (19) Kumeria, T.; Mon, H.; Aw, M. S.; Gulati, K.; Santos, A.; Griesser, H. J.; Losic, D. Advanced biopolymer-coated drug-releasing titania nanotubes (TNTs) implants with simultaneously enhanced osteoblast adhesion and antibacterial properties. Colloids Surf., B 2015, 130 (1), 255-263, DOI: 10.1016/j.colsurfb.2015.04.021. (20) Zhang, F.; Zhang, Z.; Zhu, X.; Kang, E. T.; Neoh, K. G. Silk-functionalized titanium surfaces for enhancing osteoblast functions and reducing bacterial adhesion. Biomaterials 2008, 29 (36), 4751-4759, DOI: 10.1016/j.biomaterials.2008.08.043. (21) Zhang, F.; Shi, Z. L.; Chua, P. H.; Kang, E. T.; Neoh, K. G. Functionalization of Titanium Surfaces via Controlled Living Radical Polymerization From Antibacterial Surface to Surface for Osteoblast Adhesion. Ind. Eng. Chem. Res. 2007, 46 (26), 9077-9086, DOI: 10.1021/ie070795j. (22) Lee, D. W.; Yun, Y. P.; Park, K.; Kim, S. E. Gentamicin and bone morphogenic protein-2 (BMP-2)-delivering heparinized-titanium implant with enhanced antibacterial activity and osteointegration. Bone 2012, 50 (4), 974-982, DOI: 10.1016/j.bone.2012.01.007. (23) Shi, Z.; Neoh, K. G.; Kang, E.-T.; Poh, C.; Wang., W. Titanium with Surface-Grafted Dextran and Immobilized Bone Morphogenetic Protein-2 for Inhibition of Bacterial Adhesion and Enhancement of Osteoblast Functions. Tissue Eng., Part A 2008, 15 (2), 417-426, DOI: 10.1089/ten.tea.2007.0415. (24) Shi, Z.; Neoh, K. G.; Kang, E. T.; Poh, C.; Wang, W. Bacterial adhesion and osteoblast function on titanium with surface-grafted chitosan and immobilized RGD peptide. J. Biomed. Mater. Res., Part A 2008, 86 (4), 865-872, DOI: 10.1002/jbm.a.31648. (25) Muszanska, A. K.; Rochford, E. T.; Gruszka, A.; Bastian, A. A.; Busscher, H. J.; Norde, W.; van der Mei, H. C.; Herrmann, A. Antiadhesive polymer brush coating functionalized with antimicrobial and RGD peptides to reduce biofilm formation and enhance tissue integration. Biomacromolecules 2014, 15 (6), 2019-2026, DOI: 10.1021/bm500168s. (26) Song, L.; Gan, L.; Xiao, Y.-F.; Wu, Y.; Wu, F.; Gu, Z.-W. Antibacterial hydroxyapatite/chitosan complex coatings with superior osteoblastic cell response. Mater. Lett. 2011, 65 (6), 974-977, DOI: 10.1016/j.matlet.2010.12.051. (27) Wu, F.; Meng, G.; He, J.; Wu, Y.; Wu, F.; Gu, Z. Antibiotic-loaded chitosan hydrogel with superior dual functions: antibacterial efficacy and osteoblastic cell responses. ACS Appl. Mater. Interfaces 2014, 6 (13), 10005-100013, DOI: 10.1021/am502537k. (28) 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 factors incorporated hydroxyapatite coatings on metallic implant surfaces for enhancement of osteoinductivity and antibacterial properties. ACS Appl. Mater. Interfaces 2014, 6 (11), 8580-8589, DOI: 10.1021/am501428e.


