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Dopamine Modified Organic−Inorganic Hybrid Coating for Antimicrobial and Osteogenesis Man Li,† Xiangmei Liu,† Ziqiang Xu,† K.W.K. Yeung,‡,§ and Shuilin Wu*,† †

Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry of Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei Key Laboratory of Polymer Materials, School of Materials Science & Engineering, Hubei University, Wuhan 430062, China ‡ Division of Spine Surgery, Department of Orthopaedics & Traumatology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China § Shenzhen Key Laboratory for Innovative Technology in Orthopaedic Trauma, The University of Hong Kong Shenzhen Hospital, 1 Haiyuan First Road, Futian Distract, Shenzhen, China S Supporting Information *

ABSTRACT: A hybrid coating composed of hydroxyapatite (HA), Ag nanoparticles (NPs), and chitosan (CS) was successfully prepared on a Ti substrate by a layer-by-layer assembly process. A polydopamine-assisted (PDA-assisted) coating showed a good bond with HA. Ag NPs were uniformly distributed into the hybrid coating through a solution method and ultraviolet light reduction. A CS nanofilm was deposited via spincoating to control the release of Ag+ from the hybrid coating. The results disclosed that the 3-layer CS coating could efficiently control the release of Ag+ from the hybrid coating via the Fickian diffusion mechanism and that the PDA/HA/Ag/CS-1 coating exhibited antibacterial ratios of 63.0% and 51.8% against E. coli and S. aureus, respectively. Furthermore, the normal structure of E. coli was obviously destroyed by two types of Ag doped coatings. The cell viability assay showed that CS effectively reduced the cytotoxicity of the hybrid coating after a 7 day incubation. The hybrid coating presented high ALP activities at days 3 and 14. The results reveal that hybrid coatings can endow Ti implants with good antibacterial capability as well as cell viability and osteogenic activity. KEYWORDS: titanium, poly(dopamine), HA/Ag/CS hybrid coating, release, antibacterial, biocompatibility

1. INTRODUCTION The structure and components of bones have been reported by many previous reviews or articles.1−4 The exceptional chemical and mechanical properties of natural bones draw lots of attention to imitation in designing biomimetic artificial bone tissue replacements that result from their unique hierarchical structure, in which hydroxyapatite crystals are formed and separately spatially aligned with collagen molecules to assemble into mineralized fibrils. One of the alleged triplets in bone tissue engineering is the scaffold that offers a template for cell in growth and propels the formation of bone-extracellular matrix, which can structurally support the newly formed bone tissue.5 Due to its low density, high strength, and high resistance to erosion, Ti is the most widely used metallic biomaterial for bone implants and dental fixations.6 One of the main defects of Ti is its bioinert oxide passivation layer that can quickly form on the surface of Ti implants in physiological conditions,7 which leads to a weak osteoinductivity of these implants. In view of the fact that the direct contact and subsequent biological reactions occur at the interface between tissues and implants, surface modification is © XXXX American Chemical Society

an effective strategy to trigger their desired interactions through functionalizing the surface of implants.8−10 Although some studies confirm that excellent binding can be achieved by generating reactive functional groups in chemical conjugation through some techniques such as electrochemical anodization,11 acid-etching,12 and oxidation,13,14 in order to covalently conjugate the bioactive moieties onto the Ti implant surface, these procedures are very complicated, and some intrinsic surface properties have been changed during the fabrication process. Therefore, in recent years, bioinspired coatings are attracting great attention and have been introduced onto the surface of metallic implants.15 In this work, dopamine was selected as a binding agent onto the Ti substrate to build a bridge between the additional bioactive species like hydroxyapatite (HA), chitosan (CS), and metal matrix. As a kind of catechol compound, dopamine has been proven to be suitable as a binding agent for coating on Received: July 30, 2016 Accepted: November 18, 2016

