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Preparation of multifunctional drug sustained-release system by atomic layer deposition of ZnO in mesoporous titania coating
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Preparation of multifunctional drug sustained-release system by atomic layer deposition of ZnO in mesoporous titania coating Xinghai Wu1, Litao Yao1, Mohammed A. Al-Baadani, Linchao Ping, Shuyi Wu, Abdullrahman M. Al-Bishari, Kendrick HiiRuYie, Zhennan Deng, Jinsong Liu∗, Xinkun Shen∗∗ School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, 325027, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Titanium Mesoporous Atomic layer deposition Zinc oxide Antibacterial Osteogenesis
In previous studies, it has been proven that properties of titanium mesoporous (MP) coatings have a strong bond to substrates and excellent anti-stripping property, thus making it ideal for clinical implants. Also, the high porosity and specific surface area of MP material make it excellent for drug loading and sustained release. However, compared with untreated titanium, the bioactivity of MP samples does not have a significant improvement. In this study, we deposited zinc oxide (ZnO) into/on MP structures using atomic layer deposition (ALD) technology, which would significantly improve the biological activity of MP material while maintaining its structures. The fluorescence results showed that the porosity and drug loading capacity had little change after 10 ALD cycles (MP-ALD10), but decreased significantly after 30 ALD cycles (MP-ALD30). Cell results showed that with the increase of ALD cycle (from 0 to 50), the cytocompatibility of the corresponding materials increased first and then decreased: the optimal proliferation and differentiation of MC3T3-E1 cells were observed in MP-ALD10 and MP-ALD30 groups, respectively. Moreover, it was found that the antibacterial properties of different MP samples against Escherichia coli and Staphylococcus aureus gradually enhanced with the increase of ZnO (MP < MP-ALD10 < MP-ALD30 < MP-ALD50). Overall, the study proved that 10 cycles of ZnO deposition significantly enhanced the bioactivity of MP without affecting its structure, drug-loading and drugreleasing properties, thus making MP-ALD10 more suitable for the development of drug-device combined titanium implants than MP.
1. Introduction Despite its excellent biocompatibility and mechanical properties, titanium (Ti) lacks the ability to self-integrate with the surrounding bone tissue due to its biological inert surface [1,2]. To effectively improve the bioactivity of Ti implants, the “drug-device” combination approach has been widely studied in recent years [3,4]. Some active substances (e.g., cytokines, functional peptides, antibiotics, and metal ions) will be loaded onto the surface of implants in various methods, and then released locally and slowly to promote damage repair [3–6]. Compared with the conventional Ti implants, these drug-loaded materials have more advantages, such as 1) reducing the demand for drugs; 2) locally releasing drugs to minimize systemic effects on the body; 3) prolonging drug release duration; and 4) controlling drug cytotoxicity and improving its cytocompatibility [7,8]. Titania nanotubes (TNTs) are the most common drug carriers for the
preparation of functional Ti implants because of their advantages of controllable diameter, simple preparation, regular nanotube shape, and good repeatability [8–10]. Some researchers have proven that nanotubes with small diameter (< 30 nm) were conducive to cell proliferation, whereas large diameter (> 70 nm) nanotubes could significantly promote the osteogenic differentiation of osteoblasts [11,12]. Other studies have also claimed that the most suitable size of TNTs for promoting osteogenesis was around 70 nm, so it was usually used to load drugs to prepare Ti-based “drug-device” combination implants [13–16]. However, the disadvantages of weak binding strength and easy exfoliation limit the application of drug-loaded TNT materials in clinical treatment. The debris from the peeling of the coatings may lead to chronic inflammation or implant loosening [17]. Li et al. claim that the weak adhesion (Lc value: ~4 N) of TNTs to Ti substrate compromised many promising applications of the related materials [18]. We also report that as the size increased, the bonding strength of the TNT
∗
Corresponding author. 268# Xueyuan West Road, Lucheng District, Wenzhou City, Zhejiang Province, China Corresponding author. 268# Xueyuan West Road, Lucheng District, Wenzhou City, Zhejiang Province, China E-mail addresses: [email protected] (J. Liu), [email protected] (X. Shen). 1 Co-first author: Xinghai Wu and Litao Yao contributed equally to this work. ∗∗
https://doi.org/10.1016/j.ceramint.2019.12.201 Received 26 August 2019; Received in revised form 29 November 2019; Accepted 24 December 2019 0272-8842/ © 2019 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
Please cite this article as: Xinghai Wu, et al., Ceramics International, https://doi.org/10.1016/j.ceramint.2019.12.201
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Fig. 1. (A) Schematic diagram of preparation process of ALD modified MP specimens (MP-ALD); (B) top view SEM images of different samples.
still has limitations in application. Because zinc (Zn) can effectively participate in the regulation of DNA synthesis, enzyme activity, osteoblast proliferation, and biomineralization, Zn-containing coatings have been widely used for surface modification of titanium [27–29]. Previous studies had reported that the proper concentration of zinc ions (Zn2+) could promote the formation of new bone by systematically regulating the functions of macrophages, osteoblasts, and osteoclasts [30–32]. In addition to the osteoinductive properties, the released Zn2+ could also exhibit excellent antibacterial abilities by inducing excessive production of reactive oxygen species [33,34]. Therefore, we hypothesized that the biological properties of MP materials could be further enhanced by suitable zinc oxide coatings. Atomic layer deposition (ALD) is a method of forming deposition films by alternately passing pulses of gaseous precursors into the reactor and chemically adsorbing and reacting on the deposited substrate [35,36]. Substances are deposited layer by layer on the substrate in the form of a monoatomic film. A thickness-controllable functional coating can be obtained by adjusting the number of ALD cycles. Hence, making the ALD technique is the most often used method for preparing target coatings on implant surface (especially irregular materials) [36–38]. In this study, we prepared a suitable ZnO coating within the MP structures for the first time via ALD technology. The ALD treated substrates should still have good mesoporous structures for the subsequent loading of active drugs. Meanwhile, these ZnO modified samples are expected to have better osteogenic induction and antibacterial properties than the unmodified MP substrates. The following three specific
coating to the substrate gradually decreased (15 nm TNTs: ~4 N; 125 nm TNTs: < 1 N) [19]. In order to improve the adhesion strength of TNT coatings, methods such as additional anodization and calcination of TNT materials had been developed. Still, the improvement of the bonding strength was limited (Lc value: increasing to ~ 16 N) [20,21]. Therefore, it is urgent to develop other titanium-based drug-loading materials with ideal anti-stripping properties. Comparing with TNTs, mesoporous (MP) coatings prepared by the similar anodizing method have excellent stability and drug loading properties [22,23]. In 2009, Kim et al. have successfully prepared MP structures on titanium surface using glycerol/dipotassium hydrogen phosphate electrolyte for the first time [24]. After rigorous mechanical treatment such as scratching and bending, the MP coatings did not peel off, indicating that MP coating has strong anti-stripping performance [24]. In our previous study, we had successfully used the MP structure to achieve the patterning of 125 nm TNTs, and the anti-stripping ability of the patterned coatings is significantly increased (from less than 1 N to greater than 20 N) [19]. Besides, MP samples had been proven to own a larger specific surface area and porosity, ensuring a sustained release of drugs for more than 14 d [22]. After incubation for a period of time in simulated body fluids (SBF), the amount of hydroxyapatite formed on the MP surface was much higher than that on TNTs and smooth titanium [25,26]. Although MP structures have the above advantages, they have no significant positive regulation on the proliferation and differentiation of osteoblasts when comparing with untreated Ti substrates [19,22]. The purpose of surface modification of titanium is to improve its biological activity. Therefore the MP coating 2
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Fig. 2. Representative color images and corresponding statistics of different elements on the substrates of Ti, MP, MP-ALD10, MP-ALD30, and MP-ALD50. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
issues will be addressed in this study: 1) screening ALD cycle to deposit ideal ZnO in MP structures; 2) evaluating the biological responses of osteoblasts, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) to different ALD modified samples; 3) investigating the effects of ALD treatment on the drug loading capacity of MP structures. 2. Materials and methods 2.1. Preparation of mesoporous structures Ti materials (0.2 mm thickness) were purchased from Northwest Institute for Non-ferrous Metal Research (Xi'an, China) and cut into 1 × 1 cm samples. After cleaning and drying treatments, the target MP structures were prepared on Ti surface by anodic oxidation technology according to our previous study [19,23]. Briefly, 10 wt% dipotassium hydrogen phosphate (K2HPO4) was dissolved in anhydrous glycerol and stirred using a magnetic stirrer (160 rpm) at 250 °C for electrolyte preparation. After 4 h of heat treatment to remove the excess water, Ti (anode) and platinum (cathode) were immersed in the electrolyte for the oxidation reaction. The specific parameters of anodic oxidation were as follows: the first stage was to react at 20 V and 180 °C for 4 h, and the second stage was carried out at 50 V and 180 °C for 20 min. Finally, all oxidized samples were soaked in hydrogen peroxide solution (30 wt%) for 1 h and named as MP. 2.2. ALD treatment of ZnO Fig. 3. (A) Water contact angle of different samples; (B) the released profiles of Zn2+ from MP-ALD10, MP-ALD30, and MP-ALD50 substrates within 28 d.
