Bidirectional regulation of zinc embedded titania nanorods: antibiosis and osteoblastic cell growth

Abstract

New generation bone implants with favourable biocompatibility and long-term antibacterial activity have attracted a great deal of attention due to their bifunctional regulation on osteogenesis and antibiosis, demonstrating great potential for their application in biomedical science. In this study, Zn-incorporated TiO₂ nanoarrays are prepared by a hydrothermal approach and the Zn content in the nanorod-structure scaffolds can be controlled by altering the concentration of the precursor solution, thereby tailoring the Zn incorporated scaffolds to meet the requirements in bifunctional clinical applications. Cytocompatibility of the Zn-incorporated TiO₂ nanoarrays was evaluated by MTT, LDH and ALP assays, and all the results are found to be dependent on both time and Zn content. Among TiO₂–Zn nanoarrays, TiO₂–Zn0.2 has the most remarkably stimulative effects on MC3T3-E1 cells compared to untreated controls. Meanwhile, the antibacterial tests confirm that the hybrid nanoarrays can exert inhibitory effects on Escherichia coli (E. coli, Gram-negative) and Staphylococcus aureus (S. aureus, Gram-positive) to various degrees based on time and Zn content. The bifunctional implant materials that allow sustainable Zn release with both superior biocompatibility and long-term antibacterial properties will hold significant promise for scalable productions and open the horizon for further applications as biomedical devices.

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1 Introduction

Treatments on disorders of the musculoskeletal system, such as skeletal deformities, joint disease and orthopedic trauma, frequently involve the use of orthopedic implants. Nowadays, titanium-based implants have attracted increasing interest in clinical research owing to their outstanding properties including low elastic modulus, excellent corrosion resistance, superior biocompatibility and appropriate cell–material interactions.^1,2 Cell contact and adhesion are regarded as the initial steps in cell–material interactions, which are inherently sensitive to local roughness, microscale, and nanoscale patterns of chemistry and topography.^3,4 2D or 3D biomaterials are under rapid development to serve as powerful artificial microenvironments to instruct cell fate in a precise fashion both in vitro and in vivo.⁵ A noticeable early response of cell to nanotopography is to increase its complement of filopodia and microspikes, which may elevate the perception level of cells.⁶ In general, bone implant materials with nanoscale features, such as nanotubes, nanorods, nanoposts, nanocracks and grooves are able to interact with local tissues/cells by triggering cell-specific responses such as cell attachment and proliferation.⁷ In general, bone implant materials with nanoscale features, such as nanotubes, nanorods, nanoposts, nanocracks and grooves are able to interact with local tissues/cells by triggering cell-specific responses such as cell attachment and proliferation.^7,8 Among cell responses to different nanopatterns, overwhelming evidence indicated that nanorods, when contact with bone cells, could enhance osteogenesis because they can mimic better the hybzrid architecture of natural bone extracellular matrix.⁹ However, despite the remarkably potential clinical applications of nano-patterned titanium-based implant, the use of them is always hindered by infections during and after operations. Bacterial adhesion and colonization occur frequently around implant surfaces due to accumulation of serum proteins,^10–12 which may translate into a bacterial infection and lead to severe complications and long-term implant failure.¹³ If so, secondary surgical procedures, delay of rehabilitation and additional risk may be brought to the patient. Therefore, orthopedic biomaterials that possess dual function in antibiosis and biocompatibility are in urgent need to keep implants far from this common problem.

ZnO, an inorganic metal oxide, is being increasingly used for antimicrobial application over the past decade.¹⁴ Main advantages of ZnO, when compared with organic antimicrobial agents, include its stability, robustness, and long shelf life.¹⁵ Several studies proposed that aqueous suspensions of ZnO was able to generate reactive oxygen species (ROS) such as hydroxyl radical, hydrogen peroxide, singlet oxygen and superoxide, resulting in oxidative damage to bacterial cells.¹⁶ ZnO nanoparticles were reported to cause disorganization of both cell wall and cell membrane of bacteria,¹⁷ making it exceptional to inhibit the growth of a wide range of pathogenic bacteria. Likewise, it was revealed that ZnO could destroy the membrane of E. coli by increasing membrane permeability, leading to accumulation of ZnO in the bacterial membrane.¹⁸ Our previous work also provided direct evidence that ZnO-incorporated ZrO₂ nanoarrays exhibited excellent antimicrobial activity against Staphilococcus auerus (S. aureus) and Escherichia coli (E. coli).¹⁹