A

DOI: 10.1021/acsami.6b09457 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces inorganic surfaces.12−14 In practice, the abundant presence of dopamine connects the phenomenon of the strong adhesion of mussels onto numerous wet surfaces in nature. Polydopamine (PDA) can be used as a mimic of the Mytilus edulis foot protein-5 (Mefp-5), which is a specialized adhesive foot protein in mussels,16 and various metal ions can bond through its catechol group. 17 Ryu et al. have reported a general biomineralization route consisting of two steps for simple, aqueous, and functional approaches to integrate inorganic HA crystals onto the Ti substrate.18 Artificial implant biomaterials with different components can exhibit good biofunctions such as biocompatibility, bioactivity, and antibacterial performance.19Although HA possesses good biocompatibility and osteoconduction, it has little impact on bacterial growth when it is used clinically. Moreover, implantassociated infections occur during the transplant operation with high risk,10 and postsurgical infections always demand a series of additional surgical procedures, which are very complicated.20 In the case of therapy for bacterial infections, the systemic antibiotic injection often raises several serious issues, such as inherent toxicity, low efficiency, and emergence of resistance strains.21 Therefore, in the case of HA coatings, it is crucial to further improve their bioactivity and to endow them with high antibacterial performance in order to ensure the satisfied repair of damaged bone and subsequent recovery of patients. As a natural biocompatible biopolymer with unique structure, CS exhibits a great potential for biomedical applications such as antimicrobial agents,22 biomedical devices,23 and implants for controlled drug release.24 It has been frequently introduced in conjunction with bone forming matrix.25 Some studies have reported that CS-based hydroxyapatite biocomposites have been widely used to promote the reconstruction of bone defects.26−28However, these composites showed lower antibacterial activity. Ag nanoparticles (NPs) have been proven to possess superior antibacterial property against various pathogenic bacteria (Gram-positive as well as Gram-negative bacteria).29,30 As for antibacterial mechanisms of Ag NPs, the mainstream point is that Ag inhibits the activity of respiratory enzymes, partly interferes the replication of DNA, and disrupts the normality of bacterial cell membrane.31,32 However, except for its excellent antibacteral activity, high release concentration of Ag often induces high cytotoxicity.33,34 It is widely accepted that the amino groups (NH2) functionalized on CS can chelate with Ag+ and nanosilver particles with coordination interactions;35,36 hence, it may serve as a stabilizing ligand to controlled-release Ag+ to lower the cytotoxicity. In view of these observations, in this work, we designed a bioinspired hybrid coating through the deposition of composites composed of Ag doped HA and CS (HA/Ag/ CS) onto the surface of a PDA modified Ti implant. In vitro studies were performed to determine whether the HA/Ag/CS hybrid coating could endow titanium implants with both good self-antibacterial performance and good osteoinductive ability. The process was schematically depicted in Scheme 1.

Scheme 1. Schematic Illustration of a Hybrid Coating on Ti Implant via a Layer-by-Layer Method and Its Disruptive Behavior on a Bacterial Cell Membrane