The schematic diagram of the preparation process of ALD modified MP specimens (MP-ALD) was shown in Fig. 1A. Briefly, DEZ (pumping 30 ms; stopping 4s) was filled in the instrument chamber to form the first layer on the MP surface [reaction equation: Sample-OH + Zn 3
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Fig. 4. (A) Cell viability in different groups after 1, 4 and 7 d; ALP activity (B) and mineralization level (C) of MC3T3-E1 cells on different substrates at 7 d, *p < 0.05, **p < 0.01.
(C2H5)2 → Sample-O-Zn-C2H5 + C2H6]. After removing the residual DEZ and by-product (C2H6) by N2 (100 sccm, 10 s), deionized water (pumping 30 ms; stopping 4s) was used to form the second layer in MP structures [reaction equation: Sample-O-Zn-C2H5 + H2O → Sample-OZn-OH + C2H6]. The remaining H2O molecule and C2H6 compounds also needed to be removed by N2 (100 sccm, 10 s) before the next ALD cycle. Samples prepared after 10, 30, and 50 cycles were denoted as MP-ALD10, MP-ALD30, and MP-ALD50, respectively.
2.5. Cell viability, alkaline phosphatase (ALP) activity, and mineralization MC3T3-E1 cells at 1 × 104 cells/cm2 were seeded on different substrates (6 samples in each group), and cultured with α-MEM medium supplemented with 0.05 mM vitamin C, 10 mM β-glycerophosphate sodium, and 100 mM dexamethasone (osteogenic differentiation medium). Firstly, for viability assay, the mixture of medium (180 μL) and CCK-8 (20 μL) was used to replace the old medium and incubated for another 2 h after culturing for 1, 4, and 7 d. 150 μL of the final solution was collected and measured at 450 nm using a microplate reader (SpectraMax M5, Molecular Devices, USA). Secondly, for ALP activity, the cells cultured for 7 d were lysed by 1% Triton X-100 solution. The protein concentration and ALP activity in the lysates were measured by BCA protein and ALP assay kits (Beyotime, China) at 562 and 520 nm, respectively. Thirdly, for mineralization detection, cells on different samples were fixed by 4% paraformaldehyde after 14 d. Calcium nodules were then stained with alizarin red (30 min), dissolved by 10% cetylpyridinium chloride solution (120 min), and measured at 540 nm.
2.3. Sample characterization The characterizations of surface morphology and element composition were carried out using scanning electron microscopy (SEM, HitachiS4800, Japan) and energy dispersive spectrometer (EDS, HitachiS4800, Japan), respectively. The cross-sectional morphology of MP, MP-ALD10, and MP-ALD30 was also observed by SEM. In addition, the water contact angle of different samples was detected by a contact angle apparatus (Model 200, Future Scientific, China). 2.4. Release of zinc ions
2.6. Gene expression MP-ALD10, MP-ALD30, and MP-ALD50 samples were soaked in 10 mL of PBS solution to detect the release of Zn2+. After soaking at 37 °C for 1, 3, 5, 7, 14, 21, and 28 d, the Zn2+-contained solution (10 mL) was collected one by one and detected by inductively coupled plasma/optical emission spectroscopy (ICP-OES, Vista AX, USA). Once the soaking solution was collected, 10 mL of new PBS would be immediately re-added.
MC3T3-E1 cells at 1 × 104 cells/cm2 were seeded on different substrates (6 samples in each group) for 7 d. The total RNA was then collected using an RNAiso kit (TaKaRa, Japan) and reverse transcribed into cDNA using a PrimeScript™ RT reagent kit (TaKaRa, Japan). Finally, SYBR Premix ExTMTaq II was used to determine the expression of target genes [runt-related transcription factor 2 (Runx2), ALP, 4
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Fig. 5. The relative expression of Runx2, ALP, OPN and OCN genes at 7 d, *p < 0.05.
1 mg/mL of rhodamine solution was first prepared using deionized water. Four samples of MP, MP-ALD10, and MP-ALD30 were then soaked into the rhodamine solution under dark conditions for 12 h. After cleaning with deionized water for 3 times, the specimens loaded with rhodamine were observed using a confocal laser scanning microscope (CLSM, Nikon A1, Japan) with layered fluorescence scanning (scan depth: 0–18 μm). The final fluorescence data were analyzed using NIS Elements Viewer software and presented as a 3D image.
osteopontin (OPN), and osteocalcin (OCN)] with the following primers: Runx2 (5′-GCCGTAGAGAGCAGGGAAGA-3’; 5′-CTGGCTTGGATTAGGG AGTCA-3′), ALP (5′-AGCGACACGGACAAGAAGC-3’; 5′-GGCAAAGACC GCCACATC-3′), OPN (5′-GACAGCAACGGGAAGACC-3’; 5′-CAGGCTGG CTTTGGAACT-3′), OCN (5′-AGATTGTTGGGGCACAAG-3’; 5′-CCTTCA GCAGGGAAACCG-3′). 2.7. Bacterial viability E. coli (ATCC25922) and S. aureus (ATCC35984) were seeded on different substrates (6 samples in each group) at 1 × 106 cells/cm2 and cultured using Luria-Bertani (LB) medium. After 24 h, 450 μL of new LB medium and 50 μL of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT, 5 mg/mL) were added to each well and incubated for another 4 h. Finally, the formed formazan was dissolved by dimethyl sulfoxide and detected at 490 nm using a microplate reader (SpectraMax M5, Molecular Devices, USA).
2.10. Release of rhodamine
2.8. Staining of living/dead bacteria
2.11. Statistical analysis
E. coli and S. aureus were cultured on different substrates (4 samples in each group) at 1 × 106 cells/cm2 for 24 h. After washing 3 times with PBS solution, the bacterial samples were carefully stained using a LIVE/DEAD Backlight Bacterial Viability Kit (thermos fisher, USA) and observed with a fluorescence microscope (Eclipse Ni–U, Nikon, Japan). The fluorescence images were finally analyzed using Image-Pro Plus 6.0 software.
All data were independently repeated more than 3 times, analyzed by the one-way ANOVA and SNKmultiple comparison tests with OriginPro 8.0 software, and expressed in means ± SD. The confidence levels were set as 95% (*p < 0.05) and 99% (*p < 0.01).
Five rhodamine-loaded samples in each group were soaked in 5 mL of PBS solution to detect the release of rhodamine. After 1, 3, 5, 7, 14, 21, and 28 d, the soaking solution (0.5 mL) was collected one by one and detected at the excitation wavelength of 540 nm. 0.5 mL of new PBS was re-added to keep the release solution at 5 mL. The whole experimental process needed to be carried out under dark conditions.