In addition to its established roles in antibacterial activity, the pharmaceutical effect of Zn on bone metabolism have also been studied widely.²⁰ The research concerning cellular osteogenic activities demonstrated that rBMSCs cultured on the Zn-implanted coatings displayed higher ALP activity and up-regulated expression of osteogenic-related genes (OCN, Col-I, ALP, Runx2) compared to the controls.²¹ In addition, Zn hold enormous potential for accelerating initial cell adhesion, spreading, proliferation, collagen secretion and extracellular matrix mineralization.²² What's more, Zn, an essential trace element, was historically famous for its stimulatory effects on cellular signaling pathways, immune competence and reproductive function at low concentration.^23,24

The evidences of the beneficial effects of Zn in antibiosis and biocompatibility as well as the positive stimulative effect of nanorod implants in bone growth motivated us to incorporate Zn in TiO₂ nanorods for skeletal tissue applications. Both long-term antibacterial performance and the biocompatibility potential of Zn-contained TiO₂ scaffolds were assessed in vitro and the bifunctions have highlighted our present research.

2 Experimental

2.1 Specimens fabrication and modification

Firstly, commercial pure titanium foils with the dimension of 3 cm × 3 cm were ultrasonically cleaned in acetone, ethanol and deionized water for 15 minutes sequentially followed by polishing in a solution containing H₂O, HF, and HNO₃ with a volume ratio of 5 : 1 : 4 for 5 min to remove the surface native oxide. Then TiO₂ arrays were prepared through hydrothermal approach. Specifically, 100 mL deionized water was mixed with 2 mL titanium trichloride (TiCl₃), then sodium chloride was added into mixtures solution to saturation with vigorous stirring at 30 °C. The obtained solution was further transferred into Teflon-lined stainless steel autoclave with a piece of pretreated Ti plate immersed into the reaction solution. The autoclave was sealed and maintained at 160 °C for 3 h, and then cooled down to room temperature. The sample was collected and rinsed with distilled water several times, followed by annealing at 450 °C for 3 h. The as-obtainded TiO₂ arrays were hydrothermally treated in 40 mL of zinc acetate (0.025 M, 0.05 M, 0.1 M, 0.2 M) at 190 °C for 3 h to produce the Zn incorporated TiO₂ arrays (TiO₂–Zn) samples designated as TiO₂–Zn0.025, TiO₂–Zn0.05, TiO₂–Zn0.1 and TiO₂–Zn0.2. All the samples were annealed at 500 °C for 3 h in air to promote sample crystallization.

2.2 Surface characterization

The surface morphology of obtained samples was characterized by field-emission scanning electron microscopy (JSM-6700F, Japan) and transmission electron microscopy TEM (JEM-2010FEF, 200 kV). The phase compositions were analyzed by X-ray diffraction (XRD, X'Pert PRO MRD, PANalytical, Netherlands) and Raman spectroscopy (LabRAMHR evolution, 532 nm). Quantitative elemental analysis of Ti, O, and Zn were carried on energy-dispersive X-ray spectroscopy (EDS) that was equipped on the SEM of QUANT200.

2.3 Surface wettability

The static contact angles of the flat pristine Ti, TiO₂ nanorods and TiO₂–Zn were measured on a contact angle meter using a Model 200 video-based optical system (Future Scientific Ltd. Co., Taiwan, China). Drops generated with a micrometric syringe were deposited onto the sample surface and photographed immediately by a camera, and then analyzed with software supplied by the manufacturer. Each contact angle reported here was the average value of three independent measurements.