The Ti plates were ground with SiC grinding papers of 240, 400, 800, and 1200 grits gradually. Afterward, these plates went through a 30 min ultrasonic cleaning with ethyl alcohol and deionized water successively and then were dried in air at 37 °C and stored for further use. 2.2. PDA-Assisted HA Coating. A thin PDA layer was formed on the surface of Ti substrates through the oxidative polymerization of dopamine-hydrochloride which was dissolved into 10 mM Tris buffer with a final pH value of 8.5. This oxidative polymerization was carried out for 24 h, and then dark brown solution was obtained. The dopamine monomers were subjected to self-initiated polymerization and formed PDA on Ti substrate, which is depicted in Figure 1. After the reaction proceeded for a certain time, substrates coated with PDA were rinsed with abundant deionized water and dried in the N2 gas atmosphere, and then incubated in 1.5-fold simulated body fluid (1.5-SBF) at 37 °C in a calorstat to grow into HA. 1.5-SBF was changed every 3 days. In Table 1, the ionic concentrations of blood plasma, SBF, and 1.5-SBF have been reported.37−39 The solution was freshly prepared before use. After sequential immersion for a given time, all samples were gently rinsed with deionized water to remove excess ions and dried with a stream of N2 gas. 2.3. PDA/HA/Ag/CS Hybrid Coating. The above samples with an incubation in SBF for 14 days were immersed in AgNO3 solution with different concentrations (1 mM, 2 mM, 3 mM, 4 mM) for 30 min, rinsed with deionized water, and dried in air. Then, the samples were immersed in 0.1 mol/L methanol and illuminated with 12 W UV light (λ = 253 nm) for 30 min. Finally, samples were taken out and rinsed with flowing deionized water and dried with a stream of nitrogen. These samples could be denoted as PDA/HA/Ag-1, PDA/HA/Ag-2, PDA/HA/Ag-3, and PDA/HA/Ag-4. The method of spin-coating was used to prepare the CS coating. Solutions of 2 mg mL−1 chitosan 1% (v/v) acetic acid were deposited onto the plates, spun at 800 rpm for 20 s to allow uniform spreading of the solution on the surface of substrates and then at 4000 rpm for 30 s to thin the solution layer, and finally dried in the air. The process was repeated three times for each sample. These samples could be briefly denoted as PDA/HA/Ag/CS-1, PDA/HA/Ag/CS-2, PDA/HA/Ag/ CS-3, and PDA/HA/Ag/CS-4. 2.4. Surface Characterization. A scanning electron microscope (SEM; JSM6510LV, Japan) equipped with an X-ray energy dispersive spectrometer (EDS) was employed to analyze both the morphology and composition of the pure HA, HA/Ag, and CS/Ag/HA coatings. A transmission electron microscope (TEM; Tecnai G20, FEI) was used to observe the microstructures of the HA crystal and Ag in the coatings. An X-ray diffractometer (XRD; D8A25, BRUKER) was applied to examine the structure of HA crystals on PDA modified Ti plates. Fourier transform infrared spectroscopy (FTIR; NICOLET

2. EXPERIMENTAL PROCEDURES 2.1. Starting Materials. Pure Ti (Shanghai Baosteel Co. Ltd., China) was cut into discs with a diameter of 6 mm and a thickness of 2.5 mm. 3,4-Dihydroxyphenethylamine (DA·HCl, 99%), tris(hydroxymethyl) aminomethane (Tris), and chitosan (viscosity: 100−200, 179.17 MW) were purchased from Sigma-Aldrich. All reagents were of analytical grade and used without further purification. B

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Figure 1. (a) Electron microscopy images with low signification of polydopamine modification of the Ti substrate and (b) FTIR of polydopamine coating on Ti substrate.

Table 1. Ionic Concentration of Human Blood Plasma, SBF, 1.5-SBF ion conc (mM)

Na+

K+

Ca2+

Mg2+

HCO3−

Cl−

HPO4−

SO42−

blood plasma SBF 1.5-SBF

142 142 213

5 5 7.5

2.5 2.5 3.75

1.5 1.5 2.25

27 4.2 6.3

103 148 221.7

1 1 1.5

0.5 0.5 0.75

2.6.3. Integrity of Cell Membranes. Samples were seeded with bacterial suspension. After incubation for 2 h, the samples were rinsed three times with PBS and then stained for fluorescence analysis. Equal volumes of DAPI (10 μg/mL) and PI (10 μg/mL) were used to distinguish live/dead bacterial cells. Each sample was stained for 15 min in the absence of light, and then visualized with a fluorescence microscope. 2.7. In Vitro Tests. 2.7.1. Cell Culture. Mouse calvarial cell line MC3T3-E1 was used in this work. Cells were routinely cultured in αMEM (HyClone) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin solution (HyClone) in a humidified atmosphere of 5% CO2 at 37 °C. Cells were subcultured 1:3 before reaching confluence using sterile PBS and trpsin/EDTA (Sigma). To test the surface modification of Ti implants with hybrid coatings, the as-sterilized specimens were placed in 48-well culture plates at a density of 1 × 105 cells/mL. The medium was replaced with fresh medium every 2 days. 2.7.2. Cell Viability and Proliferation. The cytocompatibility for cell cultures on samples was measured through a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. After incubation for 1 day, 3 days, and 7 days, the cells were further incubated with MTT solution (0.5 mg mL−1) for 4 h at 37 °C until a purple precipitate was visible. The medium was then removed, and 150 μL of dimethyl sulfoxide (DMSO) was subsequently added to each well to dissolve the previously formed formazan crystals. Optical density at 490 nm was read by a microplate reader. All assays were performed for 3 replicate wells for every sample and control. 2.7.3. Osteogenic Differentiation. For evaluation of the osteogenic activity, cells on substrates with 3, 7, and 14 days’ incubation were examined for ALP activity. After incubation, samples were rinsed with PBS three times, and then, 1% Triton X-100 was added for cell lysis. Stain reactions were carried out according to a commercially available ALP assay kit’s instructions in 96-well plates. After ALP from cell lysate decomposed disodium phenyl phosphate into free phenol and phosphoric acid, 4-aminoantipyrine was added to react with phenol in an alkaline environment, and red ramifications were produced by potassium ferricyanide oxidation. The ALP activity was measured by a microplate reader at a wavelength of 520 nm. All experiments were performed with 3 replicate wells for every sample and control per assay.