3. Results and discussion 3.1. Physicochemical characteristics
2.9. Fluorescence and SEM observation of rhodamine-loaded samples Firstly, to characterize the surface morphology of different samples, SEM observation was carried out and further displayed in Fig. 1B. Compared to Ti (smooth surface), MP specimens had well-distributed
In this study, rhodamine was used as a model drug to investigate the drug-loading and drug-releasing properties of different substrates. 5
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Fig. 6. (A) Bacterial viability of S. aureus and E. coli at 24 h; (B) statistics of dead/total bacteria according to the live/dead stained images (C; green: living bacteria; red: dead bacteria). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
only observed on the surfaces of ALD-treated samples: MP-ALD10 (0.20 ± 0.07 at%), MP-ALD30 (0.48 ± 0.04 at%), and MP-ALD50 (0.76 ± 0.08 at%). It suggested that ZnO coatings were successfully prepared in the MP structures and gradually thickened with the increase of ALD cycles. Thirdly, to evaluate the wettability of different samples, the water contact angle was measured and displayed in Fig. 3A. Ti substrates exhibited the most robust hydrophobic property in all groups (about 68°). After anodic oxidation treatment, the formed mesoporous structures enabled MP specimens to have hydrophilic surfaces (around 24°), which was consistent with our previous study [19]. Moreover, with the increase of ZnO addition, the contact angles of the corresponding samples gradually increased and were about 19° (MP-ALD10), 25° (MPALD30) and 32° (MP-ALD50) respectively. The wettability changes of MP-ALD samples might be attributed to the gradual decrease of mesoporous size after different cycles of ALD treatment (verified in Fig. 1B). Results of the previous study have reported that the hydrophilic surface (about 15°) could significantly increase osteoblast differentiation by regulating the expression of some specific integrin [41]. Another study also claims that the hydrophilicity might be more important than nanostructures in promoting mesenchymal stem cells differentiation into osteoblasts [42]. Thus, the hydrophilic MP-based samples (MP, MPALD10, MP-ALD30, and MP-ALD50) may have better biological activities than untreated titanium.
mesoporous structures and no obvious cracks can be observed, which was consistent with the previous studies [19,23]. After ALD treatment, we found that the pore size decreases gradually with the increase of ALD cycles (MP > MP-ALD10 > MP-ALD30 > MP-ALD50), and no apparent porous structures were observed in the MP-ALD50 group, which attributed to enough deposition of ZnO. Previous studies had claimed that the thickness-controllable coatings could be obtained by adjusting the number of ALD cycles [35–38]. According to the data provided by the instrument manufacturer and determined in our previous study [35], the thickness of the ZnO layer prepared by one ALD cycle was approximately 0.17 nm. So if the pore size was sufficient, the thickness of one-sided ZnO coatings after 10, 30, and 50 cycles should reach about 1.7, 5.1, and 8.5 nm, respectively. However, we had also found out that the pore size of MP structure was only about 14 nm (maximum thickness of one-sided ZnO: about 7 nm) [23], which further explained why the pore structure on the surface of MP-ALD50 disappeared. Secondly, to investigate the elemental compositions of different materials, the EDS test was performed and shown in Fig. 2. The carbon (C) element was observed on all sample surfaces, which might be attributed to the environmental organic pollution during drying/storage [39]. Because the titanium dioxide (TiO2) coating on untreated titanium was thinner than 10 nm and EDS detection had low sensitivity to light elements (e.g., C, O, F, etc.) [29,40], only Ti element (98.35 ± 0.48 at%) was observed in Ti group. After anodic oxidation with or without ALD treatment, the contents of Ti and O element were approximately 30–32 at% and 65–68 at%, respectively. The atomic ratio of O to Ti was close to 2:1, which suggests that the surface composition of MP was mainly TiO2 after anodic oxidation treatment and the result was consistent with previous studies [23,24]. Zn element was
3.2. Zn2+ release Due to the dose-effect of Zn2+ on osteoblasts and bacteria [43,44], we detected its release from MP-ALD10, MP-ALD30, and MP-ALD50 samples at different durations. From Fig. 3A, it was found that the Zn2+ 6
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Fig. 7. (A) Cross-sectional SEM images of MP, MP-ALD10 and MP-ALD30 specimens; (B) top view and 3D fluorescent images of rhodamine-loaded substrates (purple to blue represent rhodamine molecules from surface to bottom); (C) the released profiles of rhodamine from different substrates within 28 d. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
ALP has been identified as a zinc-containing homopolymer protein that can catalyze the degradation of monophosphates under alkaline conditions [48]. The lack of Zn2+, an important component of enzyme, can lead to the inactivation of ALP [49,50]. Other studies had also reported that the addition of an appropriate amount of zinc to implants could significantly increase the ALP activity of adherent osteoblasts [28,29,50]. In this study, similar to viability result, the ALP activity of MC3T3-E1 cells on different substrates also exhibited a trend of increasing first and then decreasing with the increase of ALD cycles (Fig. 4B). But the highest ALP activity was observed in the MP-ALD30 instead of MP-ALD10 group after 7 d of culture. The weak ALP activity in MP-ALD50 group was also most likely due to cytotoxicity caused by the high dose release of Zn2+ [29,51]. Because ALP could hydrolyze phosphate monoester to provide phosphate for osteoblast mineralization [48,52,53], mineralization results (Fig. 4C) also showed a trend similar to ALP activity after 14 d MC3T3-E1 cells on MP-ALD30 substrates had the highest (p < 0.01) mineralization level among all groups. Moreover, comparing with Ti and MP-ALD50, MP-ALP10 also significantly (p < 0.01) increased the mineralization of MC3T3-E1 cells. RT-qPCR results were used to further verify the differentiation potential of MC3T3-E1 cells on different substrates at the molecular level and showed in Fig. 5. Comparing with Ti and MP-ALD50 groups, the expression of Runx2, ALP, OPN, and OCN genes of cells on MP-ALD10 and MP-ALD30 substrates was significantly increased (p < 0.05). No significant difference was found between MP-ALD10 and MP-ALD30 groups, which was consistent with the ALP results. Runx2 had been proven to be the key gene for inducing osteogenic differentiation (collagen synthesis, ALP activity, and mineralization), which could be up-regulated by zinc ions [51,54]. Yusa et al. also claimed that the released Zn2+ from zinc-modified Ti substrates could significantly stimulate the calcium deposition and gene expression (e.g., Runx2, ALP,
released from MP-ALD50, MP-ALD30, and MP-ALD10 substrates in the first week were 5.32 ± 0.11, 3.18 ± 0.15 and 1.19 ± 0.09 ppm, respectively. In the following 3 weeks, the release rate of Zn2+ slowed down, reaching 7.15 ± 0.12, 4.17 ± 0.18, and 1.61 ± 0.09 ppm on the 28th day. The long-term release of Zn2+ was attributed to the presence of mesoporous structures, which had been proven to interwoven into a network with some irregular pore channels and slowly release zoledronic acid for up to 14 days [23]. Moreover, a fast release of Zn2+ from implants would potentially cause cell damage via oxidative stress [45], and the high release of Zn2+ in MP-ALD50 group might not only lead to the vigorous bactericidal activity on S. aureus and E. coli, but also caused cytotoxicity on osteoblasts, which was confirmed by the following results (Figs. 4–6). 3.3. Cell viability and osteogenic differentiation To investigate the proliferation and differentiation of MC3T3-E1 cells on different samples, Cell Counting Kit-8 (CCK-8), alkaline phosphatase (ALP) activity, mineralization, and real-time quantitative polymerase chain reaction (RT-qPCR) assays were carried out under the condition of osteogenic differentiation medium [46,47]. The result of the CCK-8 assay was shown in Fig. 4A. After 1 d, the viability of cells on MP-ALD50 substrates was lower than that of the MP-ALD10 group (p < 0.05), but there was no significant difference with other groups. With the increase of culture time (4 and 7 d), cells on MP-ALD50 substrates still showed the worst viability. It might be attributed to the release of excess Zn2+ from MP-ALD50 sample (Fig. 3B), which was consistent with the previous study showing that high doses of Zn2+ could significantly induce significant cytotoxicity to osteoblasts via oxidative stress [43,45]. Moreover, comparing with Ti, MP-ALP10 samples also significantly (p < 0.05 or 0.01) increased the cell viability of MC3T3-E1 cells after 4 and 7 d. 7
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collagen I, and OCN) [55]. Taken together, the results of CCK-8, ALP activity, mineralization, and RT-qPCR indicate that both MP-ALD10 and MP-ALD30 could greatly promote the osteogenic differentiation of MC3T3-E1 cells, while only MP-ALD10 was beneficial to cell proliferation.
group only within 7 d. These results suggest that the ZnO coating prepared by 10 ALD cycles has no significant effect on the drug loading and controlled release properties of mesoporous materials.
