3D printing of porous Ti6Al4V bone tissue engineering scaffold and surface anodization preparation of nanotubes to enhance its biological property

Shiqi Fan; Mohd Talha; Xia Yu; Haoyuan Lei; Yi Tan; Hui Zhang; Yuanhua Lin; Changchun Zhou; Yujiang Fan

doi:10.1515/ntrev-2023-0572

Article Open Access

3D printing of porous Ti6Al4V bone tissue engineering scaffold and surface anodization preparation of nanotubes to enhance its biological property

Shiqi Fan , Mohd Talha , Xia Yu , Haoyuan Lei , Yi Tan , Hui Zhang , Yuanhua Lin , Changchun Zhou and Yujiang Fan

Published/Copyright: July 31, 2023

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From the journal Nanotechnology Reviews Volume 12 Issue 1

Abstract

Porous structures and surface morphology of bone tissue scaffolds play an important role in improving the biocompatibility and antibacterial properties for bone repair. In this study, we investigated the effect of different anodic oxidation parameters on the nanotubes morphology in 3D printed porous titanium scaffolds. Micron-scale pores were fabricated by 3D printing first, and then the nano-scale tubes were obtained via anodizing treatments. The results demonstrated that the morphology of the nanotubes depended on the anodic oxidation time and voltage, respectively. Longer anodic oxidation led to the formation of circle-like nanotubes, and the diameter of the nanotubes increased with the voltage. The scaffolds anodized at 30 V showed the best cell proliferation potential. The presence of nanotubes on the surface of scaffold altered the adhesion of bacteria so that it improved the antibacterial properties of scaffold. The formation of nanotubes improved the drug-loading ability of the scaffold, which are used for loading of minocycline antibacterial drugs. The proposed 3D printed porous Ti6Al4V scaffold with nanotubes surface modification showed obvious antibacterial effect, which is expected to have a promising application in antibacterial bone prosthesis.

Keywords: 3D printing; Ti6Al4V scaffold; nanotubes

1 Introduction

The number of implant surgeries worldwide is increasing in recent years, posing a great challenge to the traditional implants [1]. Due to individual differences, patients have different needs for the implants, including personalized shape and size. An ideal bone tissue scaffold should resemble the natural bone in terms of structure and mechanical properties. Scaffold with a proper 3D porous structure and surface bioactivity are considered to be ideal bone tissue scaffolds [2–8]. After implantation, the interconnected micropores provide channels for transporting nutrients and excreting metabolic waste. Selective laser melting (SLM) is an additive manufacturing technology which uses a laser to instantaneously melt and solidify metal powder in a designed path to form the desired part. With the ability to form complex structures with high accuracy, SLM technology has been widely used in biomedical engineering [9,10].

Titanium and its alloys have been used as bone implants for decades due to their corrosion resistance and appropriate biomechanical properties [11]. In addition to providing mechanical support and function, bone implants must also act as a matrix for the interactions between protein and cells that determine the degree of osseointegration around the implant. Titanium is biologically inert and difficult to directly connect with human bone tissue, which may lead to implant loosening or even surgical failure. Therefore, the implant surface, as the first interface contact with the surrounding tissue, determines the fate of the implant [12–14]. Anodization is a surface treatment method which has been successfully applied to titanium alloys [15]. It has been shown that the regular nanomorphic features produced during anodizing, i.e., nanotubes can enhance the biological activity of implants and improve the adhesion and proliferation of cells [16–21]. The anodized nanotubes have been reported to show some antibacterial activity [22–24]. Compared with other types of surface treatment, the nanotube produced by anodic oxidation can improve the biomechanical stability of titanium implants in vivo, and the nanotube has great drug-loading potential due to its pinhole structure [22,25]. There are some studies on anodizing of titanium alloys, but research on 3D printed porous titanium is limited [24]. 3D printing can prepare bone-like biomimetic inner micropores, but the uniform coating technology for these internal micropores (rather than surface planes), especially for irregular inner porous channels is very difficult. Ren et al. anodized 3D printed titanium alloy plates and found that anodization can improve the bioactivity and osteogenic performance of the scaffold [26]. Yavari et al. [27] conducted experiments on the anodization of 3D printed titanium scaffolds at different voltages and times, and evaluated their ability to form hydroxyapatite in a simulated in vitro environment. They found that it is difficult to obtain uniform coating on the inner and outer surfaces of porous materials. Due to the complexity structure of 3D printed porous titanium alloy scaffold, it is difficult to obtain uniform coatings on both inner and outer porous architectures. 3D printed porous titanium alloy orthopedic has entered clinical application gradually, so it is urgent to study the surface modification of porous titanium scaffold to improve its biological properties.