Is50, Thermo Scientific) was introduced to identify the PDA and CS in the hybrid coatings. 2.5. Ag+ Release Testing. Samples were immersed in 5 mL phosphate buffer solutions (PBS) with dark surroundings. Analysis of the silver concentration was, respectively, carried out in 1, 4, 7, 10, 14, and 30 days. Incubation solution was collected and refilled with fresh 5 mL portions of PBS at each time interval. A sample without CS coating was used as a blank control group. The Ag concentrations of collected solutions were measured using inductively coupled plasma atomic emission spectroscopy (ICP-AES; Optimal 8000, PerkinElmer) by absorption at 328.07 nm. 2.6. Bacteria Response to Surfaces. 2.6.1. Antibacterial Activity. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were used to evaluate the antibacterial activity of the hybrid coatings. They were cultured in a Luria−Bertani (LB) medium. A standard procedure was utilized to prepare sterile LB broth and LB agar plates. Three parallel samples in each group were applied to study the antibacterial activities of the hybrid coatings. . Samples were placed in a 48-well plate, and 500 μL of diluted bacterial suspension (106 CFU/mL) was added into each well containing a different sample. Then, samples were incubated at 37 °C in an incubator shaker for 12 h with E. coli and S. aureus. Bacterial growth can be determined by the absorption of optical density (OD) at 600 nm via a microplate reader (SpectraMax I3, Molecular Devices). Also, the bactericidal ratio was introduced to evaluate the antibacterial activity of coatings, which was calculated according to the following equation:40,41

bacterial ratio (%) OD of control group − OD of experimental group = OD of control group × 100%

(1)

LB agar plates seeded with bacteria were used to measure the zones of inhibition. After bacterium suspension (OD ∼ 0.45) was spread directly onto each agar plate, samples were placed on the agar plates and then incubated at 37 °C for 12 h with E. coli and S. aureus. 2.6.2. Morphological Studies. After incubation for a certain time, the samples were fixed with 2.5% glutaraldehyde for 4 h, dehydrated sequentially in a series of ethanol with different concentrations (30, 50, 75, 90, 95, and 100 v/v %), and freeze-dried. Then, the morphologies of bacteria on Ti plates were observed by SEM. C

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Figure 2. Different surface topographies for the formation of calcium phosphate minerals on polydopamine. Modification of the Ti substrate and pure Ti was observed by SEM/EDX with low magnification.

Figure 3. Structural analysis of hydroxyapatite crystals on polydopamine modified Ti: (a) TEM/SAED and (b) XRD. Surface topography and structural analysis of Ag doped on the surface of HA: (c) SEM and (d) XRD.