3.4. Antibacterial ability
Zinc oxide coatings were prepared inside the mesoporous (MP) structures by atomic layer deposition (ALD) techniques in this study to improve the bioactivity of MP samples without affecting their drug loading and release potential. All ZnO modified samples showed excellent antibacterial abilities (bacteriostatic rate: MP-ALD10 < MPALD30 < MP-ALD50) against both S. aureus and E. coli when comparing with Ti and MP groups. Results of osteoblast assays further displayed that both MP-ALD10 and MP-ALD30 increased cell differentiation, but only MP-ALD10 favored the proliferation of MC3T3-E1 cells. Also, compared with MP samples, MP-ALD10 did not show a significant effect on drug loading and release properties. Thus, MPALD10 might be more suitable for the development of titanium-based “drug-device” combination implants in the future.
4. Conclusion
When a bacterial infection occurs after implant surgery, bacteria will adhere to the implant surface and rapidly form a dense biofilm, which will prevent the drug from penetrating to the infected site [23,56]. Therefore, traditional clinical antimicrobial therapy, such as oral administration or intravenous injection, is difficult to control the infection around the implant [56,57]. In recent years, researches have been working to develop functional implants that can achieve superior antibacterial/osteogenic properties through in situ sustained-release antibiotics [3–6]. Metal ions (e.g., Zn2+, Ag+, and Cu2+) are often used in the preparation of antibacterial implants because they have good spectrum antibacterial properties and are not easy to induce drug resistance [23,58]. Previous studies had proven that Zn2+ could cause bacteria death by inducing excessive production of reactive oxygen species and further destroying bacterial membranes [34, 35, 59]. To evaluate the antibacterial ability of different substrates, bacterial viability and live/dead staining assays were carried out. From Fig. 6A, it was found that there was no significant difference in the viability of bacteria on Ti and MP substrates. After ALD treatment, three ZnOmodified samples showed superior antibacterial properties against both S. aureus and E. coli when compared with Ti and MP groups. It was also found that with the increase of ALD cycles, the antibacterial properties of corresponding samples gradually increased (MP-ALD10 < MPALD30 < MP-ALD50), which was also further confirmed by the results of live/dead staining (Fig. 6B and C). The ratio of dead bacteria to total bacteria in MP-ALD10, MP-ALD30 and MP-ALD50 groups were 63.3 ± 6.5 (S. aureus)/68.4 ± 5.0 (E. coli), 83.2 ± 4.3 (S. aureus)/ 87.3 ± 3.7 (E. coli), and 96.1 ± 3.5 (S. aureus)/97.1 ± 3.0 (E. coli), respectively. The above results suggest that the three ZnO coatings prepared by 10, 30, and 50 ALD cycles had excellent antibacterial abilities against both S. aureus and E. coli.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (81870810, 81701016, and 31700827), Zhejiang Provincial Science and Technology Project for Public Welfare (2015C33139), and Zhejiang Xinmiao Talents Program (2019R413066). References [1] T. Takizawa, N. Nakayama, H. Haniu, K. Aoki, M. Okamoto, H. Nomura, M. Tanaka, A. Sobajima, K. Yoshida, T. Kamanaka, K. Ajima, A. Oishi, C. Kuroda, H. Ishida, S. Okano, S. Kobayashi, H. Kato, N. Saito, Titanium fiber plates for bone tissue repair, Adv. Mater. 30 (2018) 1703608. [2] W.E. Yang, H.H. Huang, Multiform TiO2 nano-network enhances biological response to titanium surface for dental implant applications, Appl. Surf. Sci. 471 (2019) 1041–1052. [3] P. Wu, D.W. Grainger, Drug/device combinations for local drug therapies and infection prophylaxis, Biomaterials 27 (2006) 2450–2467. [4] C. Alvarez-Lorenzo, A. Concheiro, Smart drug release from medical devices, J. Pharmacol. Exp. Ther. 370 (2019) 119. [5] D. Losic, M.S. Aw, A. Santos, K. Gulati, M. Bariana, Titania nanotube arrays for local drug delivery: recent advances and perspectives, Expert Opin. Drug Deliv. 12 (2015) 103–127. [6] T.T. Wang, X.M. Liu, Y.Z. Zhu, Z.D. Cui, X.J. Yang, H.B. Pan, K.W.K. Yeung, S.L. Wu, Metal ion coordination polymer-capped pH-triggered drug release system on titania nanotubes for enhancing self-antibacterial capability of Ti implants, ACS Biomater. Sci. Eng. 3 (2017) 816–825. [7] J.R. Porter, T.T. Ruckh, K.C. Popat, Bone tissue engineering: a review in bone biomimetics and drug delivery strategies, Biotechnol. Prog. 25 (2009) 1539–1560. [8] S. Maher, A. Mazinani, M.R. Barati, D. Losic, Engineered titanium implants for localized drug delivery: recent advances and perspectives of Titania nanotubes arrays, Expert Opin. Drug Deliv. 15 (2018) 1021–1037. [9] Y. Cheng, H. Yang, Y. Yang, J. Huang, K. Wu, Z. Chen, X. Wang, C. Lin, Y. Lai, Progress in TiO2 nanotube coatings for biomedical applications: a review, J. Mater. Chem. B 6 (2018) 1862–1886. [10] A. Hamlekhan, S. Sinha-Ray, C. Takoudis, M.T. Mathew, C. Sukotjo, A.L. Yarin, T. Shokuhfar, Fabrication of drug eluting implants: study of drug release mechanism from titanium dioxide nanotubes, J. Phys. D Appl. Phys. 48 (2015) 275401. [11] S. Oh, K.S. Brammer, Y.S. Li, D. Teng, A.J. Engler, S. Chien, S. Jin, Stem cell fate dictated solely by altered nanotube dimension, Proc. Natl. Acad. Sci. U.S.A. 106 (2009) 2130e2135. [12] Y. Yu, X. Shen, Z. Luo, Y. Hu, M. Li, P. Ma, Q. Ran, L. Dai, Y. He, K. Cai, Osteogenesis potential of different titania nanotubes in oxidative stress microenvironment, Biomaterials 167 (2018) 44–57. [13] N. Wang, H. Li, W. Lü, J. Li, J. Wang, Z. Zhang, Y. Liu, Effects of TiO2 nanotubes with different diameters on gene expression and osseointegration of implants in minipigs, Biomaterials 32 (2011) 6900–6911.
