2.10. Determination of antibacterial properties of Eno@MSN-D in vitro
The effects of Eno@MSN-D on bacterial biofilm were observed using a fluorescence inversion microscope system. ATCC25923 was diluted to 1×106 CFU/ml by TSB, and 1 ml was placed into a confocal petri dish with a glass bottom and incubated at 37°C for 24 h. After incubation, the supernatant in the dish was removed, and the biofilm was treated with 5 or 10 µg/ml Eno@MSN-D. The control group included MSN (10 µg/ml) and TSB (10 µg/ml). Petri dishes were then washed with aseptic PBS to remove loosely bound bacteria. Bacteria in the biofilm were stained with a LIVE/DEAD® BacLight bacterial viability kit (L7007) at room temperature for 15 min in the dark. The dye was cleaned with aseptic PBS, and the biofilm was observed using a fluorescence inversion microscope. Bacteria with intact and damaged cell membranes were obtained by scanning under excitation using the green (488 nm) and red (543 nm) channels, respectively.
2.11. Cell viability and osteoclast differentiation assay in vitro
To evaluate the cytotoxic effect of Eno@MSN-D, we used the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's instructions. Briefly, bone marrow macrophages (BMMs) were added to 96-well plates at 8×103 cells/well in triplicate and cultured for 24 h in alpha modification of Eagle’s medium (α-MEM, Gaithersburg, MD, USA) containing 30 ng/mL macrophage colony-stimulating factor (M-CSF; PeproTech, Rocky Hill, NJ, USA), 10% foetal bovine serum (FBS; Gibco-BRL, Sydney, Australia), and 1% penicillin/streptomycin. BMMs were then separately treated with different concentrations of Eno@MSN-D (0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160µg/ml) for 48 or 96h. Next, 10 µL of CCK-8 substrate was added to each well, and the plate was incubated at 37 ℃ under 5% CO2 for 2 h. The absorbance of each well was measured at 450 nm with an ELX800 microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). On the other hand, BMMs were seeded in a 96-well plate at a density of 8×103 cells/well in α-MEM with 30 ng/mL M-CSF, 50 ng/mL RANKL (PeproTech, Rocky Hill, NJ, USA), and different concentrations of Eno@MSN-D (0, 2.5, 5, 10µg/ml). BMMs were supplemented with fresh medium every 2 days until mature osteoclasts were observed. Next, the cells were fixed with 4% paraformaldehyde and stained for tartrate-resistant acid phosphatase (TRAP; Sigma Aldrich, St. Louis, MO, USA) activity. The number of mature osteoclasts (TRAP-positive cells with ≥ 3 nuclei) was counted, and their spread area was measured.
2.12. Bone-targeting properties of Eno@MSN-D in vivo
All animal experiments were performed in the Department of Animal Experimental Sciences of Nanchang University, under the approval and guidance of the Animal Experimental Ethics Committee of the First Affiliated Hospital of Nanchang University. Targeting of common fluorescent MSN and fluorescent Eno@MSN-D was evaluated in Sprague Dawley (SD) rats (Shanghai SLAC Laboratory Animal Co., Ltd. (China). Twelve 3-month-old female SD rats were divided into two groups: MSN control groups and Eno@MSN-D experimental groups. Six animals from each group were injected in the tail vein with identical doses of nanoparticles. After injection, all animals had free access to food and water. The animals were euthanised after 4 and 72 h, and the main organs (heart, liver, spleen, lung, kidney, femur) were removed. A fluorescence imaging system was used to detect the fluorescence of each organ in each group.
2.13. Antibacterial properties of Eno@MSN-D in vivo
Fifty specific pathogen-free-grade 12-week-old female SD rats were used and randomly assigned to five independent groups. All animals were housed in clean plastic cages with a 12h light dark cycle and free access to fresh food and water. Five groups of rats were anaesthetised by intraperitoneal injection with 10% chloral hydrate (4 ml/kg). After complete anaesthesia, the supine position was used to remove hair from the left knee joint, subsequently sterilised with 75% ethanol. A 15 mm incision was made along the lateral end of the femur. Subcutaneous tissues and muscles of the lateral femoral condyle were incised, the joint capsule and lateral collateral ligaments were retained, and the femoral condyle was fully exposed. The bone marrow cavity of the femur was opened and expanded to a depth of 10 mm using an electric drill with a diameter of 1 mm. Subsequently, a 1-mm diameter titanium rod with a length of 10 mm was implanted. ATCC25923 concentration was set to 1×106 CFU/ml, and 100 µl was injected into the bone marrow cavity. The hole in the femoral condyle was blocked with bone wax. A saline solution was used for flushing the wound, and a medical suture was used to close the wound. Berberine was then applied to the wound. In the Sham group, only the condyle of the femur was exposed before closing the incision. After the operation, rats were resuscitated under a fan heater and put back in their cages. The rats were kept in separate cages and could eat and drink at will.