Page 22 of 25

Page 23 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(29) Wu, Y.; Zitelli, J. P.; TenHuisen, K. S.; Yu, X.; Libera, M. R. Differential response of Staphylococci and osteoblasts to varying titanium surface roughness. Biomaterials 2011, 32 (4), 951-960, DOI: 10.1016/j.biomaterials.2010.10.001. (30) Gabriel Colon; Brian C. Ward; Webster, T. J. Increased osteoblast and decreased Staphylococcus epidermidis functions on nanophase ZnO and TiO2. J. Biomed. Mater. Res. 2006, 78A (3), 595-604, DOI: 10.1002/jbm.a.30789. (31) Li, J.; Tan, L.; Liu, X.; Cui, Z.; Yang, X.; Yeung, K. W. K.; Chu, P. K.; Wu, S. Balancing Bacteria-Osteoblast Competition through Selective Physical Puncture and Biofunctionalization of ZnO/Polydopamine/Arginine-Glycine-Aspartic Acid-Cysteine Nanorods. ACS nano 2017,11 (11), 11250–11263, DOI: 10.1021/acsnano.7b05620. (32) Narayanan, R.; Seshadri, S. K.; Kwon, T. Y.; Kim, K. H. Calcium phosphate-based coatings on titanium and its alloys. J. Biomed. Mater. Res., Part B 2008, 85 (1), 279-299, DOI: 10.1002/jbm.b.30932. (33) de Jonge, L. T.; Leeuwenburgh, S. C.; Wolke, J. G.; Jansen, J. A. Organic-inorganic surface modifications for titanium implant surfaces. Pharm. Res. 2008, 25 (10), 2357-2369, DOI: 10.1007/s11095-008-9617-0. (34) Connor, P. A.; McQuillan, A. J. Phosphate Adsorption onto TiO2 from Aqueous Solutions: An in Situ Internal Reflection Infrared Spectroscopic Study. Langmuir 1999, 15 (8), 2916-2921, DOI: 10.1021/la980894p. (35) Zhao, D.; Chen, C.; Wang, Y.; Ji, H.; Ma, W.; Zang, L.; Zhao, J. Surface Modification of TiO2 by Phosphate Effect on Photocatalytic Activity and Mechanism Implication. J. Phys. Chem. C 2008, 112 (15), 5993-6001, DOI: 10.1021/jp712049c. (36) Körösi, L.; Papp, S.; Bertóti, I.; Dékány, I. Surface and Bulk Composition, Structure, and Photocatalytic Activity of Phosphate-Modified TiO2. Chem. Mater. 2007, 19 (19), 4811-4819, DOI: 10.1021/cm070692r. (37) Mutin, P. H.; Lafond, V.; Popa, A. F.; Granier, M.; Markey, L.; Dereux, A. Selective Surface Modification of SiO2-TiO2 Supports with Phosphonic Acids. Chem. Mater. 2004, 16 (26), 5670-5675, DOI: 10.1021/cm035367s. (38) Randon, J.; Blanc, P.; Paterson, R. Modification of ceramic membrane surfaces using phosphoric acid and alkyl phosphonic acids and its effects on ultrafiltration of BSA protein. J. Membr. Sci. 1995, 98 (1-2), 119-129, DOI: 10.1016/0376-7388(94)00183-Y. (39) Lowe, S.; O'Brien-Simpson, N. M.; Connal, L. A. Antibiofouling polymer interfaces: poly(ethylene glycol) and other promising candidates. Polym. Chem. 2015, 6 (2), 198-212, DOI: 10.1039/c4py01356e. (40) Wang, J.; Layrolle, P.; Stigter, M.; de Groot, K. Biomimetic and electrolytic calcium phosphate coatings on titanium alloy: physicochemical characteristics and cell attachment. Biomaterials 2004, 25 (4), 583-592, DOI: 10.1016/s0142-9612(03)00559-3. (41) Michael J. Serpe, C. D. J., and L. Andrew Lyon. Layer-by-Layer Deposition of Thermoresponsive Microgel Thin Films. Langmuir 2003, 19 (21), 8759-8764, DOI: 10.1021/la034391h (42) Guerrero, G.; Mutin, P. H.; Vioux, A. Anchoring of Phosphonate and Phosphinate Coupling Molecules on Titania Particles. Chem. Mater. 2001, 13 (11), 4367-4373, DOI: 10.1021/cm001253u. (43) Giulio Alberti; Mario Casciola; Umberto Costantino; Vivani, R. Layered and pillared