2.4 Zn ion release

The release profile Zn ion was determined by soaking the Zn incorporated samples with 1 cm × 1 cm diameter in 5 mL PBS at 37 °C. At pre-determined time intervals, 2 mL of soaking solution was collected for determining the amount of Zn ion released and the remaining medium was removed and replaced with another 5 mL fresh PBS. This process was repeated for 21 days and the amounts of Zn released from different samples were analyzed by inductively-coupled plasma atomic emission spectrometry (ICP-AES, IRIS Advantage ER/S).

2.5 Cell culture

Newborn mouse calvaria-derived MC3T3-E1 cells obtained from a preservation center for typical culture in Wuhan university (Wuhan, P. R. China) were cultured in alpha-MEM medium (HyClone) supplemented with 10% (vol/vol) fetal bovine serum (FBS; Gibco), 1% penicillin/streptomycin. Cells were incubated in the presence of 5% (vol/vol) CO₂ at 37 °C in a humidified atmosphere. The culture media were changed every 3 days. Only 2–4 passage cells were used in the following experiments.

2.6 Cell morphology observation

The cells were seeded onto 96-well plates at a density of 8 × 10³ cells per well and incubated for 1, 2 and 3 days. Afterward the cells on the samples were washed with PBS and fixed with fresh 3% glutaraldehyde at 4 °C for 1 h, then dehydrated in the graded ethanol series, freeze-dried, coated with gold, and examined using a scanning electron microscopy (SEM) to evaluate the attachment behaviors of MC3T3-E1 cells on different substrates.

2.7 Cell viability

MTT assay was used to evaluate proliferation and viability of MC3T3-E1 cells. MC3T3-E1 cells were plated in 96-well at an initial seeding density of 1 × 10⁴ cells per well, respectively. After 24 h, 48 h and 72 h of culture, cells were rinsed by PBS and then incubated with fresh culture medium with 100 μL MTT at 37 °C for another 4 h. Then the MTT contained medium was removed before adding dimethyl sulfoxide (150 mL per well) into the wells to dissolve the formazane, a kind of reaction product between MTT and cells. The finally solution was measured by microplate reader (DNM-9602) at wavelength of 492 nm. In the cell viability assay, five wells were established under each experimental condition.

2.8 Lactate dehydrogenase activity assay

The activity of lactate dehydrogenase (LDH) in the culture media released by the cells was measured to investigate the cytotoxicity. In this experiment, MC3T3-E1 cells were seeded onto different samples at a density of 1 × 10⁴ cells per well. After 24 h, 48 h and 72 h of incubation, respectively, the culture media were collected and centrifuged and the activity of LDH in the supernatant was measured according to the manufacturer's instructions.

2.9 ALP activity assay

MC3T3-E1 cells were seeded onto each specimen in a 24-well plate at a density of 1 × 10⁴ cells per well. After 14 days of incubation, ALP activity was measured to assess the bone forming ability of cells. In brief, cells were washed with PBS and lysed in 1 vol% Triton X-100 and then broken by a sonic oscillator for 30 min. The cells lysates were centrifuged at 13 500 rpm at 4 °C for 4 min to remove cell debris and then the supernatants were incubated with p-nitrophenyl phosphate solution at 37 °C for 30 min to produce p-nitrophenol, a type of yellow product, which was measured using microplate reader at a wavelength of 405 nm. The Bicinchoninic acid (BCA) Protein Assay kit was ultilized to determine the intracellular total protein content, and according to which the ALP activity was normalized. Measurements were made in triplicate.