In this article, we proposed a method that combines 3D printing technology with surface nano-modification to enhance the cytoactivity of 3D printed titanium scaffold. The Solidworks was used to design a scaffold model with micron-scale pores first, and then SLM 3D printing was applied to obtain the porous scaffold. After post-treatment [28,29], the nano-scale tubes were prepared through the surface electrochemical anodization [30]. The morphology of the nanotubes may be modulated by adjusting the voltage and the oxidizing time during electrochemical anodization. Biological properties including cytocompatibility, protein adsorption, bacterial adhesion, and drug-loading ability of the prepared scaffolds were evaluated and studied. Results indicated that this scaffold shows a promising potential in antibacterial bone prosthesis.

2 Materials and methods

2.1 Design and printing of Ti6Al4V scaffold

The design of a titanium implant is developed by Solidworks. The base unit of the scaffold has a diamond structure and the bore diameter is set to 600 μm. The scaffold has a cylinder shape with a height of 3 mm and a diameter of 6 mm. It is prepared through SLM system (M2, Concept laser, Germany) with Ti6Al4V powder as the raw material. The power of the printing laser is 90 W and the scanning speed is 500 mm/s.

2.2 Surface modification of porous Ti6Al4V scaffold

First, the residual printing powder on the porous Ti6Al4V scaffolds is removed through chemical corrosion. The corrosive solution is hydrofluoric acid (HF) solution consisting of 1 mL HF (40.0 wt%, Khron Chemicals, Sichuan, China) and 50 mL deionized water. The scaffolds are placed in the solution and react at room temperature for 15 min. Afterward, the samples are placed in deionized water and sonicated for 5 min, and this process is repeated three times. After washing with deionized water, the scaffolds are placed in anhydrous ethanol and sonicated for 5 min three times and then dried in a constant temperature oven at 60°C. Nanotubes are prepared on 3D printed titanium alloy scaffolds through anodization. The anode is a printed porous titanium alloy scaffold and the cathode is a platinum plate (0.5 × 0.5 × 0.1 mm). The distance between the electrodes is 20 mm and the ethylene glycol solution (0.5 wt% NH4F, 10 vol% deionized water) is chosen as the electrolyte. The voltage is set to 20, 30, and 40 V at room temperature and the reaction time is 15 min, 30 min, and 1 h. There are nine different groups of specimens, and each group contains at least three samples to eliminate random errors. Finally, the porous scaffolds are heat-treated in a muffle furnace for 2 h to relieve the internal stress and strengthen the bonding of the anodized nanotubes to the substrate.

2.3 Characterization of the Ti6Al4V scaffold

The microstructures of the inner and outer surfaces (labeled as INSIDE and OUTSIDE in Figure 1(f)) and the crystallinity of the scaffolds are characterized separately. The scanning electron microscope (SEM; JSE-5900LV, Japan) is adopted and the voltage is set to 5.0 kV. The surface elements of the samples are determined by an energy dispersive spectrometer (EDS). The main composition of the surface is determined by an X-ray diffractometer (XRD; Philips X’Pert 1, Netherlands). An atomic force microscope (AIST-NT smart SPM, USA) is adopted to analyze the roughness of the surface. Samples after 1 h of anodization at different voltages are used for the protein adsorption experiment. We add 1 mL of bovine albumin (1 mg/mL, BSA, Sigma, USA) to each sample and place the samples in an incubator at 37°C for 2 h. After removing from the protein solution, the samples are quickly rinsed with phosphate buffered saline (PBS) to remove the unadhered protein. The protein adhered to the surface is stained with bicinchoninic acid (BCA) (Beyotime, BCA Protein Assay Kit, China) and then the adsorption quantity is evaluated using a microplate reader.

