J Mater Sci: Mater Med (2014) 25:1435–1448 DOI 10.1007/s10856-014-5190-8
Surface modification of titanium substrates with silver nanoparticles embedded sulfhydrylated chitosan/gelatin polyelectrolyte multilayer films for antibacterial application Wen Li • Dawei Xu • Yan Hu • Kaiyong Cai Yingcheng Lin
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Received: 5 September 2013 / Accepted: 8 March 2014 / Published online: 25 March 2014 Ó Springer Science+Business Media New York 2014
Abstract To develop Ti implants with potent antibacterial activity, a novel ‘‘sandwich-type’’ structure of sulfhydrylated chitosan (Chi-SH)/gelatin (Gel) polyelectrolyte multilayer films embedding silver (Ag) nanoparticles was coated onto titanium substrate using a spin-assisted layer-by-layer assembly technique. Ag ions would be enriched in the polyelectrolyte multilayer films via the specific interactions between Ag ions and –HS groups in Chi-HS, thus leading to the formation of Ag nanoparticles in situ by photo-catalytic reaction (ultraviolet irradiation). Contact angle measurement and field emission scanning electron microscopy equipped with energy dispersive X-ray spectroscopy were employed to monitor the construction of Ag-containing multilayer on titanium surface, respectively. The functional multilayered films on titanium substrate [Ti/PEI/(Gel/ChiSH/Ag)n/Gel] could efficiently inhibit the growth and activity of Bacillus subtitles and Escherichia coli onto titanium surface. Moreover, studies in vitro confirmed that Ti substrates coating with functional multilayer films remained the biological functions of osteoblasts, which was reflected by cell morphology, cell viability and ALP activity measurements. This study provides a simple, versatile and generalized methodology to design functional titanium implants with good cyto-compatibility and antibacterial activity for potential clinical applications.
W. Li D. Xu Y. Hu (&) K. Cai Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, People’s Republic of China e-mail: [email protected] Y. Lin College of Communication Engineering, Chongqing University, Chongqing 400044, People’s Republic of China
1 Introduction Titanium-based orthopedic implants have been extensively used in the medical clinical applications since they have good biocompatibility and mechanical properties. However, infection on or around titanium implants still remains a vital problem to deal with, which may result in eventual implant loosening and removal [1–3]. To inhibit the bacteria adhesions and biofilms formation, various antibacterial agents or adhesion-resistant materials were introduced into the Ti implants surfaces [4, 5]. However, most of antibacterial molecules were still susceptible to physiological environment, thus leading them to lose bioactivity in a short time [6]. Interestingly, silver (Ag) nanoparticles could long-term resist the formation of bacterial biofilm onto implants while not impairing the biological functions of mammalian cells [3, 7–11]. At present, Ag nanoparticles were usually directly coated onto or incorporated into Ti implants by using chemical vapor deposition (CVD) or physical vapor deposition (PVD) methods [12–16]. The fabrication processes tended to be time consuming and high costly. Therefore, it is necessary to design a simple and versatile method to deposit Ag nanoparticles onto Ti substrates to efficiently inhibit the growth and activity of pathogenic bacteria while not affecting bio-functions of mammalian cells [17]. Recently, layer-by-layer assembly (LBLs) technique attracts extensive attention since it provides a simple, useful and versatile methodology for material surface modifications [18, 19]. And many antibacterial agents with surface charge properties (antibiotic [20], antibacterial polymer/peptide [21, 22] or other antibacterial agents [23]) could be directly deposited onto material surfaces by using LBL technique. Moreover, Ag ions also could be incorporated into multilayered structure via ion exchange or
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electrodeposition [24, 25], thus leading to the formation of Ag nanoparticles in situ after adding external reductive agents (e.g. NaBH4) [26]. The Ag-containing films had long-lasting antimicrobial activity against microorganism, such as Staphylococcus aureus and Escherichia coli (E. coli) [27, 28]. Herein, we design and fabricate ‘‘sandwich’’ sulfhydrylated chitosan (Chi-SH)/gelatin (gel) multilayer films with Ag nanoparticles onto titanium substrates via a spinassisted LBLs technique (Fig. 1a). The sulfhydrylated chitosan (Chi-SH) molecules acted as Ag ion nanocontainers due to the specific conjugation of Ag ion to –HS groups in Chi-SH. The Ag nanoparticles would be then formed in situ within multilayer films after the
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photocatalytic reactions (ultraviolet irradiation), while not introducing the extra reductive agents. It was reported that Ag ions could be bound to the membrane of microorganism [28] and uptaken by bacteria, thus leading to the death of bacteria when its accumulation concentration reached the toxicity threshold. The potential mechanism lies in that Ag ions coordinate with electron-donating groups such as carboxylates, imidazoles, amides, indoles, hydroxyls as well as thiols [29, 30]. We validated the binding effect of sulfhydrylated chitosan to Ag ions within multilayer structure and their antibacterial activity against E. coli and Bacillus subtitles. Meanwhile, the cellular responses of osteoblasts to those substrates were also investigated.
Fig. 1 a Scheme of the fabrication of silver-containing multilayered films on titanium surface based on sulfhydrylated chitosan. b Synthesis of sulfhydrylated chitosan
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2 Materials and methods
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2.4 Characterization of silver-containing multilayer films
2.1 Materials Titanium disks (15 mm dia 9 2 mm thick) were provided by Northwest Institute for Non-ferrous Metal Research, Xi’an, China. The chemicals of N-hydroxybenzotriazole (HOBt) and N-acetyl-L-cysteine (NAC) were obtained from Alfa Aesar Co. Ltd. (Tianjin, China). And the products of chitosan, gelatin, 1-ethyl-3-(3-dimethylaminopropyl)–carbodiimide (EDC), p-nitrophenyl phosphate, 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) and Hoechst 33258 were supplied by Sigma-Aldrich (St. Louis, MO, USA). 2.2 Synthesis and characterization of sulfhydrylated chitosan Sulfhydrylated chitosan was synthesized according to a previous study [31]. Briefly, both of chitosan (500 mg) and HOBt (348.5 mg) were dispersed into 46 mL of deionized water with stirring until the solution was transparent. Then, a mixture of NAC (420 mg) and EDC (989 mg) in 4 mL deionized water were added into the above solution and stirred for another 2 h (pH 5). Subsequently, the resulting product was purified using a dialysis tube (8,000–14,000 MWCO) against 5 mM HCl containing 1 % NaCl and 1 mM HCl, respectively. Finally, the product was obtained after treatment with lyophilization, denoting as Chi-SH. The structure properties of Chi-SH were characterized by fourier transform infrared spectrometry (FTIR) and 1H nuclear magnetic resonance spectroscopy (1H NMR). 2.3 Construction of silver-containing multilayer films The silver-containing multilayered films onto Ti substance were prepared as following. Firstly, Gelatin (5 mg/mL) and polyethyleneimine (PEI) solution (5 mg/mL) were prepared with phosphate buffered saline (PBS, pH 7.4). Meanwhile, Chi-SH (5 mg/mL) and AgNO3 (0.005 and 0.1 M) solutions were prepared with deionized water, Then, 100 lL of PEI solution was deposited onto titanium substrates by spin coating at a speed of 4000-rpm for 40 s. After rinsing with deionized water, the above solution of gelatin, Chi-SH and AgNO3 were subsequently deposited onto pre-coated titanium substrates by using the above procedure. The procedures were repeated until the desired multilayer films of (Gel/Chi-SH/Ag)n onto titanium substrates were achieved. Finally, Ag nanoparticles would be formed in situ when exposed to ultraviolet lamp (100 W, 48 h). The sample was denoted as Ti/PEI/(Gel/Chi-SH/ Ag)n/Gel.
