Construction of Antibacterial and Bioactive Surface for Titanium Implant
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Titanium (Ti) and its alloy are extensively used as hard tissue implant materials in the medical field due to their good mechanical and biological properties. However, implant-associated bacterial infection and delayed osseointegration of an implant to its surrounding bone tissue are still huge challenges to clinicians and material scientists in orthopedic and dental surgery. Therefore, a novel method to construct the antibacterial and bioactive surface for titanium implant was proposed in this study. Briefly, micro/nanostructure was fabricated on the surface of titanium by sandblasting and chemical etching, and silver nanoparticles were then immobilized on the surface by polydopamine (Ag–Ti). The treated sample showed the enhanced roughness, hydrophilicity, and corrosion resistance. And silver ions were released from the treated samples. Moreover, it was observed that the Ag–Ti sample surface covered many calcium phosphate compounds after immersed in simulated body fluid for 7 days. Furthermore, the results of biological tests demonstrated that the treated sample possessed a good antibacterial activity and biocompatibility. This study may provide a new insight for constructing of the antibacterial and bioactive surface for titanium implant to satisfy clinical requirements.
KeywordsTitanium Micro/nanostructure Silver nanoparticles Antibacterial activity Biocompatibility
Titanium (Ti) and its alloy are extensively used as hard tissue implant materials in the medical field due to their good mechanical and biological properties [1, 2, 3]. However, implant-related bacterial infections are still a huge challenge in the clinic because of the lack of antibacterial activity for Ti-based biomaterials . It is reported that implant infections mainly happened in the operation sites and surgical procedures, and the incidence of infections was approximately 2–30% of percutaneous fracture fixation needle, 13% of bone filler, 2–5% of cervical infections, 2% of primary joint replacements, and 1.4–4% of total hip and knee replacements [5, 6, 7, 8, 9, 10, 11, 12]. Once adhering to Ti surface, bacteria will colonize and form a biofilm to protect microorganisms from antibiotic treatment, thus causing periprosthetic infection . If bacteria infection occurs, patients not only need to take on extra cost but undergo multiple surgeries that result in suffering . Therefore, it is necessary to endow Ti implant surface with antibacterial property for reducing bacterial infections.
Various surface modification methods have been proposed by scholars to endow implants with excellent antibacterial activity for increasing the success rates of implantation. Inorganic antibacterial agents such as silver (Ag) [4, 14], copper (Cu) [15, 16] and zinc (Zn) [17, 18, 19] exhibited excellent antibacterial activities. They have been widely incorporated on implant surface to obtain the desired antibacterial property without a risk of the emergence of bacterial resistance. Ag is believed to have a good broad-spectrum antimicrobial activity and extensively used as antimicrobials in industrial and healthcare, which plays an essential role in the treatment of infections [20, 21, 22]. In particular, Ag nanoparticles (Ag NPs) are recognized to be more reactive than the bulk metallic counterpart thanks to the larger active surface area . Recently, different Ag NPs-modified films have been constructed on implant surfaces for reducing bacterial infection [4, 24, 25, 26].
In addition to antibacterial ability, good biocompatibility is also necessary to implants. After implantation, the interactions between biological environments and biomaterials would happen at their surface . The low activity of bone formation may lead to the failure of implants. Therefore, the balance of antibacterial and biocompatibility should be considered at the design of the desired implant surface. It is reported that microstructure surface was conducive to the anchorage of implants to bone [28, 29, 30], while nanostructure was beneficial to promote cell functions and control the mineralization of the collagen matrix [31, 32]. Therefore, multi-scale structural surface with microstructure and nanostructure may be presented a more positive effect on cellular responses. For example, Li et al.  constructed micro/nanostructure by generating nanotubes on the micropitted Ti surface and these structures enhanced the multiple osteoblast functions such as cell adhesion.
Based on the above considerations, in this paper, we fabricated the micro/nanostructured Ti surface by sandblasting and chemical etching. And Ag NPs were then immobilized on micro/nanostructured Ti surface by harnessing the adhesion and reactivity of bioinspired polydopamine . By this means, the antibacterial and bioactive titanium surface was successfully obtained. The surface property and corrosion resistance of different Ti samples were evaluated. In addition, the antibacterial activity and biocompatibility of different samples were also investigated in this study.
2 Materials and Methods
2.1 Preparation and Characterization of Samples
Ti plates (10 × 10 × 1 mm3) were mechanically polished with waterproof abrasive paper from 400# to 2000# and sequentially rinsed in acetone, anhydrous ethanol, and deionized water. Then, the polished Ti samples (P) were sandblasted with alumina particles (125–150 μm) for 20 s at a constant pressure of 3.4 bars to produce micro-pits. After that, the etching of treated samples was performed in the mixed solution of HF and HCl for 150 s to eliminate residual alumina particles on the surface. Next, the samples were further etched in piranha solution for 2 h to construct nanostructure , and micro/nanostructured Ti (MN–Ti) was thus successfully obtained. For the fabrication of antibacterial and bioactive Ti samples (Ag–Ti), MN–Ti samples were soaked in dopamine solution (2 mg/ml) for 24 h and avoiding light. Finally, Ti samples were immersed in the AgNO3 solution (0.05 M) for 6 h to endow Ti surface with antibacterial activity.