Figure 1

Preparation of the scaffold: (a) SLM 3D printing, (b) acid washing, (c) surface of the printed scaffold, (d) anodizing, (e) surface of the scaffold after anodizing, and (f) inside and outside of the scaffold.

2.4 Cytocompatibility test

Fetal bovine serum (10%) and antibiotic antifungal solution (1%) are added to α medium (α-MEM, 89%) to culture MC3T3-E1 cells. Take 100 µL cell suspension with a concentration of 10⁴ cells inoculated on each scaffold and cultured for 1, 3, and 7 days to observe the adhesion pattern and cytotoxicity. The cell proliferation was detected by live cell staining and cell counting kit-8 (cck-8). Incubate the scaffold in cck-8 incubation solution for 2 h, then inhale the incubation solution into 96-well plate, and measure the absorbance value at 450 nm with microplate reader. The scaffolds are immersed in PBS solution containing fluorescein diacetate for 1 min, and then living cells are dyed green. After rapid rinsing of the scaffolds with PBS, the cell survival was observed by a confocal laser microscope (CLSM). Another group of cells is used to analyze the cell morphology. The scaffolds are rinsed with PBS and fixed with paraformaldehyde (4%) for 20 min. Then they are soaked in Triton-X-100 (0.1%) for 10 min after rinsing with PBS again. Afterward, the cells are stained with phalloidin and 4′,6-diamidino-2-phenylindole (DAPI) respectively, where phalloidin stained the fibrillar actin in red and DAPI stained the nuclei in blue. The adhesion pattern of the cells on the scaffold is then observed with the CLSM.

2.5 Antibacterial test

The presence of nanotubes on the surface of a scaffold alters the adhesion of bacteria to the scaffold. To reduce the error, the printed flat titanium is prepared by the same method as above to test the antibacterial properties of the nanotubes. Staphylococcus aureus (S. aureus) is used to evaluate the antibacterial performance of the scaffold. The scaffold is incubated in a liquid culture medium (10 mL) for 2 h where the concentration of S. aureus is 10⁵ and then it is gently rinsed with sterile liquid medium. Afterward, it is placed in a sterile medium (10 mL) and sonicated for 1 min. The medium is then cultured in sterile broth (LB, purchased from Solarbio) at 37°C for 24 h. Finally, Image J was used to count the colony on the culture dish to evaluate the amount of bacteria adhering to the scaffolds.

2.6 Drug loading and antibacterial experiment

Minocycline is a commonly used antibacterial drug. Immerse the anodized scaffolds with different voltage and untreated scaffolds in 1 mg/mL minocycline solution for 2 h. After removal, use PBS to quickly wash the surface to remove the residual drugs outside the nanotubes, and dry at room temperature. S. aureus was used to evaluate the antibacterial properties of the stent. Evenly smear the 10⁵ concentration of S. aureus on the petri dish of sterile broth (LB, purchased from Solarbio), place the scaffold in the center of the petri dish at 37°C for 24 h.

3 Results and discussions

3.1 Ti6Al4V scaffold surface characteristic

The appearance and dimensions of the printed scaffolds are shown in Figure 2(a) and the size of the titanium powder particles used for printing is shown in Figure 2(c). As shown in the figures, there is a small difference between the dimensions of the printed and the designed scaffolds. Figure 2(b) shows the appearance of the scaffolds obtained after acid washing, anodizing, and high-temperature holding (Blank represents the scaffold which is directly under high-temperature holding after printing). Figure 2(d) and (e) are the SEM images of the scaffolds at lower magnification before and after acid washing. It can be observed that the surface of the scaffolds becomes smooth after acid washing. It is because the reaction removes the incompletely melted titanium powder particles, which may cause harm to humans. Previous studies have shown that acid washing of porous scaffolds can comprehensively clean the internal and external channels [31]. So acid washing improves the quality of the scaffold surface. Figure 2(f) and (g) is the SEM images of the scaffolds before and after anodization, which illustrate the formation of a uniform array of pore-like nanotubes on the surface.