3. RESULTS AND DISCUSSION 3.1. Characterization of PDA/HA Coatings. PDA coatings are formed on Ti substrate by a universal immersion of the Ti in dopamine solution dissolved in Tris buffer; these are highly robust and have been proven to have utility conjugation with HA.42 Figure S1 (Supporting Information) schematically represents the self-assembly process of dopamine

on Ti. Briefly, the covalent oxidative polymerization and crosslinking of PDA occur on Ti through pH induced oxidization. The dopamine molecules are oxidized to form a series of middle products. Then, the 5,6-dihydroxyindole (DHI) and 5,6-indolequinone may be produced through the rearrangement of the middle products. Finally, multiple isomers of dimers and higher oligomers, DHI-DHI and dopamine-DHID

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Figure 4. (a) In vitro release profiles of Ag ions (1 mM) from substrates with or without CS covered coatings. (b) In vitro release profiles of Ag ions from different Ag concentration coating with 3 layers CS covered.

intensively decreased from 16.36 at. % (Figure S2) to 0.66 at. % (Figure S3), and that the atomic ratio of Ag, P, and O is almost close to 3.4:1:3.3. The XRD pattern shown in Figure 3d implies that the coatings are predominantly composed of silver phosphate (SP) and small amount of HA (Figure 3d), which indicate that the surface deposited HA particles react with silver nitrate to form SP NPs during the period of immersion in 1 mM AgNO3 solution.48,49 The higher Ag atomic ratio detected by EDS than that in SP indicates that there is a small amount of Ag NPs produced by the following UV irradiation. SP NPs are partly agglomerated and form small clusters on the surface of HA. There are a large number of hydrogen bonds between or within the chains of CS molecules according to the structure of CS shown in Figure S4. Owing to the different existences of the length and intensity of hydrogen bonds, the frequency values of the stretching vibration bands of functional groups of CS molecules are in a wide range. In the FTIR spectrum of CS coating shown in Figure S5, the peaks at ∼3400 and 1652 cm−1 correspond to the amide group of CS.50 The peak at 1149 cm−1 is the signal of the CC bond. The strong peaks observed at 1075 and 1029 cm−1 are associated with the stretching vibration of COC and CN bonds.51 These characteristic peaks verify the presence of CS in this hybrid coating. 3.3. Ag Release and Antibacterial Activity of Hybrid Coatings. Ag released from the LBL composite coating is evaluated by ICP-AES. Samples are immersed in PBS for up to 30 days with periodic sampling of the Ag concentration in the PBS solution. Figure 4a shows the release behaviors of Ag+ from hybrid coatings with different CS layers. It can be obviously observed that the Ag release concentration can be controlled by adjusting the number of spin-coatings of CS. Samples without CS coating (0CS) show the highest leaching concentration among all types of samples, and Ag release concentration decreases with the increasing number of CS. Compared to 0CS, other samples show a more stable and much slower silver release concentration after one-week immersion in PBS. Since the release rate of Ag+ is fast in the first 4 days and subsequently gradually slows down in the following days, the continuous and sustained Ag+ released from hybrid coatings can be achieved by building this hybrid coating on Ti implants. The relationship between initial reaction concentrations of silver nitrate and Ag+ is shown in Figure 4b. In the earlier stage, all of the samples exhibit fast Ag release rate. After 14 days, in terms of samples with lower initial concentrations of AgNO3, i.e., 1 and 2 mM, the release concentration of Ag+ tends to

DHI trimeric conjugate, may derive from these products through branching reactions.43 PDA subsequently forms crosslinked coatings on Ti. As shown in Figure 1a, the deposited PDA film is very thin and almost cannot change the surface morphologies of mechanically polished Ti plates. The specular reflection infrared spectroscopy discloses that PDA is successfully deposited on the surface of Ti (Figure 1b). Peaks at ∼1170 and ∼1730 cm−1 derive from the aromatic CO and CH bonds, respectively. Weak absorption peaks at 1400− 1600 cm−1 are attributed to CC and NH bonds of PDA. The measured spectrum of as-deposited PDA is in good agreement with the reported results.44−46 As shown in Figure 2, some crystals can rapidly nucleate on the PDA coating, and after incubation in SBF for 3 days, many white crystals appear on the entire surface area. It has been reported that free catechol groups of PDA boost the integrations of interfaces, and that the abundant part of free catechol that do not participate in substrate surface adhesion plays a critical role in the formation process of a CaP nucleus.18 After 2 weeks incubation, PDA modified Ti substrate is fully covered by spherical minerals, while there is almost no deposition on unmodified Ti even after incubation for 2 weeks. EDS results reveal that the minerals assembled on the surface of PDA-Ti are mainly composed of Ca and P (insert image) with an atomic ratio of 1.46 that is slightly less than the theoretical ratio of HA due to the trace amount of magnesium (shown in Figure S2, Supporting Information). Transmission electron microscopy (TEM) analysis showed lath-like nanocrystals (Figure 3a). The inserted selected area electron diffraction (SEAD) image confirms that the mineral is mainly composed of crystalline and amorphous HA. The diffraction pattern of the CaP agglomerates exhibits arcs corresponding to the (002) and (004) diffraction rings, suggesting that PDA directs the directional growth of HA crystals. The HA crystals grew aligned along the c-axes parallel to PDA.47 Meanwhile, the XRD pattern shown in Figure 3b further proves that the assembled minerals on PDA are HA. On the basis of the above analysis, it can be concluded that PDA on Ti can promote the formation of HA in physiological environments. 3.2. Characterization of PDA/HA/Ag/CS Coatings. As we can see in Figure 3c, after immersion in 1 mM silver nitrate solution with UV irradiation, compared with HA deposited surface shown in Figure 2, there are numerous small nanoparticles formed on the surface. In addition, EDS area scan discloses that the content of Ca on the surface is E