3.5. Drug loading and release Since MP-ALD50 had significant cytotoxicity on the proliferation and differentiation of osteoblasts, it was not suitable for the preparation of functional titanium materials as a drug carrier. Thus, only the drug loading and release properties of MP, MP-ALD10, and MP-ALD30 samples were characterized in this study. Rhodamine was used as the model drug. Firstly, the cross-sectional morphology of MP, MP-ALD10, and MP-ALD30 was imaged and shown in Fig. 7A. Similar to our previous study, there were some clear mesoporous structures in the crosssection of MP sample [23]. The pore size of mesoporous structures was significantly reduced in the MP-ALD10 and almost completely disappeared in the MP-ALD30, which was consistent with the aforementioned top view results (Fig. 1B). Secondly, rhodamine-loaded substrates were observed by CLSM and showed in Fig. 7B. From the top view images, weak fluorescence was observed in MP-ALD30 when comparing with MP and MP-ALD10 groups. The 3D fluorescence images further confirmed that the MP and MP-ALD10 groups had stronger fluorescence signals and greater penetration depth (MP/MP-ALD10: ~18 μm; MP/MP-ALD30: ~8 μm), which indicated that the MP and MPALD10 could load more rhodamine than the MP-ALD30 sample. Thirdly, from the released profiles (Fig. 7C), it was found that there was no significant difference in the release amount and release rate of rhodamine in the MP and MP-ALD10 groups at different durations. The release period of rhodamine in these two groups was more than 28 d, and the total amount at 28 d was approximately 50 μg/mL. However, the loaded rhodamine (~10 μg/mL) wholly released in the MP-ALD30 8
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Preparation of multifunctional drug sustained-release system by atomic layer deposition of ZnO in mesoporous titania coating Xinghai Wu1, Litao Yao1, Mohammed A. Al-Baadani, Linchao Ping, Shuyi Wu, Abdullrahman M. Al-Bishari, Kendrick HiiRuYie, Zhennan Deng, Jinsong Liu∗, Xinkun Shen∗∗ School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, 325027, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Titanium Mesoporous Atomic layer deposition Zinc oxide Antibacterial Osteogenesis
In previous studies, it has been proven that properties of titanium mesoporous (MP) coatings have a strong bond to substrates and excellent anti-stripping property, thus making it ideal for clinical implants. Also, the high porosity and specific surface area of MP material make it excellent for drug loading and sustained release. However, compared with untreated titanium, the bioactivity of MP samples does not have a significant improvement. In this study, we deposited zinc oxide (ZnO) into/on MP structures using atomic layer deposition (ALD) technology, which would significantly improve the biological activity of MP material while maintaining its structures. The fluorescence results showed that the porosity and drug loading capacity had little change after 10 ALD cycles (MP-ALD10), but decreased significantly after 30 ALD cycles (MP-ALD30). Cell results showed that with the increase of ALD cycle (from 0 to 50), the cytocompatibility of the corresponding materials increased first and then decreased: the optimal proliferation and differentiation of MC3T3-E1 cells were observed in MP-ALD10 and MP-ALD30 groups, respectively. Moreover, it was found that the antibacterial properties of different MP samples against Escherichia coli and Staphylococcus aureus gradually enhanced with the increase of ZnO (MP < MP-ALD10 < MP-ALD30 < MP-ALD50). Overall, the study proved that 10 cycles of ZnO deposition significantly enhanced the bioactivity of MP without affecting its structure, drug-loading and drugreleasing properties, thus making MP-ALD10 more suitable for the development of drug-device combined titanium implants than MP.
1. Introduction Despite its excellent biocompatibility and mechanical properties, titanium (Ti) lacks the ability to self-integrate with the surrounding bone tissue due to its biological inert surface [1,2]. To effectively improve the bioactivity of Ti implants, the “drug-device” combination approach has been widely studied in recent years [3,4]. Some active substances (e.g., cytokines, functional peptides, antibiotics, and metal ions) will be loaded onto the surface of implants in various methods, and then released locally and slowly to promote damage repair [3–6]. Compared with the conventional Ti implants, these drug-loaded materials have more advantages, such as 1) reducing the demand for drugs; 2) locally releasing drugs to minimize systemic effects on the body; 3) prolonging drug release duration; and 4) controlling drug cytotoxicity and improving its cytocompatibility [7,8]. Titania nanotubes (TNTs) are the most common drug carriers for the
preparation of functional Ti implants because of their advantages of controllable diameter, simple preparation, regular nanotube shape, and good repeatability [8–10]. Some researchers have proven that nanotubes with small diameter (< 30 nm) were conducive to cell proliferation, whereas large diameter (> 70 nm) nanotubes could significantly promote the osteogenic differentiation of osteoblasts [11,12]. Other studies have also claimed that the most suitable size of TNTs for promoting osteogenesis was around 70 nm, so it was usually used to load drugs to prepare Ti-based “drug-device” combination implants [13–16]. However, the disadvantages of weak binding strength and easy exfoliation limit the application of drug-loaded TNT materials in clinical treatment. The debris from the peeling of the coatings may lead to chronic inflammation or implant loosening [17]. Li et al. claim that the weak adhesion (Lc value: ~4 N) of TNTs to Ti substrate compromised many promising applications of the related materials [18]. We also report that as the size increased, the bonding strength of the TNT
∗
Corresponding author. 268# Xueyuan West Road, Lucheng District, Wenzhou City, Zhejiang Province, China Corresponding author. 268# Xueyuan West Road, Lucheng District, Wenzhou City, Zhejiang Province, China E-mail addresses: [email protected] (J. Liu), [email protected] (X. Shen). 1 Co-first author: Xinghai Wu and Litao Yao contributed equally to this work. ∗∗
https://doi.org/10.1016/j.ceramint.2019.12.201 Received 26 August 2019; Received in revised form 29 November 2019; Accepted 24 December 2019 0272-8842/ © 2019 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
Please cite this article as: Xinghai Wu, et al., Ceramics International, https://doi.org/10.1016/j.ceramint.2019.12.201
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Fig. 1. (A) Schematic diagram of preparation process of ALD modified MP specimens (MP-ALD); (B) top view SEM images of different samples.
still has limitations in application. Because zinc (Zn) can effectively participate in the regulation of DNA synthesis, enzyme activity, osteoblast proliferation, and biomineralization, Zn-containing coatings have been widely used for surface modification of titanium [27–29]. Previous studies had reported that the proper concentration of zinc ions (Zn2+) could promote the formation of new bone by systematically regulating the functions of macrophages, osteoblasts, and osteoclasts [30–32]. In addition to the osteoinductive properties, the released Zn2+ could also exhibit excellent antibacterial abilities by inducing excessive production of reactive oxygen species [33,34]. Therefore, we hypothesized that the biological properties of MP materials could be further enhanced by suitable zinc oxide coatings. Atomic layer deposition (ALD) is a method of forming deposition films by alternately passing pulses of gaseous precursors into the reactor and chemically adsorbing and reacting on the deposited substrate [35,36]. Substances are deposited layer by layer on the substrate in the form of a monoatomic film. A thickness-controllable functional coating can be obtained by adjusting the number of ALD cycles. Hence, making the ALD technique is the most often used method for preparing target coatings on implant surface (especially irregular materials) [36–38]. In this study, we prepared a suitable ZnO coating within the MP structures for the first time via ALD technology. The ALD treated substrates should still have good mesoporous structures for the subsequent loading of active drugs. Meanwhile, these ZnO modified samples are expected to have better osteogenic induction and antibacterial properties than the unmodified MP substrates. The following three specific
coating to the substrate gradually decreased (15 nm TNTs: ~4 N; 125 nm TNTs: < 1 N) [19]. In order to improve the adhesion strength of TNT coatings, methods such as additional anodization and calcination of TNT materials had been developed. Still, the improvement of the bonding strength was limited (Lc value: increasing to ~ 16 N) [20,21]. Therefore, it is urgent to develop other titanium-based drug-loading materials with ideal anti-stripping properties. Comparing with TNTs, mesoporous (MP) coatings prepared by the similar anodizing method have excellent stability and drug loading properties [22,23]. In 2009, Kim et al. have successfully prepared MP structures on titanium surface using glycerol/dipotassium hydrogen phosphate electrolyte for the first time [24]. After rigorous mechanical treatment such as scratching and bending, the MP coatings did not peel off, indicating that MP coating has strong anti-stripping performance [24]. In our previous study, we had successfully used the MP structure to achieve the patterning of 125 nm TNTs, and the anti-stripping ability of the patterned coatings is significantly increased (from less than 1 N to greater than 20 N) [19]. Besides, MP samples had been proven to own a larger specific surface area and porosity, ensuring a sustained release of drugs for more than 14 d [22]. After incubation for a period of time in simulated body fluids (SBF), the amount of hydroxyapatite formed on the MP surface was much higher than that on TNTs and smooth titanium [25,26]. Although MP structures have the above advantages, they have no significant positive regulation on the proliferation and differentiation of osteoblasts when comparing with untreated Ti substrates [19,22]. The purpose of surface modification of titanium is to improve its biological activity. Therefore the MP coating 2
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Fig. 2. Representative color images and corresponding statistics of different elements on the substrates of Ti, MP, MP-ALD10, MP-ALD30, and MP-ALD50. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
issues will be addressed in this study: 1) screening ALD cycle to deposit ideal ZnO in MP structures; 2) evaluating the biological responses of osteoblasts, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) to different ALD modified samples; 3) investigating the effects of ALD treatment on the drug loading capacity of MP structures. 2. Materials and methods 2.1. Preparation of mesoporous structures Ti materials (0.2 mm thickness) were purchased from Northwest Institute for Non-ferrous Metal Research (Xi'an, China) and cut into 1 × 1 cm samples. After cleaning and drying treatments, the target MP structures were prepared on Ti surface by anodic oxidation technology according to our previous study [19,23]. Briefly, 10 wt% dipotassium hydrogen phosphate (K2HPO4) was dissolved in anhydrous glycerol and stirred using a magnetic stirrer (160 rpm) at 250 °C for electrolyte preparation. After 4 h of heat treatment to remove the excess water, Ti (anode) and platinum (cathode) were immersed in the electrolyte for the oxidation reaction. The specific parameters of anodic oxidation were as follows: the first stage was to react at 20 V and 180 °C for 4 h, and the second stage was carried out at 50 V and 180 °C for 20 min. Finally, all oxidized samples were soaked in hydrogen peroxide solution (30 wt%) for 1 h and named as MP. 2.2. ALD treatment of ZnO Fig. 3. (A) Water contact angle of different samples; (B) the released profiles of Zn2+ from MP-ALD10, MP-ALD30, and MP-ALD50 substrates within 28 d.