The construction of silver-containing multilayer films onto titanium substrates was monitored by contact angle measurements (Future Scientific Ltd Co. Taiwan, China). Meanwhile, the morphologies of the substrates were observed by field emission scanning electron microscopy (FE-SEM) equipped with energy dispersive X-ray spectroscopy (EDS) (FEINova 400 Nano SEM, Phillips Co. Holland). 2.5 Bacteria culture E. coli (gram negative bacteria) [32] and B. subtitles (gram positive bacteria) [32] were employed to investigate the broad spectrum antibacterial activity of Ti/PEI/(Gel/ChiSH/Ag)n/Gel. Firstly, both of the two bacteria were cultured with Cation-adjusted Mueller Hinton Broth II (CMHB) at 37 °C for 12 h, respectively. The bacteriacontaining mediums were then centrifuged at 3000 rpm for 10 min. After that, the bacteria were washed with fresh CMHB medium. Finally, the bacteria were re-suspended in 1 mL of CMHB medium for the following studies. 2.6 Kirby–Bauer test Kirby–Bauer test was performed according to a previous report [26]. Briefly, an agar gel plate was firstly swabbed with 30 lL of E. coli solution at a concentration of 1 9 106 cells/mL. Then, the samples of titanium, Ti/PEI/ (Gel/Chi/Ag0.1)4/Gel, Ti/PEI/(Gel/Chi-SH/Ag0.1)4/Gel, Ti/ PEI/(Gel/Chi-SH)4/Gel and Ti/PEI/(Gel/Chi-SH/Ag0.005)n/ Gel (n = 1, 2, 3, 4) substrates were placed onto the agar plates, respectively. Subsequently, all plates were incubated at 37 °C for another 48 h. Finally, the zones of inhibition (ZOIs) around different samples were observed by an optical microscope. The sizes of ZOIs were directly correlated to the antimicrobial activity of the substrates. 2.7 Bacteria adhesion Escherichia coli and B. subtitles were cultured as described above. Titanium, Ti/PEI/(Gel/Chi-SH)4/Gel and Ti/PEI/(Gel/Chi-SH/Ag0.005)n/Gel (n = 1, 2, 3, 4) substrates were exposed to bacteria suspension at a concentration of 1 9 106 cells/mL, respectively. After incubation for 12 h, all of the substrates were taken out and washed with fresh CMHB medium. Subsequently, the remaining bacteria onto the surfaces of different samples were fixed by 2 % glutaraldehyde for 10 min and stained by 10 lg/mL of Hoechst 33258 at 4 °C for
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5 min, respectively. The stained samples were mounted with 90 % glycerinum and observed with confocal laser scanning microscopy (CLSM, 510 Metanlo, Zeiss Co., Germany). 2.8 Bacteria activity E. coli and B. subtitles were also obtained as described above. The sample of titanium, Ti/PEI/(Gel/Chi-SH)4/Gel and Ti/PEI/(Gel/Chi-SH/Ag0.005)n/Gel (n = 1, 2, 3, 4) substrates were immersed into bacteria suspension at a concentration of 1 9 106 cells/mL. After incubation for 12 h, the bacteria adhered onto the samples were removed with 2 mL of fresh CHMB medium by vigorous vortexing at room temperature for 5 min. Subsequently, agar gel plate was swabbed with 30 lL of bacteria suspension. After incubation at 37 °C for 12 h, the bacteria-containing agar gel plate was taken out and placed into 24-well plate, followed by adding 0.9 mL of fresh CHMB medium and 0.1 mL of MTT solution (5 mg/mL). After incubation at 37 °C for another 4 h, MTT-containing medium was removed and 0.5 mL of dimethyl sulfoxide (DMSO) was added to dissolve formazan crystals. After centrifugation, the optical density of supernatant was measured at a wavelength of 490 nm [33]. Each treatment was performed for 3 times. And the mean value was used as the final result. 2.9 In vitro cytocompatibility evaluation
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2.9.3 Cell viability MTT assay was used to determine the viability of osteoblasts cultured on different substrates. After culture for 48 h, 100 lL of MTT (5 mg/mL) was added to each well and incubated at 37 °C for another 4 h. MTT containing medium was then removed and 0.6 mL of dimethyl sulfoxide (DMSO) was added to dissolve formazan crystals. The optical density of the solution was measured at a wavelength of 490 nm. Each treatment was performed for 4 times. The mean value was used as the final result. 2.9.4 Alkaline phosphatase (ALP) assay The ALP assay was performed according to a previous study [34]. Osteoblasts cultured on different titanium substrates were lysed by Triton X-100 with three freeze–thaw cycles after culture for 48 h. After centrifugation, the supernatant was used to determine the total intracellular protein and ALP activity with p-nitrophenyl phosphate as substrate, respectively. The ALP activity (expressed as lmol of converted p-nitrophenol/ min) was normalized by synthesized total intracellular protein and thus reflected as lmol p-nitrophenol/min/mg protein. 2.10 Statistical analysis All data were presented as means ± standard deviations (SD). Statistical analysis was performed by Student’s T test and one-way analysis of variance (ANOVA) at confidence levels of 95 and 99 % (OriginPro version 7.5).
2.9.1 Cell culture Primary osteoblasts isolated from neonatal rat calvaria were cultured in DMEM supplemented with 10 % bovine serum (FBS) at 37 °C under 5 % CO2 atmosphere [32]. Osteoblasts at the 3rd passage were used in the following studies. Cells were seeded onto tissue culture plates (TCPS), bare Ti substrate, Ti/PEI/(Gel/Chi-SH)4/Gel and Ti/PEI/(Gel/Chi-SH/Ag0.005)n/Gel (n = 1, 2, 3, 4) substrates at an initial density of 2 9 104 cells/cm2 for all experiments. 2.9.2 Cell morphology observation After culturing for 48 h, osteoblasts on different substrates were fixed by glutaraldehyde and permeabilized with Triton X-100 at 4 °C for 15 and 2 min, respectively. Then, samples were stained with rhodamine-phalloidin at 4 °C (Invitrogen Co., USA) overnight and counterstained with 10 lg/mL of Hoechst 33258, respectively. The stained samples were mounted with 90 % glycerinum and observed with confocal laser scanning microscopy.
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3 Results 3.1 Synthesis and characterization of sulfhydrylated chitosan Sulfhydrylated chitosan (Chi-HS) could be synthesized via the reaction between chitosan and N-acetyl-L-cysteine (NAC) by using EDC/HOBt as coupling agents (Fig. 1b). FTIR measurement was firstly performed to confirm the successful conjugation of NAC onto chitosan molecules. As shown in Fig. 2 a, there was no obvious differences in characteristic peaks between chitosan (Fig. 2a1) and ChiSH (Fig. 2a2). However, the intensity of amide I characteristic peak at 1,655 cm-1 was increased and peak position slightly shifted to 1,630 cm-1 after NAC coupled to chitosan molecules. The phenomena were attributed to the reaction between –NH2 groups in chitosan molecules and –COOH groups in NAC molecules. Besides, a new peak at 2,570 cm-1 was observed on the spectrum of Chi-SH (Fig. 2a2). The result confirmed that the new groups of –SH were successfully grafted onto the chitosan molecules.
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Fig. 2 FTIR (a) and 1HNMR (b) spectra of chitosan (a1, b1) and Chi-SH (a2, b2), respectively
All of the results were consistent with a previous report [35]. For more detailed structure characterization, 1H NMR measurement was performed. As shown in Fig. 2b1, the resonance peak at d = 2.05 ppm was assigned to N-acetyl protons of N-acetylglucosamine residues in chitosan molecules [36]. After NAC molecules grafted to chitosan molecules, the intensity of peak at d = 2.05 ppm was increased obviously (Fig. 2b2). It shows that the groups of –COCH3 were introduced into chitosan molecules. Besides, a new peak at d = 2.9 ppm was also observed form the spectrum of Chi-SH (Fig. 2b2), which indicates –HS groups were grafted to the chitosan. The phenomena were consistent with previous studies [37, 38]. And the grafting ratio of NAC to chitosan was nearly to 10.1 % based on the 1H-NMR spectrum of Chi-SH. All of the results confirmed that ChiSH molecules were synthesized successfully based on the reactions between NAC and chitosan.