2.10 Bactericidal activity evaluation

In biological terminology, bacteria are classified into two types: Gram-positive and Gram-negative. Since clinically susceptible bacteria S. aureus and E. coli are representatives of Gram-positive and Gram-negative bacteria, respectively, they were chosen as the target bacteria in our study. Both E. coli and S. aureus were cultured in Lysogeny broth (LB) medium overnight at 37 °C with shaking at 200 rpm. Ti, TiO₂ and TNR-Zn were placed at the bottom of the wells of 96-well plates and incubated with 100 μL bacterial suspension. Control experiments were performed under identical conditions in the absence of any sample. After incubation in a humidified incubator at 37 °C for 24 h, the culture medium was collected and the optical density was measured using ultra-violet visible spectroscopy (PGENERAL) at 600 nm. Bacteria adhered on each specimen were fixed with 3% glutaraldehyde at 4 °C for 1 h, then dehydrated in graded ethanol series, and examined by scanning electron microscopy in accordance with the procedures described in Section 2.6. The inhibition zone test was employed to evaluate the long-term antibacterial effects. 100 μL bacterial suspension with concentration of 1 × 10⁷ CFU mL⁻¹ were spread onto agar plates evenly on LB agar plates. Then, the sample disks with different Zn content were gently placed at the LB agar plates orderly and incubated overnight at 37 °C for 24 h, 48 h and 72 h, respectively. A series of representative pictures were taken with an optical camera (Panasonic, DMC-FZ50) and the antibacterial activity was measured by calculating the diameter of the zone of inhibition around the disks.

2.11 Statistical analysis

The results were representative of at least three independent experiments and statistical analysis was carried out using the statistical software Origin 7.5. All data are presented as mean ± standard error of the mean. Statistically significant differences between groups were measured using a t-test. p-value of 0.05 or less indicates a significant difference between samples (*p < 0.05 and **p < 0.01).

3 Results and discussion

3.1 Characterization of TiO₂–Zn nanorod-patterned coatings

Schematic illustration of the two-step hydrothermal synthesis process was shown in Fig. 1. The TiO₂ nanorods array can be obtained by hydrothermal method in titanium trichloride precursor solution. The following hydrothermal treatment in zinc acetate made Zn embedded in the as-prepared TiO₂ nanorods uniformly.

Fig. 1 Illustration of the two-step hydrothermal synthesis path of TiO₂–Zn nanorods array on Ti substrate.

The surface morphology of the pure Ti, TiO₂ nanorods and Zn incorporated TiO₂ samples designated as TiO₂–Zn0.025, TiO₂–Zn0.05, TiO₂–Zn0.1 and TiO₂–Zn0.2, respectively, were demonstrated in Fig. 2. The pure Ti surface showed a flat topography whereas TiO₂ nanorods array scaffold were highly ordered and evenly distributed with dimension of 80–180 nm, mimicking the hybrid architecture of natural bone ECM.²⁵ Obviously, the surface of nanoarrays seemed rougher than that of the pure Ti, which was beneficial to stimulate cell adhesion and encourage cell differentiation.²⁶ Subsequent hydrothermal treatment in zinc acetate almost kept the main surface topography, structural characteristic and homogeneous distribution of nanorods, providing a distinct advantage in the observation of both responses of MC3T3-E1 cells and bacteria to substrates with varying Zn content.

Fig. 2 SEM images of different substrates and the insets are high magnification image.

A typical transmission electron microscopy (TEM) analysis on the hybrid nanorods consisting of quasi-continuous nanoparticles was displayed in Fig. 3a. Apparently, each ZnO subunit particle was embedded well into the TiO₂ nanorods with a dimension of several nanometers. High-resolution TEM images in Fig. 3b revealed different interplanar spacings of 0.189 nm and 0.148 nm, corresponding to (200) and (213) lattice planes of the anatase TiO₂, and the interplanar spacings of 0.162 nm is (110) lattice planes of ZnO. The highly clear crystal lattice as well as the corresponding spot pattern of the selected area electron diffraction (SAED) pattern demonstrated the high-quality polycrystalline nature of TiO₂ and ZnO, which was further supported by the XRD and Raman in Fig. 4A and B. The crystal phases of the as-prepared samples were recorded by XRD. As shown clearly in Fig. 4A, only characteristic peaks of Ti and TiO₂ were obtained in the XRD pattern of TiO₂. After the hydrothermal treatment in zinc acetate for 3 h, two diffraction peaks at 31 and 47° emerged in the pattern of TiO₂–Zn, which could be well indexed to the Wurtzite ZnO. Additional information on the structure of the as-grown TiO₂ nanorods on Ti substrate with different Zn content was obtained by Raman spectroscopy. Fig. 4B illustrated a typical Raman scattering spectra of the TiO₂ and a series of TiO₂–Zn nanorods annealed at 500 °C. As seen in the spectra, the strongest peak centered at 145 cm⁻¹ and four other peaks at 200, 396, 519, 637 cm⁻¹ matched with corresponding E_g, E_g, B_1g, A_1g and E_g phonon modes of the anatase phase, respectively.²⁷ As for the peak located at 438 cm⁻¹, it was the intrinsic characteristic of the E₂ (H) mode of Wurtzite ZnO,²⁸ the intensity of which became stronger with the increase of Zn content. Of note, this result was also in good agreement with that of the XRD measurement.