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In order to assess the bacterial killing efficiency of substrates with immersion in the initial 1 mM silver nitrate and the following 3 layers of CS deposition, suspension assays have been carried out after periodic incubation.54 The antibacterial ratios have been calculated by eq 1, and the results have been shown in Figure S6. The antibacterial ratio of the PDA/HA/ Ag-1 coating has revealed the smallest OD value and the highest antibacterial ratio of over 78.0% and 59.3% in E. coli and S. aureus, respectively, indicating that PDA/HA/Ag-1 coatings are effective to inhibit bacterial growth, which is possibly ascribed to the bactericidal action of Ag+ released from the coatings. In comparison with the PDA/HA/Ag-1 coating, the antibacterial ratio of the PDA/HA/Ag/CS-1 hybrid coating slightly drops. It is believed that the CS coating defers the Ag release from the coatings, which can be confirmed by the release curve of Ag+ shown in Figure 4a. Several possible mechanisms have been proposed for the action of Ag on bacteria. The widely accepted one is that the antimicrobial activity of implants is mainly attributed to the released Ag+ and its binding to the thiol groups of bacterial enzymes and interferes with DNA replications.55,56 It is widely accepted that the antibacterial mechanism of CS mainly stemmed from the interaction between NH3+ of chitosan which possesses positive charge and the cell membranes which are usually negative charged. This interaction may disrupt the organization of the outer membrane and increase its permeability. Finally, it may lead to the leakage of proteinaceous and intracellular constituents.57 Disc diffusion studies have been conducted in independent duplicate using E. coli and S. aureus covered agar plates. The increase in diameter of the inhibition zone around the disc has been measured after periodic incubation at 37 °C. PDA/HA/ Ag-1 and PDA/HA/Ag/CS-1 coatings display an obvious inhibition zone on both E. coli and S. aureus shown in Figure 5. The PDA/HA/Ag group exhibits a much larger diameter of the inhibition zone than the PDA/HA/Ag/CS-1 group, which is in accordance with the suspension assays (Figure S6). This further suggests that the leaching Ag+ species play a critical role for antibacterial activity of hybrid coatings; i.e., the leaching Ag+ ions can diffuse into the surrounding environment to kill bacteria. No inhibition zone has been observed in the HA

become a constant, whereas it remains increasing for samples with initial higher AgNO3 concentration of 3 and 4 mM. The mechanism for Ag release from the hybrid coatings has been elaborated in this study; a simple Ritger−Peppas model (eq 2) is suitable for fitting the release curves:

Q = kt n

(2)