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contact angle measurement was performed to monitor the buildup processes. As shown in Fig. 3 a, from the fourth layer to sixth layer, the value of contact angles were around 55°, 20° and 40°, which corresponded to Chi-SH, silver and gelatin as the outmost layers, respectively. Further multilayer coating resulted in regular alternative changes in contact angles, which indicated Ti substrates were completely covered after 4 deposition cycles. All of the results were well consistent with previous study [34]. To reveal the binding property of sulfhydryl groups to Ag ions, we firstly prepared Ti/PEI/(Gel/Chi/Ag0.1)4/Gel and Ti/PEI/(Gel/Chi-SH/Ag0.1)4/Gel samples by using AgNO3 (0.1 M) as Ag? sources, respectively. The surface morphologies of samples were characterized by SEM. As depicted in Fig. 3b1, a few Ag nanoparticles were observed on the surfaces of Ti/PEI/(Gel/Chi/Ag0.1)4/Gel substrate (control group). However, Ti/PEI/(Gel/Chi-SH/Ag0.1)4/Gel substrate was completely covered with Ag nanoparticles when the chitsoan was replaced by Chi-SH(Fig. 3b2). The amount of Ag nanoparticles onto Chi-SH/Gel multilayer films was much more than those of Chi/Gel multilayer films after the UV treatment. To understand the relationship between the layer number of Chi-SH and the Ag ions deposition onto Ti substrate, the samples of Ti/PEI/(Gel/Chi-SH/Ag0.005)n/Gel (n = 1, 2, 3, 4) were prepared. The concentration of AgNO3 solution was 0.005 M. Only a small amount of Ag nanoparticles were observed after one deposition cycle of Gel/Chi-SH/Ag onto Ti substrate (Fig. 4a3). However, Ag nanoparticles in clear round shape were visible after four deposition cycles of Gel/ Chi-SH/Ag onto Ti substrates (Fig. 4a6). Here, some Ag nanoparticles were half-hidden within the multilayered films owing to the coverage of multilayer films onto those nanoparticles. The results demonstrated Ag nanoparticles were successfully formed in situ within Chi-SH/Gel multilayer films onto Ti substrate. Besides, the loading amount of Ag nanoparticles increased with the number of deposition cycles (Fig. 4a3–a6). Moreover, EDS measurements further verified this conclusion. As shown in Fig. 4b3, the characteristic peaks for Ag elements were observed after one deposition cycles (Gel, Chi-SH and Ag ions) onto Ti substrate. Besides, the intensity of characteristic peaks for Ag elements increased with the number of deposition cycle (Fig. 4b3–b6). All of the results indicate the loading amount of Ag nanoparticles on Ti substrate increased with the number of deposition cycles, which was well consistent with previous report [26]. 3.3 Antibacterial activity
3.2 Construction and characterization of Ag-containing multilayer films
3.3.1 Kirby–Bauer test
To validate the successful deposition of silver-containing multilayered films on the surfaces of titanium substrates,
To evaluate the antibacterial activity of Ag-containing multilayer films, we firstly performed a Kirby–Bauer test
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Fig. 3 a Water contact angles as a function of the number of coating layers. The layer numbered 3, 6, 9, 12, 15 and 18 were the Gel layers; the layer numbered 4, 7, 10 13 and 16 were the Chi-SH layers; the layer numbered 5, 8, 11, 14 and 17 were Ag layers; while the layers numbered 1 and 2 were the titanium substrate and PEI, respectively (n = 4). b Representative SEM images of b1 Ti/PEI/(Gel/Chi/Ag0.1)4/ Gel, and b2 Ti/PEI/(Gel/ChiSH/Ag0.1)4/Gel, respectively. The AgNO3 concentration was 0.1 M
against E. coli in vitro. The ZOIs produced by Ti substrates, Ti/PEI/(Gel/Chi/Ag0.1)4/Gel and Ti/PEI/(Gel/Chi-SH/Ag0.1)4/ Gel substrates were observed by optical microscopic, respectively. As shown in Fig. 5a1, there was no ZOI around bare titanium after incubation for 48 h, which implies the bacteria were not inhibited. In contrast, remarkable ZOIs were observed around the samples of Ti/PEI/(Gel/Chi/Ag0.1)4/Gel (Fig. 5a2) and Ti/PEI/(Gel/Chi-SH/Ag0.1)4/Gel (Fig. 5a3), respectively. The phenomena might be interpreted that the Ag nanoparticles within multilayer films were released into surrounding gel in the form of Ag ions, thus leading to the killing of bacteria. Moreover, the ZOI size of Ti/PEI/(Gel/Chi-SH/ Ag0.1)4/Gel was much larger than that of Ti/PEI/(Gel/Chi/ Ag0.1)4/Gel (Fig. 5a3 vs a2). The results could be attributed to the fact that the loading amount of Ag nanoparticles within Ti/PEI/(Gel/Chi-SH/Ag0.1)4/Gel substrate was much more than that of Ti/PEI/(Gel/Chi/Ag0.1)4/Ge substrate (Fig. 3b), resulting in much larger ZOI size at the same culture duration. To further investigate antibacterial activity of Ti/PEI/ (Gel/Chi-SH/Ag)4/Gel substrates, we decreased the loading concentration of silver ions from 0.1 to 0.005 M and
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prepared samples of Ti/PEI/(Gel/Chi-SH)4/Gel, Ti/PEI/ (Gel/Chi-SH/Ag0.005)n/Gel (n = 1, 2, 3, 4), respectively. As shown in Fig. 5b, there was no ZOI around both of bare Ti substrate and Ti/PEI/(Gel/Chi-SH)4/Gel substrate (Fig. 5b1 and b2). However, a visible ZOI was observed around Ti/PEI/(Gel/Chi-SH/Ag0.005)1/Gel substrate when one deposition cycle of Ag ions was achieved (Fig. 5b3), This result shows the bacteria would be highly susceptible to Ag-containing multilayer films onto Ti substrate. After more deposition cycles onto Ti substrate, the larger ZOI were observed (Fig. 5b3–b6). The phenomena could be interpreted that the amount of Ag nanoparticles onto Ti substrates increased with the number of deposition cycles, thus leading to the death of bacteria. It was consistent with the results as described above. 3.3.2 Bacterial adhesion To investigate the anti-adhesion activity of Ag-containing multilayer films, CLSM was used to observe the number of bacteria adhered on substrates. After culture for 12 h, a large number of E. coli were observed onto both Ti
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bare titanium, a2, b2 Ti/PEI/(Gel/Chi-SH)4/Gel, a3, b3 Ti/PEI/(Gel/ Chi-SH/Ag0.005)/Gel, a4, b4 Ti/PEI/(Gel/Chi-SH/Ag0.005)2/Gel, a5, b5 Ti/PEI/(Gel/Chi-SH/Ag0.005)3/Gel, and a6, b6 Ti/PEI/(Gel/ChiSH/Ag0.005)4/Gel, respectively. The AgNO3 concentration was 0.005 M
substrate and Ti/PEI/(Gel/Chi-SH)4/Gel substrate (Fig. 6a1 and a2). However, the number of E. coli decreased when one deposition cycle of Ag ions was achieved onto Ti substrate (Ti/PEI/(Gel/Chi-SH/Ag0.005)1/Gel)(Fig. 6a3). Further deposition cycles of Ag ions onto titanium substrates led to the sharp decreasing of bacteria number (Fig. 6a4–a6). The results indicate that Ag-containing multilayer films could efficiently inhibit the bacterial adhesion. MTT assay further confirmed this result. As depicted in Fig. 6b, the activity of E. coli adhered onto substrates decreased against the numbers of Ag ions deposition cycles. Compared to bare Ti substrates, the average viability of E. coli decreased to 80, 68, 51 and 19 % when the number of Ag ion deposition cycles onto Ti substrates increased to 1, 2, 3 and 4, respectively. Fig. 5 Kirby–Bauer tests after culture for 2 days. a: bare titanium (a1), Ti/PEI/(Gel/Chi/ Ag0.1)4/Gel (a2) and Ti/PEI/ (Gel/Chi-SH/Ag0.1)4/Gel (a3). The AgNO3 concentration was 0.1 M. b: Bare titanium (b1), Ti/PEI/(Gel/Chi-SH)4/Gel (b2), Ti/PEI/(Gel/Chi-SH/Ag0.005)/ Gel (b3), Ti/PEI/(Gel/Chi-SH/ Ag0.005)2/Gel (b4), Ti/PEI/(Gel/ Chi-SH/Ag0.005)3/Gel (b5), and Ti/PEI/(Gel/Chi-SH/Ag0.005)4/ Gel (b6), respectively. The AgNO3 concentration was 0.005 M
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To further confirm the broad spectrum antibacterial activity of Ag-containing multilayer films, the behaviors of B. subtitles onto substrates were investigated as well. B. subtitles is a Gram-positive, catalase-positive bacterium. The same trend was found regarding bacteria adhesion and activity as those of E. coli (Fig. 7a, b). 3.4 In vitro cytocompatibility To investigate cytocompatibility of Ag-containing multilayer films onto Ti substrates, the morphologies of osteoblasts were observed with CLSM. The osteoblasts onto Ti substrate displayed well spread shape (Fig. 8a1). There was no obvious difference in cell morphology when osteoblasts were cultured onto Ag-containing multilayer films (Ti/PEI/ (Gel/Chi-SH/Ag0.005)4/Gel) and Ti substrate (Fig. 8a1 vs a3–a6). Although the number of Ag ions deposition cycles onto Ti substrate were increased, the number of osteoblasts adhered onto substrate did not change obviously (Fig. 8a1, a3–a6). It suggests that Ti/PEI/(Gel/Chi-SH/Ag0.005)n/Gel substrates had no obvious cytotoxicity effects on the proliferation of osteoblasts, which was also confirmed by MTT
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Fig. 6 CLSM images (a) and viabilities (b) of E. coli adhered to different titanium substrates after culturing for 12 h. a1 Bare titanium, a2 Ti/PEI/(Gel/ChiSH)4/Gel, a3 Ti/PEI/(Gel/ChiSH/Ag0.005)/Gel, a4 Ti/PEI/ (Gel/Chi-SH/Ag0.005)2/Gel, a5 Ti/PEI/(Gel/Chi-SH/Ag0.005)3/ Gel, and a6 Ti/PEI/(Gel/ChiSH/Ag0.005)4/Gel, respectively. The AgNO3 concentration was 0.005 M
assay. Ti/PEI/(Gel/Chi-SH)4/Gel substrates displayed similar cell viability to those of bare Ti and Ti/PEI/(Gel/ChiSH/Ag0.005)n/Gel substrates after incubation for 48 h (Fig. 8b). It was contributed to the good biocompatibility of chitosan molecules and gelatin molecules [39]. To further investigate the effects of Ag-containing multilayer films on cell functions, alkaline phosphatase (ALP) activity of osteoblasts we evaluated. Osteoblasts cultured onto Ti/PEI/(Gel/Chi-SH)4/Gel substrates displayed significantly higher ALP activity (P \ 0.05) than those of bare Ti substrate (Fig. 8c), Ti/PEI/(Gel/Chi-SH/ Ag0.005)n/Gel (n = 1, 2, 3, 4) substrates after culturing for 48 h. There was no difference in ALP activity among bare Ti and Ti/PEI/(Gel/Chi-SH/Ag0.005)n/Gel (n = 1, 2, 3, 4) substrates. The result demonstrates the introduction of Ag
nanoparticles into multilayer films did not inhibit the ALP activity of osteoblasts.