Fig. 3 (a) TEM analysis of TiO₂–Zn0.2; (b) HR-TEM lattice patterns of the encircled areas in (a); (c) the selected area electron diffraction (SAED) pattern of (a).

Fig. 4 (A) XRD patterns and (B) Raman scattering spectra of the TiO₂ and a series of TiO₂–Zn nanorods prepared by the hydrothermal method with the same condition.

EDS spectra was conducted to determine the surface elemental compositions of the as-prepared materials and results analysis of TiO₂–Zn0.025 TiO₂–Zn0.05, TiO₂–Zn0.1 and TiO₂–Zn0.2 were displayed in Fig. 5. Apparently, Ti, O and Zn were only elements detected in the samples, confirming the final products were free of impurity. The successful incorporation of Zn in TiO₂ nanorods was ambiguously certified by obvious Zn signals emerging in the EDS spectra of TiO₂–Zn. The mass percentage of TiO₂–Zn0.025, TiO₂–Zn0.05, TiO₂–Zn0.1 and TiO₂–Zn0.2 was 1.81, 3.4, 5.28 and 9.58 wt%, respectively, all raised as the concentration of zinc acetate in the precursor solution increased.

Fig. 5 EDS spectra and elemental compositions detected by EDS maps: (A) TiO₂–Zn0.025, (B) TiO₂–Zn0.05, (C) TiO₂–Zn0.1, (D) TiO₂–Zn0.2.

Recent studies have suggested that surface hydrophilicity may impact bio-functions, such as cell and bacteria adhesion, morphology and function,^21,29,30 making it an important parameter to optimize substrates for cell culture in vitro. In our present study, the surface hydrophilicity was evaluated by the static sessile drop method using distilled water. As shown obviously in Fig. 6, nanorod arrays were more hydrophilic than pristine Ti, but there was no significantly difference between TiO₂–Zn and TiO₂ due to their analogous surface morphology. Moreover, contact angles of all materials with array-structure remained about 70°.

Fig. 6 (A) Contact angles of the series of different substrates *p < 0.05. (B) Typical water droplet images on different samples.

3.2 Adhesion and morphology of MC3T3-E1 cells

The contact of cell and material is crucial for the evaluation on the cytocompatibility of biomaterial and development of appropriate material for medical applications in vivo. In this study, the morphological features of MC3T3-E1 cells on the flat Ti, TiO₂, TiO₂–Zn0.025, TiO₂–Zn0.05, TiO₂–Zn0.1 and TiO₂–Zn0.2 substrates after 1, 2 and 3 days of culture, respectively, were characterized by SEM. As displayed in Fig. 7, shapes of the MC3T3-E1 cells cultured on flat Ti and various TiO₂–Zn nanorods were remarkably different. On flat Ti, the MC3T3-E1 cells were fusiform without obvious filopodia propagation, whereas spontaneous adhesion of cells with extensive cellular filopodia and lamellipodia was observed on all nanorods substrates, especially on the TiO₂–Zn0.2. In addition, at any point time, the number of MC3T3-E1 cells raised significantly with the increase of Zn content, forming a connected layer with the characteristic polygonal shape. It could be speculated that the synergy of nanorod-pattern and Zn component allowed for enhanced cell proliferation and increased osteoblast activation, both meant a lot for reconstructive surgery.

Fig. 7 SEM micrographs of MC3T3-E1 cells (which appear dark) on flat Ti, TiO₂, TiO₂–Zn0.025, TiO₂–Zn0.05, TiO₂–Zn0.1 and TiO₂–Zn0.2 surfaces after 1, 2 and 3 days of culture incubation.