The schematic diagram of the preparation process of ALD modified MP specimens (MP-ALD) was shown in Fig. 1A. Briefly, DEZ (pumping 30 ms; stopping 4s) was filled in the instrument chamber to form the first layer on the MP surface [reaction equation: Sample-OH + Zn 3
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Fig. 4. (A) Cell viability in different groups after 1, 4 and 7 d; ALP activity (B) and mineralization level (C) of MC3T3-E1 cells on different substrates at 7 d, *p < 0.05, **p < 0.01.
(C2H5)2 → Sample-O-Zn-C2H5 + C2H6]. After removing the residual DEZ and by-product (C2H6) by N2 (100 sccm, 10 s), deionized water (pumping 30 ms; stopping 4s) was used to form the second layer in MP structures [reaction equation: Sample-O-Zn-C2H5 + H2O → Sample-OZn-OH + C2H6]. The remaining H2O molecule and C2H6 compounds also needed to be removed by N2 (100 sccm, 10 s) before the next ALD cycle. Samples prepared after 10, 30, and 50 cycles were denoted as MP-ALD10, MP-ALD30, and MP-ALD50, respectively.
2.5. Cell viability, alkaline phosphatase (ALP) activity, and mineralization MC3T3-E1 cells at 1 × 104 cells/cm2 were seeded on different substrates (6 samples in each group), and cultured with α-MEM medium supplemented with 0.05 mM vitamin C, 10 mM β-glycerophosphate sodium, and 100 mM dexamethasone (osteogenic differentiation medium). Firstly, for viability assay, the mixture of medium (180 μL) and CCK-8 (20 μL) was used to replace the old medium and incubated for another 2 h after culturing for 1, 4, and 7 d. 150 μL of the final solution was collected and measured at 450 nm using a microplate reader (SpectraMax M5, Molecular Devices, USA). Secondly, for ALP activity, the cells cultured for 7 d were lysed by 1% Triton X-100 solution. The protein concentration and ALP activity in the lysates were measured by BCA protein and ALP assay kits (Beyotime, China) at 562 and 520 nm, respectively. Thirdly, for mineralization detection, cells on different samples were fixed by 4% paraformaldehyde after 14 d. Calcium nodules were then stained with alizarin red (30 min), dissolved by 10% cetylpyridinium chloride solution (120 min), and measured at 540 nm.
2.3. Sample characterization The characterizations of surface morphology and element composition were carried out using scanning electron microscopy (SEM, HitachiS4800, Japan) and energy dispersive spectrometer (EDS, HitachiS4800, Japan), respectively. The cross-sectional morphology of MP, MP-ALD10, and MP-ALD30 was also observed by SEM. In addition, the water contact angle of different samples was detected by a contact angle apparatus (Model 200, Future Scientific, China). 2.4. Release of zinc ions
2.6. Gene expression MP-ALD10, MP-ALD30, and MP-ALD50 samples were soaked in 10 mL of PBS solution to detect the release of Zn2+. After soaking at 37 °C for 1, 3, 5, 7, 14, 21, and 28 d, the Zn2+-contained solution (10 mL) was collected one by one and detected by inductively coupled plasma/optical emission spectroscopy (ICP-OES, Vista AX, USA). Once the soaking solution was collected, 10 mL of new PBS would be immediately re-added.
MC3T3-E1 cells at 1 × 104 cells/cm2 were seeded on different substrates (6 samples in each group) for 7 d. The total RNA was then collected using an RNAiso kit (TaKaRa, Japan) and reverse transcribed into cDNA using a PrimeScript™ RT reagent kit (TaKaRa, Japan). Finally, SYBR Premix ExTMTaq II was used to determine the expression of target genes [runt-related transcription factor 2 (Runx2), ALP, 4
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Fig. 5. The relative expression of Runx2, ALP, OPN and OCN genes at 7 d, *p < 0.05.
1 mg/mL of rhodamine solution was first prepared using deionized water. Four samples of MP, MP-ALD10, and MP-ALD30 were then soaked into the rhodamine solution under dark conditions for 12 h. After cleaning with deionized water for 3 times, the specimens loaded with rhodamine were observed using a confocal laser scanning microscope (CLSM, Nikon A1, Japan) with layered fluorescence scanning (scan depth: 0–18 μm). The final fluorescence data were analyzed using NIS Elements Viewer software and presented as a 3D image.
osteopontin (OPN), and osteocalcin (OCN)] with the following primers: Runx2 (5′-GCCGTAGAGAGCAGGGAAGA-3’; 5′-CTGGCTTGGATTAGGG AGTCA-3′), ALP (5′-AGCGACACGGACAAGAAGC-3’; 5′-GGCAAAGACC GCCACATC-3′), OPN (5′-GACAGCAACGGGAAGACC-3’; 5′-CAGGCTGG CTTTGGAACT-3′), OCN (5′-AGATTGTTGGGGCACAAG-3’; 5′-CCTTCA GCAGGGAAACCG-3′). 2.7. Bacterial viability E. coli (ATCC25922) and S. aureus (ATCC35984) were seeded on different substrates (6 samples in each group) at 1 × 106 cells/cm2 and cultured using Luria-Bertani (LB) medium. After 24 h, 450 μL of new LB medium and 50 μL of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT, 5 mg/mL) were added to each well and incubated for another 4 h. Finally, the formed formazan was dissolved by dimethyl sulfoxide and detected at 490 nm using a microplate reader (SpectraMax M5, Molecular Devices, USA).
2.10. Release of rhodamine
2.8. Staining of living/dead bacteria
2.11. Statistical analysis
E. coli and S. aureus were cultured on different substrates (4 samples in each group) at 1 × 106 cells/cm2 for 24 h. After washing 3 times with PBS solution, the bacterial samples were carefully stained using a LIVE/DEAD Backlight Bacterial Viability Kit (thermos fisher, USA) and observed with a fluorescence microscope (Eclipse Ni–U, Nikon, Japan). The fluorescence images were finally analyzed using Image-Pro Plus 6.0 software.
All data were independently repeated more than 3 times, analyzed by the one-way ANOVA and SNKmultiple comparison tests with OriginPro 8.0 software, and expressed in means ± SD. The confidence levels were set as 95% (*p < 0.05) and 99% (*p < 0.01).
Five rhodamine-loaded samples in each group were soaked in 5 mL of PBS solution to detect the release of rhodamine. After 1, 3, 5, 7, 14, 21, and 28 d, the soaking solution (0.5 mL) was collected one by one and detected at the excitation wavelength of 540 nm. 0.5 mL of new PBS was re-added to keep the release solution at 5 mL. The whole experimental process needed to be carried out under dark conditions.