4 Discussions To endow Ti implants with long-term potential antibacterial activities, herein a novel ‘‘sandwich type’’ structure of Chi-SH/gelatin multilayer films containing Ag nanoparticles were deposited onto titanium substrates using a spinassisted LBLs technique. Ag ions would be spontaneously accumulated into the polyelectrolyte multilayer films via the specific coordination between Ag ions and –HS groups in Chi-HS, thus leading to formation of Ag nanoparticles in situ after ultraviolet irradiation [40]. To some
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Fig. 7 CLSM images (a) and viabilities (b) of Bacillus subtitles grown onto different titanium substrates after culturing for 12 h. a1 Bare titanium, a2 Ti/PEI/(Gel/ChiSH)4/Gel, a3 Ti/PEI/(Gel/ChiSH/Ag0.005)/Gel, a4 Ti/PEI/ (Gel/Chi-SH/Ag0.005)2/Gel, a5 Ti/PEI/(Gel/Chi-SH/Ag0.005)3/ Gel, and a6 Ti/PEI/(Gel/ChiSH/Ag0.005)4/Gel. The AgNO3 concentration was 0.005 M
extent, the loading amount of Ag nanoparticles onto the Ti substrate was greatly dependent on the interactions between Ag ions and multilayer films. For Chi/Gel multilayered films, although Ag ions might be incorporated into multilayer films by physical adsorptions and ion exchanges, most of ions still would be excluded from the multilayer films via electrostatic repulsion due to the group of amino groups in chitosan molecules. For Chi-SH/Gel multilayer films, on the contrary, the specific interactions between Ag ions and –HS groups in Chi-SH could lead to the enrichment of Ag ions within multilayer films [41]. Previous studies confirmed Ag ions were facile to form stable sulfur-Ag bonds [42, 43], which would remain silver ions within multilayer films during assembly process. Therefore, the amount of Ag nanoparticles onto Chi-SH/
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Gel multilayer films was much more than those of Chi/Gel multilayer films after the UV treatment. Besides, the loading amount of Ag nanoparticles also increased with the number of deposition cycles (Fig. 4A, c–f), which was well consistent with previous report [26]. The functional multilayered films on titanium substrate (Ti/PEI/(Gel/Chi-SH/Ag)n/Gel) could efficiently inhibit the growth and activity of B. subtitles and E. coli onto titanium surface. The components of multilayer films onto Ti substrates determined their antibacterial activity. The multilayer films without Ag deposition (Ti/PEI/(Gel/Chi-SH)4/ Gel) slightly inhibited the bacteria adhesion and growth since chitosan were able to disrupt bacteria membranes [44]. However, the antibacterial activity of chitosan was not enough to inhibit the growth of bacteria for a long
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Fig. 8 Osteoblasts functions assays. a CLSM images of osteoblasts adhered to different titanium substrates after culture for 2 days. a1 bare titanium, a2 Ti/PEI/(Gel/Chi-SH)4/Gel, a3 Ti/PEI/(Gel/Chi-SH/Ag0.005)/ Gel, a4 Ti/PEI/(Gel/Chi-SH/ Ag0.005)2/Gel, a5 Ti/PEI/(Gel/ Chi-SH/Ag0.005)3/Gel, and a6 Ti/PEI/(Gel/Chi-SH/Ag0.005)4/ Gel. b Cells viabilities of osteoblasts adhered to different titanium substrates after culture for 2 days. c Alkaline phosphatase activity of osteoblasts adhered to different substrates after culturing for 2 days. The AgNO3 concentration was 0.005 M. Error bars represent mean ± SD for n = 6, *P \ 0.05
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period. Thus, there was no significant difference in antibacterial activity between Ti/PEI/(Gel/Chi-SH)4/Gel and bare Ti substrates (Fig. 5b1, b2). Nevertheless, Ag nanoparticles within multilayered films endowed the Ti substrate with potential antibacterial activity. It could be interpreted that Ag ions derived from Ag nanoparticles within multilayer films were released into peripheral region around Ti substrate and kill bacterial until Ag nanoparticles were completely consumed. It means that the more Ag nanoparticles were formed within the multilayer films, the longer period of antibacterial activity against microorganism would be achieved. Therefore, the adhesion and activity of both B. subtitles and E coli onto Ti/PEI/(Gel/ Chi-SH/Ag0.005)n/Gel substrates were severely inhibited, especially those substrate with multiple deposition cycles of Ag ions, comparing with those of bare Ti substrates., Moreover, the loading amount of Ag nanoparticles was not only dependent on the number of deposition cycles but also the types of polyelectrolyte in multilayer films. The coordination between Chi-SH and silver significantly enhanced the loading amount of Ag nanoparticles in multilayered films, leading to its obvious antimicrobial effect after only two cycles of deposition (Fig. 5b). The study thus demonstrated its advantage in the loading of Ag ions via ChiSH molecules. Apart from the antibacterial property, Ti substrates coating with such functional multilayer films could remain biological functions of osteoblasts, which was reflected by cell morphology, cell viability and ALP activity measurements. The phenomenon was attributed to the following two reasons. On one hand, the concentration of Ag ions released from multilayer films was not enough to induce cytotoxicity to osteoblasts, while efficiently inhibited the bacteria activity. To some extent, the cytotoxicity of Ag nanoparticles within multilayer films was dominated by the concentration of released Ag ion [45–48]. Both of multilayer films and the coordination between Chi-SH to Ag elements could control the Ag ions release in a slow manner, thus leading to low cytotoxicity to osteoblasts. Besides, the loading of Ag nanoparticles on or within a biomaterial was involved with extra chemical agents [49], e. g., NaBH4. It thus results in undesired cytotoxicity to cells. Here, Ag nanoparticles within Chi-SH/Gel multilayered films were formed only by using sulfur groups (Chi-SH) as seeds for silver ions accumulation and followed by the photocatalysis of ultraviolet exposure. Therefore, there would be no toxic chemicals involving into the whole process. On the other hand, the interaction mechanism of osteoblasts to Ag ions was different from that of bacteria (B. subtitles and E. coli) to Ag ions due to their differences in size and structure [50]. The protondepleted region formed around Ag nanoparticles would, disrupt the synthesis of adenosine triphosphate, leading to
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the death of bacteria [51]. However, mammal cells could diminish the effects of proton-depleted region since they have much larger size and more complex membrane structures [52].
5 Conclusion In summary, the functional ‘‘sandwich-type’’ Chi-SH/Gel polyelectrolyte multilayer films containing Ag nanoparticles were successfully coated onto the surface of titanium substrate via a spin-assisted layer-by-layer assembly technique. Chi-SH molecular was used as nanocontainers to store Ag ions via the specific interactions between Ag ions and –SH groups. Ag nanoparticles could be formed in situ via the simple photo-catalysis reaction under conventional UV light. Antibacterial tests in vitro revealed that the Ag-containing multilayer films onto Ti substrates (Ti/PEI/(Gel/Chi-SH/Ag)n/ Gel) had potent antibacterial activity. Moreover, Ti/PEI/(Gel/ Chi-SH/Ag)n/Gel substrates displayed no cytotoxicity to the biological function of osteoblasts. The approach presented here has potential application for the development of functional titanium-based implants with antibacterial property. Acknowledgments This work was financially supported by Natural Science Foundation of Chongqing Municipal Government (CSTC, 2011JJJQ10004 and JJA10056), China Ministry of Science and Technology (973 Project No. 2009CB930000), Natural Science Foundation of China (51173216 and 31200712), National Key Technology R&D Program of the Ministry of Science and Technology (2012BAI18B04) and the ‘‘111’’ project (B06023).