Here, Q represents the fraction of total Ag released from the hybrid coatings, and k and n represent the kinetic constant and release exponent, respectively. As for the thin film model, the following limits apply: if n < 0.5, the diffusion is Fickian; if 0.5 < n < 1, released diffusion is a kind of diffusion/erosion mechanism; and if n = 1, the released profile dominated by zero order drug release mechanism.52,53 The values of n of the Ag release curves in this work are shown in Table 2. Both the Table 2. Ag+ Release Kinetics Parameters Obtained from the Peppas Model in 2 Weeks sample

k

n

R2

0CS 1CS 3CS 5CS 7CS 1 mM 2 mM 3 mM 4 mM

0.279 0.232 0.226 0.151 0.170 0.226 0.218 0.376 0.519

0.619 0.644 0.457 0.470 0.310 0.457 0.560 0.603 0.646

0.989 0.976 0.925 0.996 0.952 0.925 0.999 0.994 0.993

initial concentration of AgNO3 and thickness of CS layer obviously play important roles in the release mechanism of Ag in the first 2 weeks. The release mechanism of Ag was changing from Fickian diffusion to erosive diffusion with the increase of the initial concentration of AgNO3 and the decrease of the thickness of CS. 3.4. Antibacterial Activity of Hybrid Coatings. The antibacterial activity of PDA/HA/Ag/CS hybrid coatings has been investigated in two different bacterial strains, namely, Gram-negative bacteria E. coli and Gram-positive ones S. aureus, in suspension culture and by the disc diffusion method.

Figure 5. E. coli and S. aureus inhibition zones of different samples. F

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Figure 6. Typical SEM images of E. coli and S. aureuss morphology on (a) Ti, (b) PDA/HA, (c) PDA/HA/Ag-1, and (d) PDA/HA/Ag/CS-1.

Figure 7. (a) MTT assay of cell viability on different coatings for 1, 3, and 7 days (mean ± SD, n = 3). (b) ALP activity of cells on different coatings for 3, 7, and 14 days (mean ± SD, n = 3).

doped coatings, and an increased adhesion has been observed. The results are consistent with those that have been recorded on optical density measurements, which suggests that the hybrid coatings are efficient for bacteria killing, especially E. coli. A fluorescence staining assay has been carried out to further verify the antibacterial activities of the hybrid coatings (shown in Figures S7 and S8). Membrane-impermeable propidium iodide (PI)-labeled dead bacteria with red fluorescence while only damaged bacterial membranes are susceptible to PI. In addition, membrane permanent 4,6-diamidino 2-phenylindole (DAPI) has been used to label live bacteria with blue fluorescence. Fluorescence images of E. coli and S. aureus clearly show that most bacteria are alive in the control group, which has been shown via the DAPI blue fluorescence in the merge images of pure titanium substrates. Blue fluorescence can be observed in the image of the HA coating group as well. In the PDA/HA/Ag-1 coating group, it confirms that most bacteria are dead, as shown by the lack of blue fluorescence in Figures S7 and S8. Both DAPI-labeled blue fluorescence (live bacteria) and PI-labeled red fluorescence (dead bacteria) bacterial strains are apparent on PDA/HA/Ag/CS-1 hybrid coatings; the results are consistent with the values of optical density measurements, demonstrating that CS coating can control the release of Ag+ from substrate but not significantly attenuate the antibacterial activity of hybrid coatings. 3.6. Cytotoxicity Assay in Vitro. The cell viabilities of different samples after 1, 3, and 7 days of incubation are shown in Figure 7a. In the first day, lower cellular survival rates were

group (2 in Figure 5), while it exhibits a weak antimicrobial activity to E. coli and S. aureus with ratios of 25.3% and 6.8%, respectively, in suspension assays (in Figure S6). This phenomenon is similar to the one reported by Chen et al.58It implies, to some extent, that nanoscaled HA crystals on the coatings can kill some of them directly, but there is no diffusion of ions. On the other hand, clear zones of growth inhibition have been observed in PDA/HA/Ag-1 and PDA/HA/Ag/CS-1 (in Figure 5) which resulted from strong antibacterial activity of the released Ag+. 3.5. Bacterial Morphology and Fluorescence Assay. SEM has been employed to observe the morphology of E. coli and S. aureus after periodic incubation on PDA/HA/Ag/CS-1 coating (Figure 6) for detecting the influence of the hybrid coatings on the bacterial morphology. In the control group, untreated E. coli cells on pure Ti substrate are typically rodshaped. Their surface is very smooth, and intact cell walls can also be observed. SEM images of E. coli on HA coating demonstrate a larger amount than on pure titanium substrate, but both the cell walls and cell shapes appear normal. The surfaces of E. coli on PDA/HA/Ag-1 and PDA/HA/Ag/CS-1 coatings have become rough and wrinkled after 12-h incubation since Ag+ can destroy the bacterial membranes and be toxic to respiratory enzyme in bacterial cells.55 As for S. aureus, the results of SEM observations are similar to those of E. coli cells. As shown in Figure 6, the S. aureus cells are spherical shaped, and the surface is very smooth on the control group. However, there is no evident change in the shape of S. aureus cells on Ag G