3. Results and discussion 3.1. Physicochemical characteristics
2.9. Fluorescence and SEM observation of rhodamine-loaded samples Firstly, to characterize the surface morphology of different samples, SEM observation was carried out and further displayed in Fig. 1B. Compared to Ti (smooth surface), MP specimens had well-distributed
In this study, rhodamine was used as a model drug to investigate the drug-loading and drug-releasing properties of different substrates. 5
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Fig. 6. (A) Bacterial viability of S. aureus and E. coli at 24 h; (B) statistics of dead/total bacteria according to the live/dead stained images (C; green: living bacteria; red: dead bacteria). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
only observed on the surfaces of ALD-treated samples: MP-ALD10 (0.20 ± 0.07 at%), MP-ALD30 (0.48 ± 0.04 at%), and MP-ALD50 (0.76 ± 0.08 at%). It suggested that ZnO coatings were successfully prepared in the MP structures and gradually thickened with the increase of ALD cycles. Thirdly, to evaluate the wettability of different samples, the water contact angle was measured and displayed in Fig. 3A. Ti substrates exhibited the most robust hydrophobic property in all groups (about 68°). After anodic oxidation treatment, the formed mesoporous structures enabled MP specimens to have hydrophilic surfaces (around 24°), which was consistent with our previous study [19]. Moreover, with the increase of ZnO addition, the contact angles of the corresponding samples gradually increased and were about 19° (MP-ALD10), 25° (MPALD30) and 32° (MP-ALD50) respectively. The wettability changes of MP-ALD samples might be attributed to the gradual decrease of mesoporous size after different cycles of ALD treatment (verified in Fig. 1B). Results of the previous study have reported that the hydrophilic surface (about 15°) could significantly increase osteoblast differentiation by regulating the expression of some specific integrin [41]. Another study also claims that the hydrophilicity might be more important than nanostructures in promoting mesenchymal stem cells differentiation into osteoblasts [42]. Thus, the hydrophilic MP-based samples (MP, MPALD10, MP-ALD30, and MP-ALD50) may have better biological activities than untreated titanium.
mesoporous structures and no obvious cracks can be observed, which was consistent with the previous studies [19,23]. After ALD treatment, we found that the pore size decreases gradually with the increase of ALD cycles (MP > MP-ALD10 > MP-ALD30 > MP-ALD50), and no apparent porous structures were observed in the MP-ALD50 group, which attributed to enough deposition of ZnO. Previous studies had claimed that the thickness-controllable coatings could be obtained by adjusting the number of ALD cycles [35–38]. According to the data provided by the instrument manufacturer and determined in our previous study [35], the thickness of the ZnO layer prepared by one ALD cycle was approximately 0.17 nm. So if the pore size was sufficient, the thickness of one-sided ZnO coatings after 10, 30, and 50 cycles should reach about 1.7, 5.1, and 8.5 nm, respectively. However, we had also found out that the pore size of MP structure was only about 14 nm (maximum thickness of one-sided ZnO: about 7 nm) [23], which further explained why the pore structure on the surface of MP-ALD50 disappeared. Secondly, to investigate the elemental compositions of different materials, the EDS test was performed and shown in Fig. 2. The carbon (C) element was observed on all sample surfaces, which might be attributed to the environmental organic pollution during drying/storage [39]. Because the titanium dioxide (TiO2) coating on untreated titanium was thinner than 10 nm and EDS detection had low sensitivity to light elements (e.g., C, O, F, etc.) [29,40], only Ti element (98.35 ± 0.48 at%) was observed in Ti group. After anodic oxidation with or without ALD treatment, the contents of Ti and O element were approximately 30–32 at% and 65–68 at%, respectively. The atomic ratio of O to Ti was close to 2:1, which suggests that the surface composition of MP was mainly TiO2 after anodic oxidation treatment and the result was consistent with previous studies [23,24]. Zn element was
3.2. Zn2+ release Due to the dose-effect of Zn2+ on osteoblasts and bacteria [43,44], we detected its release from MP-ALD10, MP-ALD30, and MP-ALD50 samples at different durations. From Fig. 3A, it was found that the Zn2+ 6
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Fig. 7. (A) Cross-sectional SEM images of MP, MP-ALD10 and MP-ALD30 specimens; (B) top view and 3D fluorescent images of rhodamine-loaded substrates (purple to blue represent rhodamine molecules from surface to bottom); (C) the released profiles of rhodamine from different substrates within 28 d. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
ALP has been identified as a zinc-containing homopolymer protein that can catalyze the degradation of monophosphates under alkaline conditions [48]. The lack of Zn2+, an important component of enzyme, can lead to the inactivation of ALP [49,50]. Other studies had also reported that the addition of an appropriate amount of zinc to implants could significantly increase the ALP activity of adherent osteoblasts [28,29,50]. In this study, similar to viability result, the ALP activity of MC3T3-E1 cells on different substrates also exhibited a trend of increasing first and then decreasing with the increase of ALD cycles (Fig. 4B). But the highest ALP activity was observed in the MP-ALD30 instead of MP-ALD10 group after 7 d of culture. The weak ALP activity in MP-ALD50 group was also most likely due to cytotoxicity caused by the high dose release of Zn2+ [29,51]. Because ALP could hydrolyze phosphate monoester to provide phosphate for osteoblast mineralization [48,52,53], mineralization results (Fig. 4C) also showed a trend similar to ALP activity after 14 d MC3T3-E1 cells on MP-ALD30 substrates had the highest (p < 0.01) mineralization level among all groups. Moreover, comparing with Ti and MP-ALD50, MP-ALP10 also significantly (p < 0.01) increased the mineralization of MC3T3-E1 cells. RT-qPCR results were used to further verify the differentiation potential of MC3T3-E1 cells on different substrates at the molecular level and showed in Fig. 5. Comparing with Ti and MP-ALD50 groups, the expression of Runx2, ALP, OPN, and OCN genes of cells on MP-ALD10 and MP-ALD30 substrates was significantly increased (p < 0.05). No significant difference was found between MP-ALD10 and MP-ALD30 groups, which was consistent with the ALP results. Runx2 had been proven to be the key gene for inducing osteogenic differentiation (collagen synthesis, ALP activity, and mineralization), which could be up-regulated by zinc ions [51,54]. Yusa et al. also claimed that the released Zn2+ from zinc-modified Ti substrates could significantly stimulate the calcium deposition and gene expression (e.g., Runx2, ALP,
released from MP-ALD50, MP-ALD30, and MP-ALD10 substrates in the first week were 5.32 ± 0.11, 3.18 ± 0.15 and 1.19 ± 0.09 ppm, respectively. In the following 3 weeks, the release rate of Zn2+ slowed down, reaching 7.15 ± 0.12, 4.17 ± 0.18, and 1.61 ± 0.09 ppm on the 28th day. The long-term release of Zn2+ was attributed to the presence of mesoporous structures, which had been proven to interwoven into a network with some irregular pore channels and slowly release zoledronic acid for up to 14 days [23]. Moreover, a fast release of Zn2+ from implants would potentially cause cell damage via oxidative stress [45], and the high release of Zn2+ in MP-ALD50 group might not only lead to the vigorous bactericidal activity on S. aureus and E. coli, but also caused cytotoxicity on osteoblasts, which was confirmed by the following results (Figs. 4–6). 3.3. Cell viability and osteogenic differentiation To investigate the proliferation and differentiation of MC3T3-E1 cells on different samples, Cell Counting Kit-8 (CCK-8), alkaline phosphatase (ALP) activity, mineralization, and real-time quantitative polymerase chain reaction (RT-qPCR) assays were carried out under the condition of osteogenic differentiation medium [46,47]. The result of the CCK-8 assay was shown in Fig. 4A. After 1 d, the viability of cells on MP-ALD50 substrates was lower than that of the MP-ALD10 group (p < 0.05), but there was no significant difference with other groups. With the increase of culture time (4 and 7 d), cells on MP-ALD50 substrates still showed the worst viability. It might be attributed to the release of excess Zn2+ from MP-ALD50 sample (Fig. 3B), which was consistent with the previous study showing that high doses of Zn2+ could significantly induce significant cytotoxicity to osteoblasts via oxidative stress [43,45]. Moreover, comparing with Ti, MP-ALP10 samples also significantly (p < 0.05 or 0.01) increased the cell viability of MC3T3-E1 cells after 4 and 7 d. 7
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collagen I, and OCN) [55]. Taken together, the results of CCK-8, ALP activity, mineralization, and RT-qPCR indicate that both MP-ALD10 and MP-ALD30 could greatly promote the osteogenic differentiation of MC3T3-E1 cells, while only MP-ALD10 was beneficial to cell proliferation.
group only within 7 d. These results suggest that the ZnO coating prepared by 10 ALD cycles has no significant effect on the drug loading and controlled release properties of mesoporous materials.