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Surface modification of titanium substrates with silver nanoparticles embedded sulfhydrylated chitosan/gelatin polyelectrolyte multilayer films for antibacterial application Wen Li • Dawei Xu • Yan Hu • Kaiyong Cai Yingcheng Lin
•
Received: 5 September 2013 / Accepted: 8 March 2014 / Published online: 25 March 2014 Ó Springer Science+Business Media New York 2014
Abstract To develop Ti implants with potent antibacterial activity, a novel ‘‘sandwich-type’’ structure of sulfhydrylated chitosan (Chi-SH)/gelatin (Gel) polyelectrolyte multilayer films embedding silver (Ag) nanoparticles was coated onto titanium substrate using a spin-assisted layer-by-layer assembly technique. Ag ions would be enriched in the polyelectrolyte multilayer films via the specific interactions between Ag ions and –HS groups in Chi-HS, thus leading to the formation of Ag nanoparticles in situ by photo-catalytic reaction (ultraviolet irradiation). Contact angle measurement and field emission scanning electron microscopy equipped with energy dispersive X-ray spectroscopy were employed to monitor the construction of Ag-containing multilayer on titanium surface, respectively. The functional multilayered films on titanium substrate [Ti/PEI/(Gel/ChiSH/Ag)n/Gel] could efficiently inhibit the growth and activity of Bacillus subtitles and Escherichia coli onto titanium surface. Moreover, studies in vitro confirmed that Ti substrates coating with functional multilayer films remained the biological functions of osteoblasts, which was reflected by cell morphology, cell viability and ALP activity measurements. This study provides a simple, versatile and generalized methodology to design functional titanium implants with good cyto-compatibility and antibacterial activity for potential clinical applications.
W. Li D. Xu Y. Hu (&) K. Cai Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, People’s Republic of China e-mail: [email protected] Y. Lin College of Communication Engineering, Chongqing University, Chongqing 400044, People’s Republic of China
1 Introduction Titanium-based orthopedic implants have been extensively used in the medical clinical applications since they have good biocompatibility and mechanical properties. However, infection on or around titanium implants still remains a vital problem to deal with, which may result in eventual implant loosening and removal [1–3]. To inhibit the bacteria adhesions and biofilms formation, various antibacterial agents or adhesion-resistant materials were introduced into the Ti implants surfaces [4, 5]. However, most of antibacterial molecules were still susceptible to physiological environment, thus leading them to lose bioactivity in a short time [6]. Interestingly, silver (Ag) nanoparticles could long-term resist the formation of bacterial biofilm onto implants while not impairing the biological functions of mammalian cells [3, 7–11]. At present, Ag nanoparticles were usually directly coated onto or incorporated into Ti implants by using chemical vapor deposition (CVD) or physical vapor deposition (PVD) methods [12–16]. The fabrication processes tended to be time consuming and high costly. Therefore, it is necessary to design a simple and versatile method to deposit Ag nanoparticles onto Ti substrates to efficiently inhibit the growth and activity of pathogenic bacteria while not affecting bio-functions of mammalian cells [17]. Recently, layer-by-layer assembly (LBLs) technique attracts extensive attention since it provides a simple, useful and versatile methodology for material surface modifications [18, 19]. And many antibacterial agents with surface charge properties (antibiotic [20], antibacterial polymer/peptide [21, 22] or other antibacterial agents [23]) could be directly deposited onto material surfaces by using LBL technique. Moreover, Ag ions also could be incorporated into multilayered structure via ion exchange or
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electrodeposition [24, 25], thus leading to the formation of Ag nanoparticles in situ after adding external reductive agents (e.g. NaBH4) [26]. The Ag-containing films had long-lasting antimicrobial activity against microorganism, such as Staphylococcus aureus and Escherichia coli (E. coli) [27, 28]. Herein, we design and fabricate ‘‘sandwich’’ sulfhydrylated chitosan (Chi-SH)/gelatin (gel) multilayer films with Ag nanoparticles onto titanium substrates via a spinassisted LBLs technique (Fig. 1a). The sulfhydrylated chitosan (Chi-SH) molecules acted as Ag ion nanocontainers due to the specific conjugation of Ag ion to –HS groups in Chi-SH. The Ag nanoparticles would be then formed in situ within multilayer films after the
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photocatalytic reactions (ultraviolet irradiation), while not introducing the extra reductive agents. It was reported that Ag ions could be bound to the membrane of microorganism [28] and uptaken by bacteria, thus leading to the death of bacteria when its accumulation concentration reached the toxicity threshold. The potential mechanism lies in that Ag ions coordinate with electron-donating groups such as carboxylates, imidazoles, amides, indoles, hydroxyls as well as thiols [29, 30]. We validated the binding effect of sulfhydrylated chitosan to Ag ions within multilayer structure and their antibacterial activity against E. coli and Bacillus subtitles. Meanwhile, the cellular responses of osteoblasts to those substrates were also investigated.
Fig. 1 a Scheme of the fabrication of silver-containing multilayered films on titanium surface based on sulfhydrylated chitosan. b Synthesis of sulfhydrylated chitosan
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2 Materials and methods
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2.4 Characterization of silver-containing multilayer films
2.1 Materials Titanium disks (15 mm dia 9 2 mm thick) were provided by Northwest Institute for Non-ferrous Metal Research, Xi’an, China. The chemicals of N-hydroxybenzotriazole (HOBt) and N-acetyl-L-cysteine (NAC) were obtained from Alfa Aesar Co. Ltd. (Tianjin, China). And the products of chitosan, gelatin, 1-ethyl-3-(3-dimethylaminopropyl)–carbodiimide (EDC), p-nitrophenyl phosphate, 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) and Hoechst 33258 were supplied by Sigma-Aldrich (St. Louis, MO, USA). 2.2 Synthesis and characterization of sulfhydrylated chitosan Sulfhydrylated chitosan was synthesized according to a previous study [31]. Briefly, both of chitosan (500 mg) and HOBt (348.5 mg) were dispersed into 46 mL of deionized water with stirring until the solution was transparent. Then, a mixture of NAC (420 mg) and EDC (989 mg) in 4 mL deionized water were added into the above solution and stirred for another 2 h (pH 5). Subsequently, the resulting product was purified using a dialysis tube (8,000–14,000 MWCO) against 5 mM HCl containing 1 % NaCl and 1 mM HCl, respectively. Finally, the product was obtained after treatment with lyophilization, denoting as Chi-SH. The structure properties of Chi-SH were characterized by fourier transform infrared spectrometry (FTIR) and 1H nuclear magnetic resonance spectroscopy (1H NMR). 2.3 Construction of silver-containing multilayer films The silver-containing multilayered films onto Ti substance were prepared as following. Firstly, Gelatin (5 mg/mL) and polyethyleneimine (PEI) solution (5 mg/mL) were prepared with phosphate buffered saline (PBS, pH 7.4). Meanwhile, Chi-SH (5 mg/mL) and AgNO3 (0.005 and 0.1 M) solutions were prepared with deionized water, Then, 100 lL of PEI solution was deposited onto titanium substrates by spin coating at a speed of 4000-rpm for 40 s. After rinsing with deionized water, the above solution of gelatin, Chi-SH and AgNO3 were subsequently deposited onto pre-coated titanium substrates by using the above procedure. The procedures were repeated until the desired multilayer films of (Gel/Chi-SH/Ag)n onto titanium substrates were achieved. Finally, Ag nanoparticles would be formed in situ when exposed to ultraviolet lamp (100 W, 48 h). The sample was denoted as Ti/PEI/(Gel/Chi-SH/ Ag)n/Gel.