Figure 2

Appearance of the 3D printed Ti6Al4V scaffold: (a) untreated scaffolds, (b) scaffolds after anodizing at different voltages for 1 h and high temperature holding, (c) Ti6Al4V powders for 3D printing. (d and e) SEM images of the surface of the scaffold before and after acid washing. High magnification SEM images of the Ti6Al4V scaffold surface before (f) and after anodization (g).

3.2 Characterization of the porous Ti6Al4V scaffolds

The scaffold anodized at 30 V for 1 h is tested for its surface properties, as shown in Figure 3(a), and Blank is the scaffold without any treatment. The XRD image shows that compared to the untreated scaffold, anodized scaffold generates anatase structured titanium dioxide nanotubes on the surface, as shown in Figure 3(b). Decha-umphai et al.’s report also confirms this result [32]. Nanotubes with anatase structure are shown to have better physicochemical properties. And the inherent negative electricity of anatase structure titanium dioxide can absorb calcium ions in the human environment, which is conducive to bone reconstruction [33]. The EDS image illustrates that the surface of the scaffold contains oxygen, which is because the main component of the nanotubes is titanium dioxide, as shown in Figure 3(c). The nanotubes produced after anodizing at different voltages for 1 h are measured using Image J, as shown in Figure 3(d). The diameters of the nanotubes generated at 20, 30, and 40 V are about 26, 57, and 77 nm, respectively. This result is slightly larger than the previous report by Gong et al. [34]. This may be due to the use of 0.5 wt% NH4F in the electrolyte instead of 0.25 wt%. In Xie et al.’s report, the anodizing electrolyte used 1 wt% NH4F, and the resulting diameter of the nanotubes was also larger than the data presented in this study [35]. The surface roughness of the scaffold at 30 V/1 h is tested using AFM, as shown in Figure 3(e). The results reveal that by generating nanotubes on the surface, anodization can significantly enhance the surface roughness of the scaffolds, and greater roughness is more favorable for cell adhesion [34,36]. Figure 3(f) shows the adsorption capacity of the samples to bovine serum. The absorbance indicates that the nanotubes can improve the adhesion of proteins and the sample anodized at 30 V has the highest adsorption capacity which may result from the balance of protein molecules dimension and interaction energy because physical adsorption is their main driving force [37]. Compared with the untreated scaffold, the adsorption of BSA on the surface of these scaffolds with nanotubes was significantly increased, which indicated that nanotubes can promote the adsorption of protein. It benefits from the improvement of coarseness [38]. The surface with high roughness possesses larger specific surface area, and it also provides larger contact area and more adsorption sites for protein adsorption. The adsorbed protein layer with nanoscale morphology has a significant impact on cell activity and spreading morphology for their bridging role in cell migration on larger than 100 nm nanotube pore size, which is the critical dimension of cell filopodia (50–100 nm) [37]. The improved adsorption capacity of the scaffolds leads to enhanced osseointegration properties, which makes implant scaffolds more beneficial for patients’ postoperative recovery [39].

Figure 3

Characterization of the scaffold: (a) appearance of nanotubes anodized at 30 V for 1 h, (b) XRD image of the sample anodized at 30 V, (c) EDS image of the scaffold anodized at 30 V, (d) comparison of the diameter of nanotubes on the samples prepared at 20, 30, and 40 V, respectively, (e) surface roughness of the scaffold before and after anodizing, and (f) assessment of the adsorption capacity of the scaffold to protein.

3.3 Effect of different anodization parameters on the morphology of nanotubes

Figure 4 shows the SEM images of the nanotubes generated on the inner and outer surfaces of the scaffold after 15 min of anodizing at different voltages. It can be observed that the nanotubes on the outside of the scaffold are formed faster than those on the inside. Due to the viscosity of glycol, the pores cannot exchange electrons quickly with the external solution, which retards the formation of nanotubes. This phenomenon is alleviated after 1 h of anodizing. As can be seen in Figure 5, the appearance of the nanotubes generated on the inside and outside of the scaffold tends to be uniform after 1 h of anodic oxidation.