In the first week post-operation, body temperature and weight were examined every day. After one week of observation, each experimental group was injected with different drugs. Group A: Sham group (4 mg/kg of normal saline was injected); group B: NS group (no treatment); group C: MSN group (4 mg/kg of MSN was injected); group D: Eno group (4 mg/kg of enoxacin was injected); group E: Eno@MSN-D group (4 mg/kg of Eno@MSN-D was injected). Then, body weight and temperature were recorded every three days. The drugs were then intraperitoneally injected every day for a total of four weeks, and the animals were euthanised four weeks later. The femurs were separated from the skin and subcutaneous tissue under aseptic conditions. Soft tissues were removed, and the femurs were prepared for further experiments. All titanium rods were collected and processed for analysis.
Bacteria attached to titanium rods were detected by SEM, fluorescence staining, plate colony counting method, and plasma coagulase test. After the titanium rods were removed from the distal femur and washed with PBS, five bars of each group were randomly placed in 2.5% glutaraldehyde for 12h. Subsequently, they were dehydrated with an ethanol series (50, 60, 70, 80, 90, 100%). The samples were freeze-dried and gold-sputtered. The surfaces of the titanium rods were then observed by SEM. Five titanium rods were randomly selected and fixed for 12h according to the steps above. After washing with PBS, they were stained with a LIVE/DEAD ®BacLight bacterial viability kit (L7007) at room temperature for 15 min in the dark. They were then observed using a fluorescence inversion microscope. Meanwhile, titanium rods were washed, put into 1 ml of TSB, and ultrasonicated for 15 min. After a 10-fold dilution, 100 µl of suspension was evenly applied to a TSA plate and incubated at 37°C for 24 h. Then, the CFU was calculated according to the colony count on the plates. Additionally, a plasma coagulase test was performed to determine whether the bacteria attached to the titanium rods were S. aureus. A single colony was picked from the TSA plate and suspended in 50 µl PBS before adding 50 µl rabbit plasma. The occurrence of agglutination indicated that the bacteria were S. aureus.
2.14. Micro-computed tomography
After euthanasia, the femur of each group was removed entirely under sterile conditions. After the titanium rod was removed, the femur was fixed with 4% paraformaldehyde for two days and washed with tap water for 24 h. The peripheral bone structure of the distal femoral implant was then evaluated 28 days after the injection of different drugs. The femurs were examined using a desktop micro-X-ray computed tomography (micro-CT Skyscan1076, Aartselaar, Belgium) machine equipped with a 40 kV X-ray source with a camera pixel size of 12.60µm. A reconstructed data set with an image pixel size of 18.26 µm was generated via scanning. To determine the axial trabecular volume of interest, we selected a region of interest (ROI) with a length of 5.578 mm closest to the growth plate edge. Micro-CT images of the transverse, sagittal, and coronal sections of the area around the implants were obtained. Object volume, total VOI volume, bone evolution fraction, bone mineral density, trabecular thickness, trabecular spacing, and trabecular number were used as indices to measure trabecular bone mass and its distribution.
2.15. Histology and histomorphometry
Bone histology was used to assess infection and bone structure changes around the distal femoral implants. The femurs of the rats were fixed with 4% paraformaldehyde at room temperature for 24 h. Subsequently, 10% of ethylenediamine tetra-acetic acid was fully decalcified and dehydrated with an ethanol series (50, 75, 80, 85, 90, 95, 100%). The bone tissue, after transparent treatment in xylene, was embedded in paraffin. The sample was longitudinally cut into 5-µm-thick slices. After slicing, histological sections were prepared for TRAP and H&E staining. Slices were observed with an optical microscope. Representative images were randomly obtained from the distal femur implanted with titanium rods. We used Image-Pro Plus 6.0 software (Media Cybernetics, MD, USA) to process TRAP-stained pictures and counted the number and area of osteoclasts per field of view.
2.16. Statistical analysis
IBM SPSS statistics 22 (SPSS Inc., USA) software was used for statistical analysis. The results are presented as means ± SD. Experiments were conducted at least three times. One-way analysis of variance (ANOVA) with Bonferroni post hoc test was performed to determine group differences. p-values < 0.05 were considered statistically significant.