The construction of silver-containing multilayer films onto titanium substrates was monitored by contact angle measurements (Future Scientific Ltd Co. Taiwan, China). Meanwhile, the morphologies of the substrates were observed by field emission scanning electron microscopy (FE-SEM) equipped with energy dispersive X-ray spectroscopy (EDS) (FEINova 400 Nano SEM, Phillips Co. Holland). 2.5 Bacteria culture E. coli (gram negative bacteria) [32] and B. subtitles (gram positive bacteria) [32] were employed to investigate the broad spectrum antibacterial activity of Ti/PEI/(Gel/ChiSH/Ag)n/Gel. Firstly, both of the two bacteria were cultured with Cation-adjusted Mueller Hinton Broth II (CMHB) at 37 °C for 12 h, respectively. The bacteriacontaining mediums were then centrifuged at 3000 rpm for 10 min. After that, the bacteria were washed with fresh CMHB medium. Finally, the bacteria were re-suspended in 1 mL of CMHB medium for the following studies. 2.6 Kirby–Bauer test Kirby–Bauer test was performed according to a previous report [26]. Briefly, an agar gel plate was firstly swabbed with 30 lL of E. coli solution at a concentration of 1 9 106 cells/mL. Then, the samples of titanium, Ti/PEI/ (Gel/Chi/Ag0.1)4/Gel, Ti/PEI/(Gel/Chi-SH/Ag0.1)4/Gel, Ti/ PEI/(Gel/Chi-SH)4/Gel and Ti/PEI/(Gel/Chi-SH/Ag0.005)n/ Gel (n = 1, 2, 3, 4) substrates were placed onto the agar plates, respectively. Subsequently, all plates were incubated at 37 °C for another 48 h. Finally, the zones of inhibition (ZOIs) around different samples were observed by an optical microscope. The sizes of ZOIs were directly correlated to the antimicrobial activity of the substrates. 2.7 Bacteria adhesion Escherichia coli and B. subtitles were cultured as described above. Titanium, Ti/PEI/(Gel/Chi-SH)4/Gel and Ti/PEI/(Gel/Chi-SH/Ag0.005)n/Gel (n = 1, 2, 3, 4) substrates were exposed to bacteria suspension at a concentration of 1 9 106 cells/mL, respectively. After incubation for 12 h, all of the substrates were taken out and washed with fresh CMHB medium. Subsequently, the remaining bacteria onto the surfaces of different samples were fixed by 2 % glutaraldehyde for 10 min and stained by 10 lg/mL of Hoechst 33258 at 4 °C for
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5 min, respectively. The stained samples were mounted with 90 % glycerinum and observed with confocal laser scanning microscopy (CLSM, 510 Metanlo, Zeiss Co., Germany). 2.8 Bacteria activity E. coli and B. subtitles were also obtained as described above. The sample of titanium, Ti/PEI/(Gel/Chi-SH)4/Gel and Ti/PEI/(Gel/Chi-SH/Ag0.005)n/Gel (n = 1, 2, 3, 4) substrates were immersed into bacteria suspension at a concentration of 1 9 106 cells/mL. After incubation for 12 h, the bacteria adhered onto the samples were removed with 2 mL of fresh CHMB medium by vigorous vortexing at room temperature for 5 min. Subsequently, agar gel plate was swabbed with 30 lL of bacteria suspension. After incubation at 37 °C for 12 h, the bacteria-containing agar gel plate was taken out and placed into 24-well plate, followed by adding 0.9 mL of fresh CHMB medium and 0.1 mL of MTT solution (5 mg/mL). After incubation at 37 °C for another 4 h, MTT-containing medium was removed and 0.5 mL of dimethyl sulfoxide (DMSO) was added to dissolve formazan crystals. After centrifugation, the optical density of supernatant was measured at a wavelength of 490 nm [33]. Each treatment was performed for 3 times. And the mean value was used as the final result. 2.9 In vitro cytocompatibility evaluation
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2.9.3 Cell viability MTT assay was used to determine the viability of osteoblasts cultured on different substrates. After culture for 48 h, 100 lL of MTT (5 mg/mL) was added to each well and incubated at 37 °C for another 4 h. MTT containing medium was then removed and 0.6 mL of dimethyl sulfoxide (DMSO) was added to dissolve formazan crystals. The optical density of the solution was measured at a wavelength of 490 nm. Each treatment was performed for 4 times. The mean value was used as the final result. 2.9.4 Alkaline phosphatase (ALP) assay The ALP assay was performed according to a previous study [34]. Osteoblasts cultured on different titanium substrates were lysed by Triton X-100 with three freeze–thaw cycles after culture for 48 h. After centrifugation, the supernatant was used to determine the total intracellular protein and ALP activity with p-nitrophenyl phosphate as substrate, respectively. The ALP activity (expressed as lmol of converted p-nitrophenol/ min) was normalized by synthesized total intracellular protein and thus reflected as lmol p-nitrophenol/min/mg protein. 2.10 Statistical analysis All data were presented as means ± standard deviations (SD). Statistical analysis was performed by Student’s T test and one-way analysis of variance (ANOVA) at confidence levels of 95 and 99 % (OriginPro version 7.5).
2.9.1 Cell culture Primary osteoblasts isolated from neonatal rat calvaria were cultured in DMEM supplemented with 10 % bovine serum (FBS) at 37 °C under 5 % CO2 atmosphere [32]. Osteoblasts at the 3rd passage were used in the following studies. Cells were seeded onto tissue culture plates (TCPS), bare Ti substrate, Ti/PEI/(Gel/Chi-SH)4/Gel and Ti/PEI/(Gel/Chi-SH/Ag0.005)n/Gel (n = 1, 2, 3, 4) substrates at an initial density of 2 9 104 cells/cm2 for all experiments. 2.9.2 Cell morphology observation After culturing for 48 h, osteoblasts on different substrates were fixed by glutaraldehyde and permeabilized with Triton X-100 at 4 °C for 15 and 2 min, respectively. Then, samples were stained with rhodamine-phalloidin at 4 °C (Invitrogen Co., USA) overnight and counterstained with 10 lg/mL of Hoechst 33258, respectively. The stained samples were mounted with 90 % glycerinum and observed with confocal laser scanning microscopy.
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3 Results 3.1 Synthesis and characterization of sulfhydrylated chitosan Sulfhydrylated chitosan (Chi-HS) could be synthesized via the reaction between chitosan and N-acetyl-L-cysteine (NAC) by using EDC/HOBt as coupling agents (Fig. 1b). FTIR measurement was firstly performed to confirm the successful conjugation of NAC onto chitosan molecules. As shown in Fig. 2 a, there was no obvious differences in characteristic peaks between chitosan (Fig. 2a1) and ChiSH (Fig. 2a2). However, the intensity of amide I characteristic peak at 1,655 cm-1 was increased and peak position slightly shifted to 1,630 cm-1 after NAC coupled to chitosan molecules. The phenomena were attributed to the reaction between –NH2 groups in chitosan molecules and –COOH groups in NAC molecules. Besides, a new peak at 2,570 cm-1 was observed on the spectrum of Chi-SH (Fig. 2a2). The result confirmed that the new groups of –SH were successfully grafted onto the chitosan molecules.
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Fig. 2 FTIR (a) and 1HNMR (b) spectra of chitosan (a1, b1) and Chi-SH (a2, b2), respectively
All of the results were consistent with a previous report [35]. For more detailed structure characterization, 1H NMR measurement was performed. As shown in Fig. 2b1, the resonance peak at d = 2.05 ppm was assigned to N-acetyl protons of N-acetylglucosamine residues in chitosan molecules [36]. After NAC molecules grafted to chitosan molecules, the intensity of peak at d = 2.05 ppm was increased obviously (Fig. 2b2). It shows that the groups of –COCH3 were introduced into chitosan molecules. Besides, a new peak at d = 2.9 ppm was also observed form the spectrum of Chi-SH (Fig. 2b2), which indicates –HS groups were grafted to the chitosan. The phenomena were consistent with previous studies [37, 38]. And the grafting ratio of NAC to chitosan was nearly to 10.1 % based on the 1H-NMR spectrum of Chi-SH. All of the results confirmed that ChiSH molecules were synthesized successfully based on the reactions between NAC and chitosan.