Surface topography and chemical composition of the samples were examined by scanning electron microscopy (SEM, SUPRATM55 SAPPHIRE, Carl Zeiss, Germany) with the energy-dispersive spectroscopy (EDS). Three-dimensional profile images were determined by a laser scanning microscope (LSM, VK-X200 K, Japan). Contact angle goniometer (SL200KS, America) was used to measure the contact angle of samples at room temperature by a sessile drop method. And the liquid drops of doubly distilled water and dichloromethane were applied in measurement. The Owens–Wendt-Kaeble’s equation was employed to calculate surface free energy [36, 37].
2.2 Protein Adsorption Assay
Different Ti samples were put into bovine albumin (BSA, 1 mg/ml) solution for 2 h at 37 °C. Then, the samples were rinsed in PBS. Next, the adsorbed proteins on different samples were extracted by 2% sodium dodecyl sulfate with shaking for 1 h at room temperature. The concentration of protein was determined by BCA assay kit.
The experimental data were determined by statistical analysis software GraphPad Prism 5. P < 0.05 was considered to be significant.
2.3 Measurement of Silver Ions Release
The specimens were immersed in 6 ml α-MEM culture medium at 37 °C for 1, 4, 7 days without stirring, respectively. According to predetermined time points, the solution was collected and fresh α-MEM was added. The released ions were determined by inductively coupled plasma atomic emission spectrometry.
2.4 Corrosion Resistance Measurement
The corrosion resistance of different Ti samples was measured in NaCl solution at ambient temperature by the electrochemical workstation. The test area of samples was 1 cm2. The specimens were soaked in NaCl solution for several minutes to obtain stabilized open circuit potential. Then, the potential was scanned in the range from -1200 to 1200 mV at the rate of 1 mV/s.
2.5 In Vitro Test of Biomineralization
The biomineralization of different Ti samples was investigated by soaking in simulated body fluid (SBF). The plates were immersed in 15 ml of SBF solutions for 7 days at 37 °C. Every two days, the old solution was removed, and the new solution was added. The samples were rinsed with deionized water after 7 days. Finally, the surface morphologies of different samples were observed by SEM.
2.6 Antibacterial Assessment
The antibacterial activity of P, MN–Ti, and Ag–Ti samples was investigated in this study. One milliliter of bacterial suspensions (Staphylococcus aureus, 106 CFU/ml) was introduced into each well in 24-well plates containing the sterilized samples and cultured at 37 °C for 12 h. Then, the samples were put into the test tube with 4 ml of sterile physiological saline. Next, the bacteria were separated from the surface of samples using a vortex mixer. The dissociated bacterial suspension was dilute 5 × 103 folds, and 20 µl of the diluted bacterial suspension was coated on agar plates incubated at 37 °C for 12 h. The spread-plate method was used to evaluate the antibacterial activity of different samples.
2.7 Cell Morphology
MC3T3s were cultured α-MEM supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C. In order to investigate the effect of different sample surfaces on cell morphology, MC3T3s were seeded on P, MN–Ti, and Ag–Ti surface (2 × 104 cell/ml) and cultured for 1 day at 37 °C. After that, the samples were washed with PBS and fixed with 4% paraformaldehyde for 25 min at 4 °C. Next, 0.2% Triton X-100 was used to permeabilized samples at 4 °C for 10 min. Subsequently, the cells were stained with phalloidin for 90 min and counterstained with Hoechst 33,258 (10 µg/ml) for 15 min at room temperature. Finally, the samples were observed with a confocal laser scanning microscope (CLSM, LSM 780, Germany).
3 Results and Discussion
3.1 Surface Characterization
Contact angle measurements and surface free energy of different samples
Contact angle θ (°)
Surface energy (nJ/cm2)
3.2 Protein Adsorption
3.3 Silver Ions Release Test
As we all known, the bacterial infection risk of the implant was the highest on the first day. Therefore, it is important to control the release of Ag ions for inhibiting bacteria biofilm formation in a short time. Figure 4b displayed the Ag ions release time profile from Ag–Ti in α-MEM. Apparently, the release rate of Ag ions was the highest at the first day (0.447 ppm), which would benefit to inhibit bacterial biofilm formation. After that, the release rate of Ag ions was gradually decreased with time. In addition, Ag-MN samples were also considered to non-toxic because the concentration of Ag+ (0.5053 ppm) was lower than the threshold value of cell apoptosis (0.78-1.56 ppm) .
3.4 Corrosion Resistance Measurement
3.5 In Vitro Test of Biomineralization
3.6 In Vitro Antibacterial Ability
3.7 Cell Morphology
In this paper, the micro/nanostructure was produced on Ti surface by sandblasting and chemical etching. By harnessing the adhesion and reactivity of bioinspired polydopamine, silver ions were reduced to nanoparticles and immobilized on micro/nanostructured Ti surface. The roughness and hydrophilicity of Ag–Ti sample were increased compared to polished Ti. In addition, Ag–Ti sample also showed excellent corrosion resistance and bioactivity. More importantly, Ag–Ti sample exhibited a good antibacterial activity due to the effect of Ag nanoparticles. And MC3T3 s grew on Ag–Ti surface presented spreading cell morphology with a polygonal shape. Therefore, the designed surface exhibited a good antibacterial activity and biocompatibility. This study provides a preliminary insight into the balance of antibacterial and biocompatibility, which may be helpful guidance to implant developers on the design of antibacterial and bioactive surface in the further.
This study was funded by National Natural Science Foundation of China (51575320), Taishan Scholar Foundation (TS20130922).
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