Chemicals
Sodium chloride (NaCl), potassium chloride (KCl), dimethyl sulfoxide (DMSO), ethylene glycol((CH2OH)2), and sodium citrate were from Sinopharm Chemical Reagent Co., Ltd. L-ascorbic acid, MTT were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Trypsin-EDTA, penicillin and streptomycin, TRITC-conjugated phalloidin (actin), and 4′, 6-diamidino-2-phenylindole (DAPI) were from yeasen. Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were from Gibco. The alkaline phosphatase assay kit (AKP Microplate test kit) was purchased from Nanjing JianCheng Bioengineering Institute. BCA Protein Assay kit goat serum was purchased from Solarbio (Beijing, China). 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was purchased from the Beyotime Institute of Biotechnology. (Shanghai, China). The total cell RNA kit, total bacteria RNA kit, and total DNA kit were from Omega. PrimeScript RT Master Mix and 2×SYBR Premix Ex Taq II were from TaKaRa.
Synthesis of different samples
First, the Ti6Al4V plates (Ti6) were polished via different grits (240, 400, and 800) of silicon carbide (SiC) sandpapers. Next, the polished plates were washed with absolute ethanol and deionized water for 15 min, respectively. HA (10 mg mL−1), MoS2 (10 mg mL−1), and HA/MoS2 solutions were dropped on the Ti6 surface before vigorous ultrasonication for half an hour. The ratio (w/w) of HA and MoS2 in the HA/MoS2 solution was 50:50, which were called the precoated samples. Following this, the different precoated samples were treated by laser cladding before being dried in an oven at 60 °C for 1 h.
Regarding the laser cladding process, the precoated samples were placed on the holder of the instrument (JHM-1GY-300B; Lumonics). With a wavelength of 1.06 µm, laser cladding was carried out by CW 2 kW Nd: YAG laser, with optimal parameters of laser current = 90 A, pulse width = 2 ms, frequency = 20 Hz, spot diameter = 0.6 mm, and scanning speed = 5 mm s−1. This was the process of preparation of HA–Ti6. After laser cladding, both MoS2–Ti6 and HA/MoS2–Ti6 samples were sulfureted using the CVD method. Specifically, 0.3 g of sulfur powder and the samples were put in a quartz tube filled with nitrogen at 1 atm pressure. Then, the tube furnace was heated to 750 °C at a heating rate of 10 °C min−1 and maintained for 1 h, and then naturally cooled down to room temperature. This was the process of preparation of MoS2–Ti6 and HA/MoS2–Ti6.
Characterization of different samples
SEM images were obtained using a JSM-6510LV and a JEM-2100F microscope (JEOL, Tokyo, Japan). TEM images were recorded by a Talos F200x electron microscope (FEI Co., USA). XRD (D8A25; Bruker, Germany) was used to determine the crystal structure of the samples. XPS (ESCALAB 250Xi; Thermo Scientific, USA) was employed to disclose the surface elemental compositions of the samples. UPS was measured using an ESCALAB 250Xi instrument with a monochromatic He I light source (21.2 eV). An InVia reflex system (Renishaw) operating at 532 nm was employed to obtain Raman spectra. The JC2000D Contact Angle System (POWEREACH, China) was used to measure the water contact angles of the samples at room temperature. A UV-Vis-NIR spectrometer (UV-3600; Shimadzu, Tokyo, Japan) was used to obtain the spectra of samples.
Calculation of CB and VB of MoS2-Ti6 and HA/MoS2-Ti6
The values of CB minimums and VB minimums were obtained through combination of UV-Vis-NIR spectra and UPS spectra. UV-Vis-NIR spectroscopy was used to get the bandgap of material. UPS was used to determine the highest occupied molecular orbital (HOMO) from the Fermi level. Then, we could the predict lowest unoccupied molecular orbital (LUMO) from the observed bandgap. A complete expression of the calculation process of CB and VB was descripted as following:
The work function (φ) could be calculated using Eq. (1): φ = hν − ESEO. Here, hν = 21.20 eV, which represented the energy of the monochromatic ionizing light, while ESEO was the secondary electron onset, which was obtained from the linear extrapolation of the UPS spectrum.
The Fermi level (EF) was obtained from the work function using Eq. (2): EF = −φ.
The position of the VB maximum (EVB) was obtained from Eq. (3): EVB = EF − X, in which X was obtained from the extrapolation of the onsets in the UPS spectrum.
The CB minimum potential (ECB) was obtained from Eq. (4): ECB = EVB + EBG = EF − X + EBG. Here, the bandgap energy EBG was obtained by Tauc plots.
The CB position of MoS2–Ti6 and HA/MoS2–Ti6 was determined by the UPS spectra. The work function of MoS2–Ti6 and HA/MoS2–Ti6 was calculated to be 4.77 and 4.70 eV, respectively, by using the method of a linear approximation to the UPS spectra. The Fermi level of MoS2–Ti6 and HA/MoS2–Ti6 was calculated to be −4.77 and −4.70 eV, respectively. Next, the EVB level of MoS2–Ti6 and HA/MoS2–Ti6 was −5.92 and −5.58 eV separately. The average bandgap energy value was 1.73 eV for MoS2–Ti6 and 1.38 eV for HA/MoS2–Ti6, which was obtained from the Tauc plots. According to Eq. (4), the calculated ECB level of MoS2–Ti6 and HA/MoS2–Ti6 was −4.19 and −4.20 eV, respectively.
Culturing of bacteria
Gram-positive methicillin-susceptible S. aureus (ATCC 25923), Gram-positive methicillin-resistant S. aureus (MRSA) (43300), Gram-negative E. coli (ATCC 8099), and Gram-negative P. aeruginosa (ATCC 15692) were cultured in a sterile Luria–Bertani (LB) medium (10 g L−1 of back to-tryptone, 10 g L−1 of NaCl, and 5 g L−1 of bacto-yeast extract). The bacterial counts were obtained from the spread plate of different samples. P. aeruginosa was a strictly aerobic Gram-negative bacterium51.
In vitro antibacterial experiment
The antimicrobial efficiency of Ti6, HA–Ti6, MoS2–Ti6, and HA/MoS2–Ti6 against E. coli methicillin-susceptible S. aureus ATCC 25923, MRSA, and P. aeruginosa was evaluated by the spread plate method. The samples were sterilized before experiments. The 96-well plates were used to place the specimens separately, and then 200 μL of bacterial suspension (both E. coli, S. aureus, MRSA, and P. aeruginosa) with 2.5 × 107 CFU mL−1 was added into each well, followed by incubation at 37 °C for 6 h (S. aureus), 6 h (MRSA), 6 h or 12 h (P. aeruginosa) or 12 h (E. coli) in an orbital shaker at 200 r.p.m. Ti6 group was used as the control group, while HA–Ti6, MoS2–Ti6, and HA/MoS2–Ti6 groups were separately used as the experimental groups. After incubation, bacterial suspension was diluted 40,000 times for S. aureus and 10,000 times for E. coli with phosphate-buffered saline (PBS). Following this, 20 μL of bacterial suspension was coated on standard LB agar plates and cultivated in a furnace at 37 °C for 24 h to count the number of colonies on the plate. In addition, the LB medium was centrifugated at 12,000 × g for 20 min for further texting oxygen content.
To further illustrate the antibacterial activity, SEM was used to qualitatively examine the bacteria. The samples were fixed with 2.5% glutaraldehyde for 2 h after incubation, and then washed three times with PBS, subsequently dehydrated in turn with different concentrations of ethanol (10, 30, 50, 70, 90, and 100 v/v%) for 15 min each, and then freeze-dried. The bacterial morphologies of different groups were observed by SEM.
For live/dead staining, the samples with S. aureus and E. coli were incubated at 37 °C for 12 h. Then, they were soaked in blended dyes (live/dead baclight bacterial viability kit) in the dark for 15 min, followed by rinsing with PBS. Finally, pictures were taken with a fluorescent microscope (IX73; Olympus, Tokyo, Japan).
For the measurement of MIC, the MIC values of these nanomaterials were obtained using a series of diluted samples. A dilution series of HA/MoS2 (0 mg mL−1, 0.3125 mg mL−1, 0.625 mg mL−1, 1.25 mg mL−1, 2.5 mg mL−1, 5 mg mL−1, 10 mg mL−1, 20 mg mL−1, and 40 mg mL−1) were prepared, in which the steps were identical to the process of synthesis of HA/MoS2-Ti6. S. aureus was diluted to 1.7 × 107 cells mL−1 with LB broth, and 200 μL of the diluted solution was added into the surface of different samples in the 96-well plate, followed by incubation at 37 °C for 6 h. Optical density (OD) was obtained with a microplate reader.
For the measurement of MBC, the MBC values of these nanomaterials were obtained using a series of diluted samples. After the same antibacterial process as MIC, the bacterial suspension was diluted 60,000 times, and 20 μL bacterial suspension was coated on LB agar plates and cultured at 37 °C for 24 h. Finally, MBC was obtained from the number of colonies on the plate.
RNA sequence for S. aureus
S. aureus were cultured with Ti6 and HA/MoS2–Ti6 for 6 h and then S. aureus were collected to extract the total RNA using TRIzol reagent (Invitrogen, CA, USA). RNA sequencing was performed via the HiSeq 4000 SBS Kit (300 cycles; Illumina, CA, USA). Data analysis was performed by FastqStat.jar (v0.11.4) and RSeQC (v2.6.4). Gene Ontology (http://www.geneontology.org) and Kyoto Encyclopedia of Genes and Genome (http://www.genome.jp/kegg/) were used to analyze the gene functions. Differential gene expression analysis was performed using the R package edgeR (v3.24), and those genes conformed to |log2FC | > 1 (p-value < 0.05) were considered to be differentially expressed genes.
Detection of ROS
Fluorescence imaging was performed to investigate the intracellular ROS level. In brief, each sample (including Ti6, HA–Ti6, MoS2–Ti6, and HA/MoS2–Ti6) was placed in 96-well plates and filled with 200 μL of bacterial suspension (5 × 107~108 CFU mL−1 in LB medium or pure LB medium), followed by incubation at 37 °C for 12 h for E. coli and 6 h for S. aureus in an orbital shaker at 200 r.p.m. Next, the medium was removed and the samples were washed twice with PBS. Next, the cells on the sample surface were stained for 30 min in the dark by 200 μL DCFH-DA (10 μM), and then the samples were washed twice with PBS to remove the excess dye. In the following step, an inverted fluorescent microscope (IFM; IX73) was used to observe the samples as mentioned above.
Cell potential of MSCs and S. aureus
DiBAC4(3) was used to assess the cell membrane potential of S. aureus and MSCs. As for MSCs, 104 cells per well were seeded on Ti6 and HA/MoS2–Ti6 surfaces. After culturing for 24 h, the cell growth medium was discarded, and the new growth medium with DiBAC4(3) (2 μM) was added and cultured for 30 min at 37 °C. Following this, the cell potential was assessed via fluorescence images that were obtained via an IFM (570 Olympus, IX73). Concerning S. aureus, 5 × 107 CFU mL−1 S. aureus were added to the Ti6 and HA/MoS2–Ti6 surfaces. After culturing for 6 h, the LB medium was discarded and transferred into a new LB medium with DiBAC4(3) (5 μM) for 30 min at 37 °C. Subsequently, the bacterial potential was assessed via fluorescence images that were obtained through 570 Olympus (IX73).
Cell culture
MSCs were obtained from Tongji Hospital (Wuhan, China). The cells were cultured in a growth medium (minimum essential medium eagle alpha modification: fetal bovine serum: antibiotics penicillin/streptomycin [100 U/mL] = 89:10:1 [v/v]) at 37 °C in a 5% CO2 environment.
Mitochondrial membrane potential of MSCs
A mitochondrial membrane potential assay kit (JC-1; SBJbio life sciences, Nanjing, China) was used to obtain the mitochondrial membrane potential of MSCs. First, 104 cells per well were seeded on Ti6 and HA/MoS2–Ti6 surfaces. After this, the mitochondrial membrane potential of MSCs was evaluated via the instruction manual of JC-1.
Ca2+ fluorescence imaging of MSCs
104 cells per well were seeded on Ti6 and HA/MoS2–Ti6 surfaces and incubated for 24 h. Next, Ca2+ indicator (Flu-3 AM, 5 μM) was added into the fresh medium. And then, the cells were washed with Hanks’ Balanced Salt Solution (HBSS) three times. Finally, the pictures were obtained with IFM (Olympus, IX73) at 488 nm.
Cell proliferation assays
Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Briefly, MSCs (105 cells mL−1) were seeded on the surface of different samples and cultured for 1, 3, and 7 days. Following this, the MTT solution (0.5 mg mL−1) was added to each well and incubated for 4 h to form purple precipitates. Finally, the optical density (OD) of the liquid was tested at 490 nm with a microplate reader after dissolving it with dimethyl sulfoxide.
Osteoblastic differentiation assays
The cell osteoblastic differentiation assays were assessed via ALP, RT-qPCR, and Alizarin Red S (ARS) staining assays. The cells were cultured in an osteogenesis-inducing medium (growth medium with 10 mM β-glycerol phosphate, 10 × 10−9 M dexamethasone, and 50 μg mL−1 l-ascorbic acid). As for the ALP assay, the cells were lysed with 1% Triton X-100 at 37 °C for 2 h after culturing for 7 and 14 days. Finally, ALP activity was evaluated via an ALP assay kit. In addition, a BCA protein assay kit (Solarbio, China) was used to detect the protein content to obtain the ALP activity per unit protein. Regarding RT-qPCR, the cells were collected to extract the total RNA using a total RNA kit after culturing for 14 days. Following this, the total RNA was converted into cDNA through the PrimeScript RT Master Mix. Finally, 2×SYBR Premix Ex Taq II was used to perform RT-qPCR with cDNA using the CFX Connect real-time system. As for ARS staining, the cells were fixed with 4% formaldehyde for 20 min after culturing for 14 days. Next, the cells were stained with 2% Alizarin Red (pH 4.2) for 10 min. Finally, cetylpyridinium chloride in 10% w/v 10 mM sodium phosphate (pH 7.0) was used for further quantitative analysis at 562 nm with a microplate reader.
In vivo biological evaluation
Sprague Dawley rats (300–350 g) were bought from the Beijing Huafukang Bioscience Cojnc. The study was performed following the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The ethical aspects of the animal experiments were approved by the Animal Ethical and Welfare Committee (AEWC) of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (Approval No. YSY-DWLL-2021016). The 64 male rats were separated into eight sections (n = 8 per section), including (1) Ti6 with S. aureus after culturing 2 weeks, (2) Ti6 with no S. aureus after culturing 2 weeks, (3) HA/MoS2–Ti6 with S. aureus after culturing 2 weeks, (4) HA/MoS2–Ti6 with no S. aureus after culturing 2 weeks, (5) Ti6 with S. aureus after culturing 4 weeks, (6) Ti6 with no S. aureus after culturing 4 weeks, (7) HA/MoS2–Ti6 with S. aureus after culturing 4 weeks, and (8) HA/MoS2–Ti6 with no S. aureus after culturing 4 weeks. As for S. aureus infection group, bacterial suspension (20 µL) with a density of 107 CFU mL−1 S. aureus was uniformly coated on the surface of Ti6 and HA/MoS2–Ti6 rods before use. For surgery, pentobarbital sodium salt solution (30 mg kg−1, 1% w/w) was used to anesthetize the rats by injection. Then, the different samples were implanted into the tibia near the knee joint. The rats were fed in the same way, and after 2–4 weeks, they were euthanized via an overdose of chloral hydrate.
Spread plate analysis and histological analysis
To determine the antibacterial efficiency of Ti6 and HA/MoS2–Ti6 rods, the rods (Ti with S. aureus and HA/MoS2–Ti6 with S. aureus) were removed after 2 weeks, rolled on a standard agar plate for four rounds, and then cultured for 24 h at 37 °C. The rods after rolling on a standard plate were plated in small glass vials and then cultured for 24 h at 37 °C. The bacterial colonies and glass vials were photographed on a rolling trace using a digital camera. Meanwhile, H&E and Giemsa staining were used to determine the bacterial contamination of bone-tissue and bone implants after 2 weeks. The H&E and Giemsa staining of samples without bacteria were used as the control group, which was used to assess the influence of bacteria on bone tissue. A fluorescence microscope was used to analyze the histopathological microtomography.
Bone micro-CT and histopathological evaluation
A micro-CT system (USDA Grand Forks Human Nutrition Research Center, Grand Forks, ND, USA) was used to analyze the quantification of gross bone morphology and microarchitecture. To determine the newly formed bone around the implants, the bone volume for each total sample volume (BV/TV) was calculated.
Simultaneously, Safranin-O/Fast Green staining was used to process the samples, which was used to evaluate the osteogenic or chondrogenic differentiation on the surface of implants. The osteogenesis was represented by green while the cartilage was marked by red or orange. The osteogenesis ratio defined the percentage of osteogenesis with a region extending 20 μm from the implant surface. Methylene blue-acid fuchsin staining was used to analyze the mineralized bone tissue (red) around the implant/bone interface. The images were obtained using a fluorescence microscope.
Statistical analysis
All the quantitative data were analyzed by the t-test, one-way ANOVA with Dunnett’s multiple comparison test, or two-way ANOVA with Dunnett’s multiple comparison test. And the data were presented as mean with s.d. Values of *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 were considered statistically significant.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