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contact angle measurement was performed to monitor the buildup processes. As shown in Fig. 3 a, from the fourth layer to sixth layer, the value of contact angles were around 55°, 20° and 40°, which corresponded to Chi-SH, silver and gelatin as the outmost layers, respectively. Further multilayer coating resulted in regular alternative changes in contact angles, which indicated Ti substrates were completely covered after 4 deposition cycles. All of the results were well consistent with previous study [34]. To reveal the binding property of sulfhydryl groups to Ag ions, we firstly prepared Ti/PEI/(Gel/Chi/Ag0.1)4/Gel and Ti/PEI/(Gel/Chi-SH/Ag0.1)4/Gel samples by using AgNO3 (0.1 M) as Ag? sources, respectively. The surface morphologies of samples were characterized by SEM. As depicted in Fig. 3b1, a few Ag nanoparticles were observed on the surfaces of Ti/PEI/(Gel/Chi/Ag0.1)4/Gel substrate (control group). However, Ti/PEI/(Gel/Chi-SH/Ag0.1)4/Gel substrate was completely covered with Ag nanoparticles when the chitsoan was replaced by Chi-SH(Fig. 3b2). The amount of Ag nanoparticles onto Chi-SH/Gel multilayer films was much more than those of Chi/Gel multilayer films after the UV treatment. To understand the relationship between the layer number of Chi-SH and the Ag ions deposition onto Ti substrate, the samples of Ti/PEI/(Gel/Chi-SH/Ag0.005)n/Gel (n = 1, 2, 3, 4) were prepared. The concentration of AgNO3 solution was 0.005 M. Only a small amount of Ag nanoparticles were observed after one deposition cycle of Gel/Chi-SH/Ag onto Ti substrate (Fig. 4a3). However, Ag nanoparticles in clear round shape were visible after four deposition cycles of Gel/ Chi-SH/Ag onto Ti substrates (Fig. 4a6). Here, some Ag nanoparticles were half-hidden within the multilayered films owing to the coverage of multilayer films onto those nanoparticles. The results demonstrated Ag nanoparticles were successfully formed in situ within Chi-SH/Gel multilayer films onto Ti substrate. Besides, the loading amount of Ag nanoparticles increased with the number of deposition cycles (Fig. 4a3–a6). Moreover, EDS measurements further verified this conclusion. As shown in Fig. 4b3, the characteristic peaks for Ag elements were observed after one deposition cycles (Gel, Chi-SH and Ag ions) onto Ti substrate. Besides, the intensity of characteristic peaks for Ag elements increased with the number of deposition cycle (Fig. 4b3–b6). All of the results indicate the loading amount of Ag nanoparticles on Ti substrate increased with the number of deposition cycles, which was well consistent with previous report [26]. 3.3 Antibacterial activity
3.2 Construction and characterization of Ag-containing multilayer films
3.3.1 Kirby–Bauer test
To validate the successful deposition of silver-containing multilayered films on the surfaces of titanium substrates,
To evaluate the antibacterial activity of Ag-containing multilayer films, we firstly performed a Kirby–Bauer test
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Fig. 3 a Water contact angles as a function of the number of coating layers. The layer numbered 3, 6, 9, 12, 15 and 18 were the Gel layers; the layer numbered 4, 7, 10 13 and 16 were the Chi-SH layers; the layer numbered 5, 8, 11, 14 and 17 were Ag layers; while the layers numbered 1 and 2 were the titanium substrate and PEI, respectively (n = 4). b Representative SEM images of b1 Ti/PEI/(Gel/Chi/Ag0.1)4/ Gel, and b2 Ti/PEI/(Gel/ChiSH/Ag0.1)4/Gel, respectively. The AgNO3 concentration was 0.1 M
against E. coli in vitro. The ZOIs produced by Ti substrates, Ti/PEI/(Gel/Chi/Ag0.1)4/Gel and Ti/PEI/(Gel/Chi-SH/Ag0.1)4/ Gel substrates were observed by optical microscopic, respectively. As shown in Fig. 5a1, there was no ZOI around bare titanium after incubation for 48 h, which implies the bacteria were not inhibited. In contrast, remarkable ZOIs were observed around the samples of Ti/PEI/(Gel/Chi/Ag0.1)4/Gel (Fig. 5a2) and Ti/PEI/(Gel/Chi-SH/Ag0.1)4/Gel (Fig. 5a3), respectively. The phenomena might be interpreted that the Ag nanoparticles within multilayer films were released into surrounding gel in the form of Ag ions, thus leading to the killing of bacteria. Moreover, the ZOI size of Ti/PEI/(Gel/Chi-SH/ Ag0.1)4/Gel was much larger than that of Ti/PEI/(Gel/Chi/ Ag0.1)4/Gel (Fig. 5a3 vs a2). The results could be attributed to the fact that the loading amount of Ag nanoparticles within Ti/PEI/(Gel/Chi-SH/Ag0.1)4/Gel substrate was much more than that of Ti/PEI/(Gel/Chi/Ag0.1)4/Ge substrate (Fig. 3b), resulting in much larger ZOI size at the same culture duration. To further investigate antibacterial activity of Ti/PEI/ (Gel/Chi-SH/Ag)4/Gel substrates, we decreased the loading concentration of silver ions from 0.1 to 0.005 M and
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prepared samples of Ti/PEI/(Gel/Chi-SH)4/Gel, Ti/PEI/ (Gel/Chi-SH/Ag0.005)n/Gel (n = 1, 2, 3, 4), respectively. As shown in Fig. 5b, there was no ZOI around both of bare Ti substrate and Ti/PEI/(Gel/Chi-SH)4/Gel substrate (Fig. 5b1 and b2). However, a visible ZOI was observed around Ti/PEI/(Gel/Chi-SH/Ag0.005)1/Gel substrate when one deposition cycle of Ag ions was achieved (Fig. 5b3), This result shows the bacteria would be highly susceptible to Ag-containing multilayer films onto Ti substrate. After more deposition cycles onto Ti substrate, the larger ZOI were observed (Fig. 5b3–b6). The phenomena could be interpreted that the amount of Ag nanoparticles onto Ti substrates increased with the number of deposition cycles, thus leading to the death of bacteria. It was consistent with the results as described above. 3.3.2 Bacterial adhesion To investigate the anti-adhesion activity of Ag-containing multilayer films, CLSM was used to observe the number of bacteria adhered on substrates. After culture for 12 h, a large number of E. coli were observed onto both Ti
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bare titanium, a2, b2 Ti/PEI/(Gel/Chi-SH)4/Gel, a3, b3 Ti/PEI/(Gel/ Chi-SH/Ag0.005)/Gel, a4, b4 Ti/PEI/(Gel/Chi-SH/Ag0.005)2/Gel, a5, b5 Ti/PEI/(Gel/Chi-SH/Ag0.005)3/Gel, and a6, b6 Ti/PEI/(Gel/ChiSH/Ag0.005)4/Gel, respectively. The AgNO3 concentration was 0.005 M
substrate and Ti/PEI/(Gel/Chi-SH)4/Gel substrate (Fig. 6a1 and a2). However, the number of E. coli decreased when one deposition cycle of Ag ions was achieved onto Ti substrate (Ti/PEI/(Gel/Chi-SH/Ag0.005)1/Gel)(Fig. 6a3). Further deposition cycles of Ag ions onto titanium substrates led to the sharp decreasing of bacteria number (Fig. 6a4–a6). The results indicate that Ag-containing multilayer films could efficiently inhibit the bacterial adhesion. MTT assay further confirmed this result. As depicted in Fig. 6b, the activity of E. coli adhered onto substrates decreased against the numbers of Ag ions deposition cycles. Compared to bare Ti substrates, the average viability of E. coli decreased to 80, 68, 51 and 19 % when the number of Ag ion deposition cycles onto Ti substrates increased to 1, 2, 3 and 4, respectively. Fig. 5 Kirby–Bauer tests after culture for 2 days. a: bare titanium (a1), Ti/PEI/(Gel/Chi/ Ag0.1)4/Gel (a2) and Ti/PEI/ (Gel/Chi-SH/Ag0.1)4/Gel (a3). The AgNO3 concentration was 0.1 M. b: Bare titanium (b1), Ti/PEI/(Gel/Chi-SH)4/Gel (b2), Ti/PEI/(Gel/Chi-SH/Ag0.005)/ Gel (b3), Ti/PEI/(Gel/Chi-SH/ Ag0.005)2/Gel (b4), Ti/PEI/(Gel/ Chi-SH/Ag0.005)3/Gel (b5), and Ti/PEI/(Gel/Chi-SH/Ag0.005)4/ Gel (b6), respectively. The AgNO3 concentration was 0.005 M
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To further confirm the broad spectrum antibacterial activity of Ag-containing multilayer films, the behaviors of B. subtitles onto substrates were investigated as well. B. subtitles is a Gram-positive, catalase-positive bacterium. The same trend was found regarding bacteria adhesion and activity as those of E. coli (Fig. 7a, b). 3.4 In vitro cytocompatibility To investigate cytocompatibility of Ag-containing multilayer films onto Ti substrates, the morphologies of osteoblasts were observed with CLSM. The osteoblasts onto Ti substrate displayed well spread shape (Fig. 8a1). There was no obvious difference in cell morphology when osteoblasts were cultured onto Ag-containing multilayer films (Ti/PEI/ (Gel/Chi-SH/Ag0.005)4/Gel) and Ti substrate (Fig. 8a1 vs a3–a6). Although the number of Ag ions deposition cycles onto Ti substrate were increased, the number of osteoblasts adhered onto substrate did not change obviously (Fig. 8a1, a3–a6). It suggests that Ti/PEI/(Gel/Chi-SH/Ag0.005)n/Gel substrates had no obvious cytotoxicity effects on the proliferation of osteoblasts, which was also confirmed by MTT
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Fig. 6 CLSM images (a) and viabilities (b) of E. coli adhered to different titanium substrates after culturing for 12 h. a1 Bare titanium, a2 Ti/PEI/(Gel/ChiSH)4/Gel, a3 Ti/PEI/(Gel/ChiSH/Ag0.005)/Gel, a4 Ti/PEI/ (Gel/Chi-SH/Ag0.005)2/Gel, a5 Ti/PEI/(Gel/Chi-SH/Ag0.005)3/ Gel, and a6 Ti/PEI/(Gel/ChiSH/Ag0.005)4/Gel, respectively. The AgNO3 concentration was 0.005 M
assay. Ti/PEI/(Gel/Chi-SH)4/Gel substrates displayed similar cell viability to those of bare Ti and Ti/PEI/(Gel/ChiSH/Ag0.005)n/Gel substrates after incubation for 48 h (Fig. 8b). It was contributed to the good biocompatibility of chitosan molecules and gelatin molecules [39]. To further investigate the effects of Ag-containing multilayer films on cell functions, alkaline phosphatase (ALP) activity of osteoblasts we evaluated. Osteoblasts cultured onto Ti/PEI/(Gel/Chi-SH)4/Gel substrates displayed significantly higher ALP activity (P \ 0.05) than those of bare Ti substrate (Fig. 8c), Ti/PEI/(Gel/Chi-SH/ Ag0.005)n/Gel (n = 1, 2, 3, 4) substrates after culturing for 48 h. There was no difference in ALP activity among bare Ti and Ti/PEI/(Gel/Chi-SH/Ag0.005)n/Gel (n = 1, 2, 3, 4) substrates. The result demonstrates the introduction of Ag
nanoparticles into multilayer films did not inhibit the ALP activity of osteoblasts.