Figure 4

SEM images of the scaffold surface after 15 min of anodizing at different voltages. (a–c) Outside nanotube morphology of the scaffold which anodized at 20, 30, and 40 V, respectively. (d–f) Inside nanotube morphology of the scaffold which anodized at 20, 30, and 40 V, respectively.

Figure 5

SEM images of the scaffold surface after 1 h of anodizing at 20, 30, and 40 V, respectively. (a–c) Outside nanotube morphology of the scaffold which anodized at 20, 30, and 40 V, respectively. (d–f) Inside nanotube morphology of the scaffold which anodized at 20, 30, and 40 V, respectively.

Figure 6 shows the SEM images of the outside of the samples prepared at different anodization voltages and times. It can be observed that the nanotubes are gradually formed on the surface of the scaffold as the oxidation time increases. It is worth noting that increasing the voltage does not accelerate the generation of the nanotubes, which can be obtained by a horizontal comparison of the images. As we can see in Figure 6(g)–(i), the diameter of the nanotubes increases with the voltage. It is of great significance to control the anodizing process parameters to obtain different sizes of nanotubes for the subsequent function of the scaffold. Our result indicated that the anodization voltage and time effects on the nanotubes morphology are consistent with previous literature on flat titanium [34].

Figure 6

Outside nanotube morphologies SEM images of the porous Ti6Al4V scaffold obtained at different anodic oxidation parameters. (a–c) Outside nanotube morphologies SEM images of the porous Ti6Al4V scaffold obtained at different voltages for 15 min anodic oxidation. (d–f) Outside nanotube morphologies SEM images of the porous Ti6Al4V scaffold obtained at different voltages for 30 min anodic oxidation. (g–i) Outside nanotube morphologies SEM images of the porous Ti6Al4V scaffold obtained at different voltages for 60 min anodic oxidation.

3.4 Cytocompatibility and antibacterial properties test

Figure 7 shows the living cell staining images of cells on scaffolds anodized at different voltages for 1 h after 1, 3, and 7 days of culture. CLSM is used to observe the scaffold surface. Quantitatively, the cells proliferated significantly after 7 days of culture. A side-by-side comparison shows that more cells survived on the anodized scaffold than on the untreated one. This is probably because when the cell suspension was added, the cells were washed to the bottom of the scaffold due to the lack of attachment sites on the untreated scaffold. The surface of the scaffold anodized at 30 V has more cells and is more densely distributed than the other groups. The cytoskeleton is selected for F-actin staining and CLSM observation after 3 days of culture on the scaffold. Figure 8(a) shows the results of cell cck-8 experiment. The results still showed that 30 V scaffold was the most favorable for cell proliferation. Figure 8(b) displays that the cells are spread out in a flat structure and the pseudopods are used to attach to the scaffold. This finding aligns with the previous report by Liu et al. [40]. Comparing the four sets of images, it concludes that the cells spread best on the scaffold anodized at 30 V, indicating that the nanotubes generated at 30 V are more favorable for cell adhesion. Micro-topological structure can regulate the behavior of the cells [38,41]. The diameter of nanotubes generated under different voltage conditions directly affects the migration and spreading morphology of cells, and on the other hand, nanotubes with different diameters indirectly change the migration and spreading morphology of cells through protein adsorption which has been illustrated in protein adsorption experiment. First, cells can adhere to these functional surfaces through non-receptor binding forces, thereby enhancing cell proliferation. The nanotube surface carries a negative charge, which promotes cell adhesion and proliferation. As the surface of smaller diameter nanotubes carries more negative charges, it allows more cells to attach and proliferate on its surface. Second, nanotubes with larger diameters provide a larger surface area, increasing the opportunity for cell attachment. Larger diameter nanotubes typically have a greater attachment surface, allowing them to accommodate more cells and thereby increasing the likelihood of cell adhesion. Therefore, selecting scaffolds with nanotubes of appropriate diameters is crucial for the effectiveness of bone repair after implantation.