3.3 Proliferation and viability of MC3T3-E1 cells

The number of adherent MC3T3-E1 cells on different substrates as a function of incubation time and content of Zn was plotted in Fig. 8A, in which the adherent cells increased significantly in a manner dependent on both time and Zn content. Noticeably, TiO₂–Zn0.2 accomodated the largest number of cells among all experimental substrates. Fig. 8B depicted the optical density (OD) of reaction product from the MTT working solution with MC3T3-E1 cells cultured on flat Ti, TiO₂ nanorods and a series of TiO₂–Zn nanoarrays after 24, 48, and 72 h of incubation. Results of MTT assay could be more intuitive to reflect the density of viable cells on tested surfaces. In accord with Fig. 8A, cell viability of MC3T3-E1 cells was enhanced gradually on different substrates with the increase of Zn content as well as the culturing time. Noticeably, cells on the TiO₂–Zn0.2 substrate displayed the highest cell viability level, suggesting favorable effect of Zn on MC3T3-E1 cell growth. In addition, after 72 h, cell viability level was also found to be significantly higher on TiO₂ nanorods array than that on the pure Ti. Therefore, based on above results, it could be interpreted that promotion effects in cell proliferation and viability of TiO₂–Zn nanorods could be attributed to both the nanotopography and the increase of Zn content. Numerous studies providing direct evidence that the nanotopography of nanorod arrays with larger surface roughness and adequate surface hydrophilicity could facilitate cell growth in many different ways, such as cell adhesion, cell morphology and proliferation,^8,31 have lent support to this conclusion. Tight attachment and rapid proliferation of bistiocyte around the nanorods surface, further, offer prerequisites for reconstructive surgery.³² What's more, in a recent research, Hu et al. demonstrated that Zn-incorporated TiO₂ coatings could regulate the biological functions of MSCs including proliferation and differentiation, proving that Zn played an positive role on cell activity and cytocompatibility.³³

Fig. 8 (A) Adherent MC3T3-E1 number on different substrates measured by counting cells after culturing for 24 h, 48 h and 72 h; (B) cell viability assessed by MTT assay (C) LDH toxicity assessed by LDH release for MC3T3-E1 cells on different substrates (D) alkaline phosphatase (ALP) activity of MC3T3-E1 cells after 14d of incubation. Data are presented as mean ± SD (n = 5 per group, *p < 0.05, **p < 0.01 compared to Ti and ^#p < 0.05 and ^##p < 0.01 compared to TiO₂).

3.4 Cytotoxicity

An indispensable requirement for biomaterials is bio-safety without inhibiting the growth and function of healthy cells. The cytosolic enzyme lactate dehydrogenase (LDH) released from cells with impaired cell membranes, is regarded as an important biomarker to assess cytotoxicity.³⁴ Compared with the pure Ti and TiO₂, there was no statistically significant difference in LDH activity for all Zn-incorporated TiO₂ nanorod arrays at any time (Fig. 8C), providing a hint that Zn content in our research was within the range of safe concentration for cell growth so that all tested substrates were nontoxic to MC3T3-E1 cells. The results was consistent with previous studies that Zn has no adverse effect on healthy cells at low dosage.^35,36