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observed on HA, PDA/HA/Ag-1, and PDA/HA/Ag/CS-1 groups compared with that of the control group. After 3 day incubation, HA began to show cytocompatibility while coatings with PDA/HA/Ag-1 exhibited lower cell viability. However, after one-week incubation, both HA and PDA/HA/Ag/CS-1 groups showed excellent cytocompatibility. The lowest cellular survival rate of PDA/HA/Ag-1 coating substrate was clearly observed. In summary, CS can control the release of Ag+ to reduce cytotoxicity of hybrid coatings in the early cultivation stage, and improve the cytocompatibility after one-week cultivation. 3.7. ALP Activity Assay. ALP is an enzyme which is produced by the cells during osteogenic differentiation. The ALP activity of MC3T3-E1 on samples is shown in Figure 7b. All samples presented notably high ALP expression at day 3, and the ALP activities reduced at day 7, and then increased at day 14. Taking into account the Ag+ released concentrations, the lower initial released concentrations of Ag+ (samples with 1 mM as shown in Figure 4a) in the first 3 days are believed to promote the MC3T3-E1 osteogenic proliferation. Hence, the ALP activities on hybrid coatings with Ag doped samples are larger than that on controlled pure Ti samples. Ag+ plays an important role in ALP expressions. With the increase of Ag+ released concentration at day 7, less ALP is generated on hybrid coatings. PDA/HA/Ag/CS-1 presented the highest ALP activity at days 3 and 14 compared with that of other experimental groups due to the addition of the CS coating, which could improve the ossification59 and reduce the cytotoxicity induced by Ag+.60 The ALP activity increases to higher levels at day 14, and MC3T3-E1 on PDA/HA/Ag/CS-1 secretes more ALP than other samples. Hydrolytic CS coordinates with Ag+ can efficiently decrease the silver metal ion toxicity,60and Ag+ could effectively stimulate the formation of bone tissue after inflammatory reaction. It can be inferred that the hybrid coating efficiently enhances the long-term osteogene capability of the Ti substrate.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] or [email protected]. Phone: +86-27-88661729. Fax: +86-27-88665610. ORCID

Shuilin Wu: 0000-0002-1270-1870 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is jointly supported by Special Prophase Program for Key Basic Research of the Ministry of Science and Technology of China (973 Program) No. 2014CB660809, and the National Natural Science Foundation of China, Nos. 51422102, 51671081, 81271715, and 21603067.

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4. CONCLUSIONS PDA accelerates the formation of HA crystals and bridges the binding strength between HA and Ti substrate. SP NPs with sizes ranging from 50 to 100 nm are distributed uniformly on HA. Both the concentration of AgNO3 and CS layer thickness play important roles in Ag+ release from hybrid coatings. With the cover of CS, the release of Ag+ lasts for more than 2 weeks. In antibacterial tests, HA exhibits weak antibacterial activity while the Ag doped hybrid coating actually has a higher antimicrobial effect (63.0% and 51.8% on E. coli and S. aureus, respectively) and can damage the shape of bacterial cells, especially E. coli. Ag+ shows disruptive behavior on bacterial cell activity, which is proven by fluorescence assay. Although Ag+ is toxic to bone cells, CS effectively reduces the cytotoxicity of hybrid coating and significantly improves the cell viability after 7 day cultivation. Moreover, the hybrid coating presents a longterm osteogenesis.

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DOI: 10.1021/acsami.6b09457 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX