Graphdiyne-modified TiO2 nanofibers with osteoinductive and enhanced photocatalytic antibacterial activities to prevent implant infection


Materials preparation

First, 4 g of tetrabutyl titanate (TBT) and 1.5 g of Polyvinyl pyrrolidine (PVP, MW = 1.3 × 106) were added to a mixed solution of ethanol (20 g) and acetic acid (4 g) under magnetic stirring for 5 h. Then, The obtained transparent pale-yellow solution was transferred into a 20 mL syringe. The voltage of electrospinning setup and the feeding rate of solution was 20 kV and 2.5 mL h−1, respectively. The distance of needle-to-static collector was 10 cm. The collected TiO2 fiber precursor was heat-treated at 550 °C for 2 h in air to fabricate TiO2 nanofibers.

GDY used in this work was provided by Institute of Chemistry, Chinese Academy of Science65. A zeta potential analyzer was used to measure the zeta potential of TiO2 and GDY in water at pH 4, which are +15.1 mV and −19.2 mV, respectively. Typically, TiO2 nanofibers (200 mg) were dispersed in water at pH = 4, then, magnetically stirred for 1 h at room temperature. Meanwhile, 10 mg of GDY was dispersed in a mixture solution of water (30 mL) and ethanol (60 mL) under sonication irradiation for 2 h. Afterwards, a certain amount of GDY solution was added into the TiO2 suspension by stirring vigorously for 1 h. TiO2 and GDY were electrostatically self-assembled. Subsequently, the solvents were removed. the obtained composites were calcined at 350 °C for 2 h. The x in “TGx” represents the added GDY weight ratio. The TiO2/GDY applied in the following experiments was the typical TG 0.5.

Material characterization and photocatalytic properties

The microstructure of TiO2/GDY was characterized by TEM (Titan G2 60-300). The molecular structure was detected by Raman spectroscopy (Renishaw Co. RM1000). The surface contact angles of TiO2/GDY and TiO2 nanofibers were detected by a contact angle meter (Powereach® JC2000C1).

A RhB degradation assay was carried out to detect the photocatalytic activity of TiO2/GDY and TiO2 nanofibers. 1 mL of 8 μg mL−1 RhB solution and 100 μg of photocatalysts (TiO2/GDY or TiO2) were added into ep tubes and exposed to 365 nm UV light (2 W cm−2 for up to 60 min) (Yinzhu, UVEC-4II, China). Each sample was centrifuged for 5 min at 1000 × g. The absorbance of the supernatants was detected by an UV spectrophotometer (SOPTOP, UV2800, China).

The ability of TiO2/GDY and TiO2 nanofibers to generate ·OH and ·O2 was respectively measured in water and methanol under 300 W Xe lamp irradiation using ESR (Bruke, emx nano) at room temperature and 5,5-Dimethyl-1-Pyrroline N-oxide (DMPO) as spin trapping reagent. For measuring the generation of 1O2 and ·OOH, 2,2,6,6-tetramethyl-piperidine (TEMP) and DMPO were used as trapping reagent. The measurement was performed in water using ESR (Bruke, a300) at room temperature. The generation of H2O2 was characterized by ferric ion titration method. Fifteen milligrams of photocatalysts were suspended in 15 mL of H2O and ultrasonicated for 5 min. The suspension was firstly bubbled with high-purity O2 for 30 min and irradiated with 300 W Xe lamp for 3 h. The H2O2 concentration was measured every 1 h. After turning off the light, H2O2 level was also tested continuously in 30 and 60 min.

Cell morphology and viability

The murine-derived osteoblast-like cells MC3T3-E1 were cultured in α-minimal essential medium (Hyclone, Thermo Fisher Scientific Inc.) supplemented with 10% FBS (FBS, Gibco) and maintained at 37 °C in 5% CO2 and 95% air in a humidified incubator.

The MC3T3-E1 cells were incubated on confocal dishes at a density of 6 × 104 cell per dish with 20 μg mL−1 TiO2/GDY or TiO2 nanofibers for 24 h. After washed with phosphate buffered saline (PBS) solution, cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. Removed PFA and washed with PBS, then cells were permeabilized with −20 °C acetone for 5 min. The F-actin and nuclei were stained with FITC Phalloidin (Yeasen, 40735ES75) and DAPI, respectively and observed by laser scanning confocal microscope (LSCM, Leica-LCS-SP8-STED). 25 of random adhered cell areas of each group were calculated through Image J. Adhered cell number was analyzed by DAPI staining of cell nucleuses.

For SEM scanning, MC3T3-E1 cells were seeded on 24-well cell culture plates at a density of 2 × 104 cells per well with 20 μg mL−1 TiO2/GDY or TiO2 nanofibers and incubated for 24 h. After washing with PBS, cells were fixed with 2.5% glutaraldehyde overnight at 4 °C. The samples were washed 3 times with water, then freeze-dried. Followed by sputter coated with gold, samples were observed by SEM (TESCAN VEGA 3 LMU) with an accelerating voltage of 20 kV and a working distance of 11.7 mm.

The live/dead staining assay was carried out with Calcein-AM/PI Double Stain Kit (Yeasen, 40747ES80). In brief, cells were seeded onto glass coverslips in 24-well cell culture plates at a density of 2 × 104 cells per well. 20 μg mL−1 TiO2/GDY or TiO2 nanofibers were added to cells without UV irradiation, received UV (365 nm, 2 W cm−2) irradiation together with cells, or received UV irradiation before addition. After 24 h incubation, the glass coverslips were rinsed with 1 × Assay Buffer for 3 times. Afterwards, the coverslips were incubated with 2 μM Calcein-AM and 1 μM propidium iodide (PI) in 1 × Assay Buffer for 15 min at 37 °C. Images were taken using a fluorescence microscope (Zeiss Axio Imager A2). The numbers of live and dead cells were counted by Image J.

Cell proliferation was evaluated by the cell counting kit-8 (CCK-8) assay. MC3T3-E1 cells were seeded in 96-well cell culture plate with 5 × 103 cells per well and incubated overnight for attachment. Then cells were exposed to various concentrations of TiO2/GDY or TiO2 nanofibers from 1 to 50 μg mL−1. At the time points of 1, 3, 5-day incubation, cells were incubated with the CCK-8 agent (Dojindo, Japan) diluted 1:10 with culture medium for 2 h. Then the absorbances were measured by a microplate reader scanning at 450 nm (PowerWave XS2, BioTek, Winooski, VT, USA). The assays were implemented triplicated with three independent experiments.

Antibacterial properties in vitro

MRSA USA 300 were cultured in an aerophilic environment in LB broth at 37 °C. The bacterial suspension was diluted to 109 colony forming units (CFU mL−1) for experimental use after measuring the absorbance at 600 nm using the UV spectrophotometer.

For bacterial standard plate counting assay, MRSA cells were prepared in 20 mL liquid LB medium into 109 CFU mL−1 and then added with various ratios of TiO2/GDY, TiO2, TiO2/GO (100 μg mL−1), GO, GDY (50 μg mL−1) or PBS as control. After 1 h incubation, cells were irradiated with UV (365 nm, 2 W cm−2) for 5 min, then 25 μL bacterial suspensions were spread onto LB agar plates. The images were taken, and the colonies were counted using Image J software after 24 h incubation.

The property of biofilm destruction by photocatalysis was determined through live/dead staining. Five hundred microliters 109 CFU mL−1 MRSA suspensions were seeded onto 10% Poly-L-Lysine pretreated glass coverslips and incubated 24 h to obtain biofilms in advance. TiO2/GDY or TiO2 nanofibers were added onto the biofilms with UV irradiation for 5 min. Then the live/dead staining was carried out with Calcein-AM/PI Double Stain Kit. The glass coverslips were rinsed with 1 × Assay Buffer for 3 times. Afterwards, the coverslips were incubated with 2 μM Calcein-AM and 1 μM propidium iodide (PI) in 1 × Assay Buffer for 15 min at 37 °C. Images were taken using a fluorescence microscope (Zeiss Axio Imager A2). The numbers of live and dead cells were counted by Image J.

The effect on the bacterial biofilm formation was investigated through SEM scanning. 109 CFU mL−1 MRSA cells were mixed with 100 μg mL−1 TiO2/GDY or TiO2 nanofibers and exposed to UV for 5 min. Five hundred microliters bacterial suspensions were seeded onto a 10% Poly-L-Lysine pretreated glass coverslip and incubated 24 h to form bacterial biofilm. Then the biofilms were washed with PBS and fixed with 2.5% glutaraldehyde overnight at 4 °C. The samples were washed 3 times with water, then freeze-dried. Followed by sputter coated with gold, samples were observed by SEM (TESCAN VEGA 3 LMU) with an accelerating voltage of 20 kV and a working distance of 10.5 mm.

Crystal violet staining was carried out for semi-amount evaluation of biofilm formation. 109 CFU mL−1 MRSA cells were mixed with 100 μg mL−1 TiO2/GDY or TiO2 nanofibers and exposed to UV (2 mW cm−2) for 5 min. Ten microliters bacterial suspensions were added to 100 μL LB medium in 96-well plate. After 24 h incubation in 37 °C, the biofilms were rinsed and fixed with 4% of PFA for 10 min. Then crystal violet (Servicebio, China) was added 50 μL per well. After 20 min incubation in room temperature, removed the dye liquor and rinsed with ddH2O. Ethyl alcohol was added 100 μL per well for dissolution. Absorbance at 590 nm of each well was measured after 20 min shaking (Molecular Devicesmd, spectramax i3x).

ROS generation in MRSA was tested using a ROS assay kit (Beyotime, China). MRSA cells (109 CFU mL−1) were loaded with the fluorescence probe (DCFH-DA) in 37 °C for 20 min. Then the bacteria were incubated with 100 μg mL−1 TiO2/GDY or TiO2 nanofibers and exposed to UV for 5 min. The fluorescence intensity of DCF, denoted the intracellular ROS generation, was measured 5, 20, 60 min after UV irradiation (Molecular Devicesmd, spectramax i3x). Images of fluorescence were observed using the fluorescence microscope (Zeiss Axio Imager A2).

109 CFU mL−1 MRSA cells were incubated with 100 μg mL−1 TiO2/GDY or TiO2 nanofibers and exposed to UV for 5 min. ATP levels evaluation was carried out in accordance with the manufacturer’s protocol (Promega, G8320). For bacterial structure observation, 109 CFU mL−1 MRSA cells were incubated with 100 μg mL−1 TiO2/GDY or TiO2 nanofibers and exposed to UV for 1 h. Cells were fixed and observed with SEM 0, 1 h after treatment.

Osteogenesis properties in vitro

MC3T3-E1 cells were cultured in 24-well plates at a density of 5 × 104 cells per well. After 24 h incubation, the culture medium was replaced by the osteogenic differentiation media consisting of 10 mM of β-glycerophosphate, 10 nM of dexamethasone and 0.2 mM of L-ascorbic acid 2-phosphate (Sigma-Aldrich). Then 20 μg mL−1 of TiO2/GDY, TiO2/GO or TiO2 nanofibers, 10 μg mL−1 of GDY or GO were added into the culture medium. Medium was changed every 2–3 days. After 14 days of culture, ALP staining was performed by an ALP staining kit (Beyotime, China) and the positive cells were counted by the Image J software.

ARS staining was carried out on day 14 after osteoinduction. Using 95% alcohol to fix cells for 10 min at room temperature. Cells were stained with 2% ARS at pH of 4.1 for 30 min in 37 °C. Then washed the cells with ddH2O for 3 times. The stained mineralized nodules were observed under a light microscope. Semiquantitative analysis of ARS staining was implemented with Image J software by measuring the red staining areas.

Real time-qPCR assay on ALP, OCN, OSX, Col1a mRNA expression was conducted to explore osteogenesis property. MC3T3-E1 cells were cultured on 10 μg mL−1 of GO, GDY, TiO2/GO, TiO2/GDY or PBS, then cultured in osteogenic differentiation media for 7 days. Total RNA from cultured cells was extracted. After reverse transcription, Real time-qPCR analysis was performed for detection of gene transcriptional levels. The amount of mRNA was normalized to GAPDH. The primer sequences were shown in Table 1.

Table.1 Real time-qPCR primer sequences.

Loading capacity

To determine the loading capacity of the nanofibers, dexamethasone (Aladdin, China) with concentrations ranging from 1 to 10 μM were prepared in phosphate buffer. Each concentration of the solution (0.5 mL) was mixed with TiO2/GDY 0.5, TiO2/GDY 0.25 and TiO2 nanofibers (1 mg mL−1, 0.5 mL) separately, and vortexed in a shaker for equilibration at room temperature. After 24 h, the mixtures were centrifuged (12,000 × g, 5 min) to collect the supernatant for spectrophotometric measurement. The adsorption isotherm of dexamethasone was obtained using UV–vis spectroscopy (SOPTOP, UV2800, China).

In vivo implant infection model

All animal surgical procedures were approved by the Ethics Committee for Animal Research of Wuhan University in China. The mouse implant infection model was operated on Kunming mice (8-week-old, female). MRSA prepared for infection were in mid-exponential grown phase of 107 CFU mL−1. After anesthesia with isoflurane, the hindlimb of mice were shaved and disinfected with povidone iodine and alcohol. A small incision was made on the lateral aspect along the femur. The femur was exposed with blunt dissection. A cortical bone defect in 1-mm diameter was made close to the lower end of femur by trephination with MANI needle. Meanwhile, MRSA (1 × 107 CFU mL−1) infected 100 μg of TiO2/GDY, TiO2, TiO2/GO nanofibers or 50 μg of GDY or GO were implanted into the defect on femur and then received UV (365 nm, 2 W cm−2) irradiation for 90 s or not. Afterwards, carefully sutured and closed muscle fasciae and skin. Five days and 4 weeks after surgery, the femurs were harvested after euthanasia by sodium pentobarbital. The samples were ground in sterile PBS (5 mL) for standard plate counting or fixed in 4% PFA for 24 h at room temperature for histomorphological study. Three animal samples were operated for each experiment per group.

Histomorphological analysis

H&E staining and Masson staining were carried out for histomorphological analysis. Fixed femur samples were decalcified in 10% EDTA for 4 weeks and changed twice a week. Decalcified samples were dehydrated with alcohol gradient and embedded in paraffin. Serial sections in 4 μm thickness were mounted on slides. Samples were deparaffinized and rehydrated before subjected to H&E staining (MXB, China) and Masson staining (MXB, China) in accordance with the manufacturer’s protocol. The quantitative analysis of Masson staining was performed by the Image J software on 5 regions of interest.

For immunohistochemical staining, slices were incubated with the primary antibody of OPN (Santa cruz, sc-21742, 1:100) overnight at 4 °C and then stained with immunohistochemical kit (MXB, China) according to manufacturer’s protocol. The images were visualized by the light microscope. The quantitative analysis was performed by counting positive cells in 5 of the observed areas with Image J software.

Statistical analysis

All data were presented as means with standard deviations (SD). The significance analysis between two groups was tested with ANOVA and t test with GraphPad Prism software (6.0).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.



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