3.5 Alkaline phosphatase (ALP) assay

It is widely accepted that accelerated differentiation of osteoblast on the surface of implant materials is of great significance to regenerate the injured tissues. ALP, one of the early markers of cell differentiation, was utilized to assess the ability of bone regeneration. After MC3T3-E1 cells were incubated on different substrates for 14 days, the ALP activity normalized according to total amount of protein content were depicted in Fig. 8D. Noteworthily, the ALP activities of cells grown on all substrates showed a time and Zn content-dependent pattern. In detail, cells grown on the Zn-embedded TiO₂ nanorods proved to have higher ALP activity than those grown on pure Ti and TiO₂, and the ALP activity was proportional to the Zn content, leading to the best cell growth on the TiO₂–Zn0.2 nanorods. The ever-increasing ALP values in Fig. 8D indicated that the Zn-incorporated TiO₂ nanorods as bone implant material had overwhelming advantages over pure Ti and TiO₂ nanorods owing to the better stimulatory effects of Zn on the early differentiation and osteogenesis. Similar results were obtained from previous researches^37–39 that Zn-modified tricalcium phosphate exhibited excellent biocompatibility, enhanced cell attachment, differentiation and up-regulated ALP expression for the human bone derived cells. In the latest research, Xinkun Shen et al.⁴⁰ demonstrated that Zn-incorporated coating on microrough titanium could regulate the biological function of osteoblasts, and also provide evidence for favorable effects of Zn on osteogenesis.

3.6 Antimicrobial activity

Examination on the antibacterial effect of as-prepared substrates was conducted on two bacterial species: S. aureus and E. coli. Structural changes of two bacteria incubated with Ti, TiO₂, TiO₂–Zn0.025, TiO₂–Zn0.05, TiO₂–Zn0.1, TiO₂–Zn0.2 substrates for 18 h were observed by SEM. As shown in Fig. 9A, the cell surface of S. aureus that cultured on pure Ti was integrated, exhibiting typical binary fission whereas those on TiO₂ displayed a certain degree of abnormalities with deformation morphology. Moreover, the bacteria treated with different TiO₂–Zn composites were all severely impaired with obviously cytoplasm emanating from the cell wall. Similarly, E. coli cells (Fig. 9B) had rodlike morphology and rather smooth surface on pure Ti but irregular and undulating appearance on TiO₂ substrate. In contrast, on TiO₂–Zn, the membrane of E. coli suffered grievous distortion and twist, which deteriorated gradually with the increase of Zn content. Previous research had provided direct evidences that the active oxygen radicals, an important member of ROS, produced by ZnO could modulate biological processes in the bacterial cells and damage cell membranes, DNA, as well as cellular proteins and even lead to cell death.⁴¹ Likewise, TiO₂, one of the important photocatalytic materials,⁴² had also been documented to generate ROS which could oxidize cell membrane, resulting in the death of the microorganisms.⁴³ Thus, a conclusion could be reached that it was the synergy of TiO₂ and ZnO that disrupted the integrity of membrane and caused the death of both E. coli and S. aureus.

Fig. 9 SEM images of (A) S. aureus and (B) E. coli after incubation with Ti, TiO₂, TiO₂–Zn0.025, TiO₂–Zn0.05, TiO₂–Zn0.1, TiO₂–Zn0.2 for 18 h, respectively.

To investigate the anti-bacterial activity of Ti, TiO₂ and TiO₂–Zn, various substrates were exposed to bacterial suspensions for 12 h. As seen in Fig. 10A and B, the OD₆₀₀ values of S. aureus and E. coli suspension, directly proportional to the number of viable bacteria, all reduced gradually with the increase of Zn content. For S. aureus, a trace of Zn (TiO₂–Zn0.025) showed remarkable antiblastic effect (Fig. 10A) whereas for E. coli, the OD₆₀₀ values dramatically decreased at the high Zn dosage (TiO₂–Zn0.2), compared with that of the control group (Fig. 10B). Besides, it was the OD₆₀₀ value of S. aureus but not E. coli suspension grown on TiO₂ nanorods that was significantly decreased than those of Ti and control group, implying that the TiO₂ nanorods exhibited a stronger antimicrobial effect against S. aureus than E. coli. Different antibacterial activity against two bacteria can be attributed to their different structures of cell wall and cell membrane.⁴⁴ S. aureus, a typical Gram-positive bacterium with one membrane and thick wall, is composed of multilayers of peptidoglycan.⁴⁵ Distinctively, E. coli, a representative Gram-negative bacterium, has a more complex cell wall and two cell membranes with a layer of peptidoglycan between them.^46,47 Therefore, S. aureus is more sensitive to the exogenous inhibitors than E. coli.