3.4. Antibacterial ability
Zinc oxide coatings were prepared inside the mesoporous (MP) structures by atomic layer deposition (ALD) techniques in this study to improve the bioactivity of MP samples without affecting their drug loading and release potential. All ZnO modified samples showed excellent antibacterial abilities (bacteriostatic rate: MP-ALD10 < MPALD30 < MP-ALD50) against both S. aureus and E. coli when comparing with Ti and MP groups. Results of osteoblast assays further displayed that both MP-ALD10 and MP-ALD30 increased cell differentiation, but only MP-ALD10 favored the proliferation of MC3T3-E1 cells. Also, compared with MP samples, MP-ALD10 did not show a significant effect on drug loading and release properties. Thus, MPALD10 might be more suitable for the development of titanium-based “drug-device” combination implants in the future.
4. Conclusion
When a bacterial infection occurs after implant surgery, bacteria will adhere to the implant surface and rapidly form a dense biofilm, which will prevent the drug from penetrating to the infected site [23,56]. Therefore, traditional clinical antimicrobial therapy, such as oral administration or intravenous injection, is difficult to control the infection around the implant [56,57]. In recent years, researches have been working to develop functional implants that can achieve superior antibacterial/osteogenic properties through in situ sustained-release antibiotics [3–6]. Metal ions (e.g., Zn2+, Ag+, and Cu2+) are often used in the preparation of antibacterial implants because they have good spectrum antibacterial properties and are not easy to induce drug resistance [23,58]. Previous studies had proven that Zn2+ could cause bacteria death by inducing excessive production of reactive oxygen species and further destroying bacterial membranes [34, 35, 59]. To evaluate the antibacterial ability of different substrates, bacterial viability and live/dead staining assays were carried out. From Fig. 6A, it was found that there was no significant difference in the viability of bacteria on Ti and MP substrates. After ALD treatment, three ZnOmodified samples showed superior antibacterial properties against both S. aureus and E. coli when compared with Ti and MP groups. It was also found that with the increase of ALD cycles, the antibacterial properties of corresponding samples gradually increased (MP-ALD10 < MPALD30 < MP-ALD50), which was also further confirmed by the results of live/dead staining (Fig. 6B and C). The ratio of dead bacteria to total bacteria in MP-ALD10, MP-ALD30 and MP-ALD50 groups were 63.3 ± 6.5 (S. aureus)/68.4 ± 5.0 (E. coli), 83.2 ± 4.3 (S. aureus)/ 87.3 ± 3.7 (E. coli), and 96.1 ± 3.5 (S. aureus)/97.1 ± 3.0 (E. coli), respectively. The above results suggest that the three ZnO coatings prepared by 10, 30, and 50 ALD cycles had excellent antibacterial abilities against both S. aureus and E. coli.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (81870810, 81701016, and 31700827), Zhejiang Provincial Science and Technology Project for Public Welfare (2015C33139), and Zhejiang Xinmiao Talents Program (2019R413066). References [1] T. Takizawa, N. Nakayama, H. Haniu, K. Aoki, M. Okamoto, H. Nomura, M. Tanaka, A. Sobajima, K. Yoshida, T. Kamanaka, K. Ajima, A. Oishi, C. Kuroda, H. Ishida, S. Okano, S. Kobayashi, H. Kato, N. Saito, Titanium fiber plates for bone tissue repair, Adv. Mater. 30 (2018) 1703608. [2] W.E. Yang, H.H. Huang, Multiform TiO2 nano-network enhances biological response to titanium surface for dental implant applications, Appl. Surf. Sci. 471 (2019) 1041–1052. [3] P. Wu, D.W. Grainger, Drug/device combinations for local drug therapies and infection prophylaxis, Biomaterials 27 (2006) 2450–2467. [4] C. Alvarez-Lorenzo, A. Concheiro, Smart drug release from medical devices, J. Pharmacol. Exp. Ther. 370 (2019) 119. [5] D. Losic, M.S. Aw, A. Santos, K. Gulati, M. Bariana, Titania nanotube arrays for local drug delivery: recent advances and perspectives, Expert Opin. Drug Deliv. 12 (2015) 103–127. [6] T.T. Wang, X.M. Liu, Y.Z. Zhu, Z.D. Cui, X.J. Yang, H.B. Pan, K.W.K. Yeung, S.L. Wu, Metal ion coordination polymer-capped pH-triggered drug release system on titania nanotubes for enhancing self-antibacterial capability of Ti implants, ACS Biomater. Sci. Eng. 3 (2017) 816–825. [7] J.R. Porter, T.T. Ruckh, K.C. Popat, Bone tissue engineering: a review in bone biomimetics and drug delivery strategies, Biotechnol. Prog. 25 (2009) 1539–1560. [8] S. Maher, A. Mazinani, M.R. Barati, D. Losic, Engineered titanium implants for localized drug delivery: recent advances and perspectives of Titania nanotubes arrays, Expert Opin. Drug Deliv. 15 (2018) 1021–1037. [9] Y. Cheng, H. Yang, Y. Yang, J. Huang, K. Wu, Z. Chen, X. Wang, C. Lin, Y. Lai, Progress in TiO2 nanotube coatings for biomedical applications: a review, J. Mater. Chem. B 6 (2018) 1862–1886. [10] A. Hamlekhan, S. Sinha-Ray, C. Takoudis, M.T. Mathew, C. Sukotjo, A.L. Yarin, T. Shokuhfar, Fabrication of drug eluting implants: study of drug release mechanism from titanium dioxide nanotubes, J. Phys. D Appl. Phys. 48 (2015) 275401. [11] S. Oh, K.S. Brammer, Y.S. Li, D. Teng, A.J. Engler, S. Chien, S. Jin, Stem cell fate dictated solely by altered nanotube dimension, Proc. Natl. Acad. Sci. U.S.A. 106 (2009) 2130e2135. [12] Y. Yu, X. Shen, Z. Luo, Y. Hu, M. Li, P. Ma, Q. Ran, L. Dai, Y. He, K. Cai, Osteogenesis potential of different titania nanotubes in oxidative stress microenvironment, Biomaterials 167 (2018) 44–57. [13] N. Wang, H. Li, W. Lü, J. Li, J. Wang, Z. Zhang, Y. Liu, Effects of TiO2 nanotubes with different diameters on gene expression and osseointegration of implants in minipigs, Biomaterials 32 (2011) 6900–6911.
3.5. Drug loading and release Since MP-ALD50 had significant cytotoxicity on the proliferation and differentiation of osteoblasts, it was not suitable for the preparation of functional titanium materials as a drug carrier. Thus, only the drug loading and release properties of MP, MP-ALD10, and MP-ALD30 samples were characterized in this study. Rhodamine was used as the model drug. Firstly, the cross-sectional morphology of MP, MP-ALD10, and MP-ALD30 was imaged and shown in Fig. 7A. Similar to our previous study, there were some clear mesoporous structures in the crosssection of MP sample [23]. The pore size of mesoporous structures was significantly reduced in the MP-ALD10 and almost completely disappeared in the MP-ALD30, which was consistent with the aforementioned top view results (Fig. 1B). Secondly, rhodamine-loaded substrates were observed by CLSM and showed in Fig. 7B. From the top view images, weak fluorescence was observed in MP-ALD30 when comparing with MP and MP-ALD10 groups. The 3D fluorescence images further confirmed that the MP and MP-ALD10 groups had stronger fluorescence signals and greater penetration depth (MP/MP-ALD10: ~18 μm; MP/MP-ALD30: ~8 μm), which indicated that the MP and MPALD10 could load more rhodamine than the MP-ALD30 sample. Thirdly, from the released profiles (Fig. 7C), it was found that there was no significant difference in the release amount and release rate of rhodamine in the MP and MP-ALD10 groups at different durations. The release period of rhodamine in these two groups was more than 28 d, and the total amount at 28 d was approximately 50 μg/mL. However, the loaded rhodamine (~10 μg/mL) wholly released in the MP-ALD30 8
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