4 Discussions To endow Ti implants with long-term potential antibacterial activities, herein a novel ‘‘sandwich type’’ structure of Chi-SH/gelatin multilayer films containing Ag nanoparticles were deposited onto titanium substrates using a spinassisted LBLs technique. Ag ions would be spontaneously accumulated into the polyelectrolyte multilayer films via the specific coordination between Ag ions and –HS groups in Chi-HS, thus leading to formation of Ag nanoparticles in situ after ultraviolet irradiation [40]. To some
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Fig. 7 CLSM images (a) and viabilities (b) of Bacillus subtitles grown onto different titanium substrates after culturing for 12 h. a1 Bare titanium, a2 Ti/PEI/(Gel/ChiSH)4/Gel, a3 Ti/PEI/(Gel/ChiSH/Ag0.005)/Gel, a4 Ti/PEI/ (Gel/Chi-SH/Ag0.005)2/Gel, a5 Ti/PEI/(Gel/Chi-SH/Ag0.005)3/ Gel, and a6 Ti/PEI/(Gel/ChiSH/Ag0.005)4/Gel. The AgNO3 concentration was 0.005 M
extent, the loading amount of Ag nanoparticles onto the Ti substrate was greatly dependent on the interactions between Ag ions and multilayer films. For Chi/Gel multilayered films, although Ag ions might be incorporated into multilayer films by physical adsorptions and ion exchanges, most of ions still would be excluded from the multilayer films via electrostatic repulsion due to the group of amino groups in chitosan molecules. For Chi-SH/Gel multilayer films, on the contrary, the specific interactions between Ag ions and –HS groups in Chi-SH could lead to the enrichment of Ag ions within multilayer films [41]. Previous studies confirmed Ag ions were facile to form stable sulfur-Ag bonds [42, 43], which would remain silver ions within multilayer films during assembly process. Therefore, the amount of Ag nanoparticles onto Chi-SH/
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Gel multilayer films was much more than those of Chi/Gel multilayer films after the UV treatment. Besides, the loading amount of Ag nanoparticles also increased with the number of deposition cycles (Fig. 4A, c–f), which was well consistent with previous report [26]. The functional multilayered films on titanium substrate (Ti/PEI/(Gel/Chi-SH/Ag)n/Gel) could efficiently inhibit the growth and activity of B. subtitles and E. coli onto titanium surface. The components of multilayer films onto Ti substrates determined their antibacterial activity. The multilayer films without Ag deposition (Ti/PEI/(Gel/Chi-SH)4/ Gel) slightly inhibited the bacteria adhesion and growth since chitosan were able to disrupt bacteria membranes [44]. However, the antibacterial activity of chitosan was not enough to inhibit the growth of bacteria for a long
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Fig. 8 Osteoblasts functions assays. a CLSM images of osteoblasts adhered to different titanium substrates after culture for 2 days. a1 bare titanium, a2 Ti/PEI/(Gel/Chi-SH)4/Gel, a3 Ti/PEI/(Gel/Chi-SH/Ag0.005)/ Gel, a4 Ti/PEI/(Gel/Chi-SH/ Ag0.005)2/Gel, a5 Ti/PEI/(Gel/ Chi-SH/Ag0.005)3/Gel, and a6 Ti/PEI/(Gel/Chi-SH/Ag0.005)4/ Gel. b Cells viabilities of osteoblasts adhered to different titanium substrates after culture for 2 days. c Alkaline phosphatase activity of osteoblasts adhered to different substrates after culturing for 2 days. The AgNO3 concentration was 0.005 M. Error bars represent mean ± SD for n = 6, *P \ 0.05
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period. Thus, there was no significant difference in antibacterial activity between Ti/PEI/(Gel/Chi-SH)4/Gel and bare Ti substrates (Fig. 5b1, b2). Nevertheless, Ag nanoparticles within multilayered films endowed the Ti substrate with potential antibacterial activity. It could be interpreted that Ag ions derived from Ag nanoparticles within multilayer films were released into peripheral region around Ti substrate and kill bacterial until Ag nanoparticles were completely consumed. It means that the more Ag nanoparticles were formed within the multilayer films, the longer period of antibacterial activity against microorganism would be achieved. Therefore, the adhesion and activity of both B. subtitles and E coli onto Ti/PEI/(Gel/ Chi-SH/Ag0.005)n/Gel substrates were severely inhibited, especially those substrate with multiple deposition cycles of Ag ions, comparing with those of bare Ti substrates., Moreover, the loading amount of Ag nanoparticles was not only dependent on the number of deposition cycles but also the types of polyelectrolyte in multilayer films. The coordination between Chi-SH and silver significantly enhanced the loading amount of Ag nanoparticles in multilayered films, leading to its obvious antimicrobial effect after only two cycles of deposition (Fig. 5b). The study thus demonstrated its advantage in the loading of Ag ions via ChiSH molecules. Apart from the antibacterial property, Ti substrates coating with such functional multilayer films could remain biological functions of osteoblasts, which was reflected by cell morphology, cell viability and ALP activity measurements. The phenomenon was attributed to the following two reasons. On one hand, the concentration of Ag ions released from multilayer films was not enough to induce cytotoxicity to osteoblasts, while efficiently inhibited the bacteria activity. To some extent, the cytotoxicity of Ag nanoparticles within multilayer films was dominated by the concentration of released Ag ion [45–48]. Both of multilayer films and the coordination between Chi-SH to Ag elements could control the Ag ions release in a slow manner, thus leading to low cytotoxicity to osteoblasts. Besides, the loading of Ag nanoparticles on or within a biomaterial was involved with extra chemical agents [49], e. g., NaBH4. It thus results in undesired cytotoxicity to cells. Here, Ag nanoparticles within Chi-SH/Gel multilayered films were formed only by using sulfur groups (Chi-SH) as seeds for silver ions accumulation and followed by the photocatalysis of ultraviolet exposure. Therefore, there would be no toxic chemicals involving into the whole process. On the other hand, the interaction mechanism of osteoblasts to Ag ions was different from that of bacteria (B. subtitles and E. coli) to Ag ions due to their differences in size and structure [50]. The protondepleted region formed around Ag nanoparticles would, disrupt the synthesis of adenosine triphosphate, leading to
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the death of bacteria [51]. However, mammal cells could diminish the effects of proton-depleted region since they have much larger size and more complex membrane structures [52].
5 Conclusion In summary, the functional ‘‘sandwich-type’’ Chi-SH/Gel polyelectrolyte multilayer films containing Ag nanoparticles were successfully coated onto the surface of titanium substrate via a spin-assisted layer-by-layer assembly technique. Chi-SH molecular was used as nanocontainers to store Ag ions via the specific interactions between Ag ions and –SH groups. Ag nanoparticles could be formed in situ via the simple photo-catalysis reaction under conventional UV light. Antibacterial tests in vitro revealed that the Ag-containing multilayer films onto Ti substrates (Ti/PEI/(Gel/Chi-SH/Ag)n/ Gel) had potent antibacterial activity. Moreover, Ti/PEI/(Gel/ Chi-SH/Ag)n/Gel substrates displayed no cytotoxicity to the biological function of osteoblasts. The approach presented here has potential application for the development of functional titanium-based implants with antibacterial property. Acknowledgments This work was financially supported by Natural Science Foundation of Chongqing Municipal Government (CSTC, 2011JJJQ10004 and JJA10056), China Ministry of Science and Technology (973 Project No. 2009CB930000), Natural Science Foundation of China (51173216 and 31200712), National Key Technology R&D Program of the Ministry of Science and Technology (2012BAI18B04) and the ‘‘111’’ project (B06023).
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