Fig. 10 Effect of different substrates on the antibacterial properties against (A) S. aureus and (B) E. coli. (n = 5 per group, *p < 0.05, **p < 0.01 compared to Ti and ^#p < 0.05 and ^##p < 0.01 compared to TiO₂).

In addition, the inhibition zone test was further carried out to investigate the long-term antibacterial properties of Ti, TiO₂ and TiO₂–Zn against S. aureus and E. coli after 24 h, 48 h and 72 h of culture. As shown in Fig. 11, diameters of the inhibition zone around TiO₂–Zn0.05, TiO₂–Zn0.1 and TiO₂–Zn0.2, whether for S. aureus or E. coli, extended gradually with the increase of both the Zn content and the culture time, illustrating sustainable efficacious antibacterial activity. However, there was no inhibition zone around TiO₂ nanorods and TiO₂–Zn0.025 at any time for both bacteria. One possible reason for this phenomenon may be that the rate of sterilization caused by pure TiO₂ and TiO₂–Zn0.025 was slower than that of bacteria multiplication.

Fig. 11 Images showing the inhibition zone of S. aureus and E. coli cultured on Luria-Bertani medium after 24 h, 48 h, 72 h at 37 °C, (a) Ti, (b) TiO₂, (c) TiO₂–Zn0.025, (d) TiO₂–Zn0.05, (e) TiO₂–Zn0.1, (f) TiO₂–Zn0.2.

3.7 Zn ion release

Zn, an essential trace element for bone formation, has been reported to affect bone metabolism by stimulating osteoblast proliferation, calcium deposition and osteoblast marker gene expressions.⁴⁸ Previous studies have also documented that Zn-incorporated coatings had eminent antibacterial effect on bacteria without introducing undesired side-effect.⁴⁹ Notwithstanding, it has been reported that Zn could inhibit healthy osteoblast function via oxidative stress when its concentration in the cell culture medium exceed 3 μg mL⁻¹ (3 ppm).⁵⁰ In our experiment, the non-accumulated Zn ion released from the TiO₂–Zn0.025, TiO₂–Zn0.05, TiO₂–Zn0.1 and TiO₂–Zn0.2 were measured by ICP-AES (Fig. 12). During 21 days, as expected, all substrates modified by Zn remained durative Zn ion release profiles, which were crucial for long-term pharmacological effect and antibacterial activity. 0.36 ppm, the highest Zn ion concentration released from TiO₂–Zn in Fig. 12, fell in the range of safe concentration (<3 ppm), which was consistent with the results of LDH. In brief, the Zn content of all Zn-modified TiO₂ nanorods prepared through simple two-step hydrothermal method was controlled by varying the concentration of precursor solution, allowing a sustainable Zn release and making it possible for scaffolds to be tailored towards dual function of osteogenesis and antibiosis.

Fig. 12 Non-cumulative Zn ion release from 0.1 M PBS solution with immersion of TiO₂–Zn0.025, TiO₂–Zn0.05, TiO₂–Zn0.1 and TiO₂–Zn0.2 for 21 days.

4 Conclusion

In this study, TiO₂–Zn with a nanorod array structure had been successfully fabricated by a simple hydrothermal method. A series of analysis of the biocompatibility and osteogenesis confirmed that TiO₂–Zn nanorods could boost proliferation and differentiation of MC3T3-E1 cells in a manner dependent on both Zn dose and time, as testified by increased MTT values and ALP activity. Meanwhile, these as-prepared materials also showed long-term antibacterial property against both S. aureus and E. coli compared to pure Ti and TiO₂, and the antibacterial activity was enhanced with the increase of Zn content and the culture time. Therefore, the above-mentioned results indicated that the novel bone implant material with both antibacterial and biocompatibility has opened new horizon for their further multifunctional biomedical.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 50802032, 21103059 and 11275082), the Key Project of Natural Science Foundation of Hubei Province (no. 2011CDA092), the Key Scientific Project of Wuhan City (no. 2013011801010598), the Scientific Project of AQSIQ (no. 2013IK093) and self-determined research funds of CCNU from the colleges' basic research and operation of MOE (no. CCNU13A05007).

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Footnote

† Contributed equally.

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