Figure 7

Living cell staining on porous Ti6Al4V scaffold anodized at different voltages after 1, 3, and 7 days of culturing. (a–d) Proliferation of MC3T3-E1 cells on Ti6Al4V scaffold after 1 day culturing. (e–h) Proliferation of MC3T3-E1 cells on Ti6Al4V scaffold after 3 days culturing. (i–l) Proliferation of MC3T3-E1 cells on Ti6Al4V scaffold after 7 days culturing.

Figure 8

Cck-8 and cytoskeleton results on different Ti6Al4V scaffolds: (a) Cck-8 experimental results and (b) cytoskeleton staining of cells on scaffolds anodized at different voltages after 3 days of culturing.

The result of the bacterial adhesion test is shown in Figure 9. The images show that there are fewer bacteria on the scaffold after anodization than on the untreated one. The scaffold anodized at 30 V has the least colony and thus the best antibacterial effect. The influencing diameter factors of the nanotubes on cell adhesion are diverse which remain undetermined [42].

Figure 9

Results of the bacterial adhesion test on different Ti6Al4V scaffolds: (a) colonies cultured from bacteria on the blank scaffold, (b) colonies of bacteria cultured on 20 V anodized scaffold, (c) colonies of bacteria cultured on 30 V anodized scaffold, (d) colonies of bacteria cultured on 40 V anodized scaffold, and (e) quantitatively count the colonies on the cultured bacteria dish through Image J.

The experiment of drug-loaded antibacterial results is shown in Figure 10. Compared with the blank scaffold, after loading minocycline, the scaffolds appear pale yellow, which may be due to the attachment of minocycline in the nanotubes. The images show that all anodic oxidation scaffolds show obvious antibacterial effect after drug loading compared with untreated scaffolds. However, there is no difference in the antibacterial effect of anodic oxidation samples with different voltages after drug loading. This shows that the nanotubes formed on the surface of titanium alloy scaffold by anodic oxidation have good drug-loading potential. Indeed, there have been numerous studies on utilizing drug-loaded anodized nanotubes. Feng et al. successfully loaded gentamicin sulfate into nanotubes and coated the surface with chitosan, achieving excellent antibacterial effects [43]. The drug-loading potential of nanotubes is significant, and combining nanotubes with porous metal scaffolds is highly necessary.

Figure 10

Experimental results of drug-loaded antibacterial effects of different Ti6Al4V scaffolds: (a) antibacterial effect of blank scaffold loaded with drugs on bacterial culture medium, (b) 20 V anodized drug-loaded scaffold, (c) 30 V anodized drug-loaded scaffold, and (d) 40 V anodized drug-loaded scaffold.

4 Conclusions

This article proposed a method to modify the surface of 3D printed porous titanium alloy scaffolds by regulating anodization parameters. The results demonstrated that the micron- and nano-scale tubes were prepared successfully on the inner and outer surface of the porous scaffolds. SEM images showed that the diameter of the obtained nanotubes could be controlled by adjusting the anodization voltage and time, individually. Biological experiments demonstrated that the cytocompatibility of the scaffolds was improved after anodization treatment, with the scaffolds treated at 30 V showing the best biofunctional performance. The nanotube produced by anodic oxidation improved the drug-loading capacity of the scaffolds, which is crucial to improve the cytocompatibility and antibacterial properties of the scaffolds. This surface modification technology on 3D printed porous titanium implants has great potential for customized orthopedic implants clinical application.

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Funding information: This work was partially supported by the National Natural Science Foundation of China (31971251). Science and Technology Project of Tibet Autonomous Region (XZ202202YD0013C). Yingcai Scheme, Chengdu Women’s and Children’s Central Hospital (YC2021004, YC2022001). Sichuan Science and Technology Program (2022YFG0066, 2022NSFSC0815). Project of Chengdu Science and Technology Bureau (2021-RC05-00022-CG).
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Conflict of interest: The authors state no conflict of interest.

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Received: 2022-11-20

Revised: 2023-06-14

Accepted: 2023-06-26

Published Online: 2023-07-31

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/ntrev-2023-0572

Keywords for this article

3D printing; Ti6Al4V scaffold; nanotubes

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