metal(IV) phosphates and phosphonates. Adv. Mater. 1996, 8 (4), 291-303, DOI: 10.1002/adma.19960080405. (44) Katsikogianni, M.; Missirlis, Y. F. Concise review of mechanisms of bacterial adhesion to biomaterials and of techniques used in estimating bacteria-matierial interactions. Eur. Cells Mater. 2004, 8, 37-57, DOI: 10.22203/eCM.v008a05. (45) An., Y. H.; Friedman, R. J. Concise Review of Mechanisms of Bacterial Adhesion to Biomaterial Surfaces. J. Biomed. Mater. Res. 1998, 43 (3), 338-348, DOI: 10.1002/(SICI)1097-4636(199823)43:33.0.CO;2-B. (46) Cui, X.; Koujima, Y.; Seto, H.; Murakami, T.; Hoshino, Y.; Miura, Y. Inhibition of Bacterial Adhesion on Hydroxyapatite Model Teeth by Surface Modification with PEGMA-Phosmer Copolymers. ACS Biomater. Sci. Eng. 2016, 2 (2), 205-212, DOI: 10.1021/acsbiomaterials.5b00349. (47) Cai, Y.; Biais, N.; Giannone, G.; Tanase, M.; Jiang, G.; Hofman, J. M.; Wiggins, C. H.; Silberzan, P.; Buguin, A.; Ladoux, B.; Sheetz, M. P. Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow. Biophys. J. 2006, 91 (10), 3907-3920, DOI: 10.1529/biophysj.106.084806. (48) Cuvelier, D.; Thery, M.; Chu, Y. S.; Dufour, S.; Thiery, J. P.; Bornens, M.; Nassoy, P.; Mahadevan, L. The universal dynamics of cell spreading. Curr. Biol. 2007, 17 (8), 694-699, DOI: 10.1016/j.cub.2007.02.058. (49) Loosli, Y.; Luginbuehl, R.; Snedeker, J. G. Cytoskeleton reorganization of spreading cells on micro-patterned islands: a functional model. Philos. Trans. R. Soc., A 2010, 368 (1920), 2629-2652, DOI: 10.1098/rsta.2010.0069. (50) Boru Zhu; Thomas Eurell; Rico Gunawan; Leckband, D. Chain-length dependence of the protein and cell resistanceof oligo(ethylene glycol)-terminated self-assembledmonolayers on gold. J. Biomed. Mater. Res. 2001, 56 (3) , 406-416, DOI:10.1002/1097-4636(20010905)56:33.0.CO;2-R. (51) Chung, T.-W.; Liu, D.-Z.; Wang, S.-Y.; Wang, S.-S. Enhancement of the growth of human endothelial cells by surface roughness at nanometer scale. Biomaterials 2003, 24 (25), 4655-4661, DOI: 10.1016/s0142-9612(03)00361-2. (52) Feng, B.; Weng, J.; Yang, B. C.; Qu, S. X.; Zhang, X. D. Characterization of titanium surfaces with calcium and phosphate and osteoblast adhesion. Biomaterials 2004, 25 (17), 3421-3428, DOI: 10.1016/j.biomaterials.2003.10.044. (53) Pankov, R. Fibronectin at a glance. J. Cell Sci. 2002, 115 (20), 3861-3863, DOI: 10.1242/jcs.00059. (54) Hayakawa, T.; Yoshida, E.; Yoshimura, Y.; Uo, M.; Yoshinari, M. MC3T3-E1 Cells on Titanium Surfaces with Nanometer Smoothness and Fibronectin Immobilization. Int. J. Biomater. 2012, 2012, 743465-743471, DOI: 10.1155/2012/743465. (55) Fukazawa, K.; Ishihara, K. Fabrication of a cell-adhesive protein imprinting surface with an artificial cell membrane structure for cell capturing. Biosens Bioelectron 2009, 25 (3), 609-614, DOI: 10.1016/j.bios.2009.02.034. (56) Limin Sun; Christopher C. Berndt; Karlis A. Gross; Kucuk, A. Material fundamentals and clinical performance of plasma-sprayed hydroxyapatite coatings: A review. J. Biomed. Mater. Res. 2001, 58 (5), 570-592, DOI: 10.1002/jbm.1056. (57) Jiang, P.; Liang, J.; Song, R.; Zhang, Y.; Ren, L.; Zhang, L.; Tang, P.; Lin, C. Effect of


Page 24 of 25

Page 25 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Octacalcium-Phosphate-Modified Micro/Nanostructured Titania Surfaces on Osteoblast Response. ACS Appl. Mater. Interfaces 2015, 7 (26), 14384-14396, DOI: 10.1021/acsami.5b03172. (58) de Jonge, L. T.; Leeuwenburgh, S. C.; van den Beucken, J. J.; te Riet, J.; Daamen, W. F.; Wolke, J. G.; Scharnweber, D.; Jansen, J. A. The osteogenic effect of electrosprayed nanoscale collagen/calcium phosphate coatings on titanium. Biomaterials 2010, 31 (9), 2461-2469, DOI: 10.1016/j.biomaterials.2009.11.114.

Graphical abstract


Bacterial Inhibition and Osteoblast Adhesion on Ti Alloy Surfaces

Recommend Documents