Synthesis of PBNPs
PBNPs were prepared by adding 495 mg K3Fe(CN)6 (SCR) and 5 g PVP (K-30, Urchem) to a 40 mL 1M HCL solution, and stirred vigorously until the solution was clarified. It was put at 80℃ for 20 h, centrifuged at 1400 r/min for 15 min and the supernatant was discarded. Then the material was cleaned with double stilled water using an ultrasonic cleaning machine and centrifuged again. The materials were mixed up with 5 mL saline for storage.
Characterization of PBNPs
The particle size and morphology of the PBNPs obtained above were characterized by transmission electron microscopy (TEM, FEI TECNAI G2 F20, FEI, USA), scanning electron microscopy (SEM, FEI Nova Nano 450, FEI, USA) and X-ray diffractometer (PANalytical X'Pert PRO, PANalytical, Holland). Both the average size and the average zeta potential of PBNPs were measured by using a Nano-size potentiometer (Zetasizer Nano ZS90, Malvern, UK).
New Zealand rabbits were purchased from Chongqing Medical University and sacrificed after air injection through the ear vein. The knee articular cartilage was separated under aseptic conditions; the fascia and cartilage membrane of the wrapped cartilage tissue was stripped off and placed in a dish containing PBS solution. The isolated cartilage tissue was cut into blocks of 0.3-0.5 mm, transferred into a 25 cm2 culture flask and washed with PBS solution containing double antibodies 3 times. 0.25% of trypsin was added at a dosage of 10-15 times the volume of cartilage, and digestion was terminated after being treated at 37°C for 1-2 h. Cartilage tissue was incubated with 0.02% type II collagenase at 37°C overnight. Cells were observed using an inverted microscope. In the absence of single cells, DMEM medium was added to dilute the type II collagenase to terminate digestion. The cell mass was blown into a single cell and then filtered with a 200 mesh sieve to collect the filtrated. Next, the filtrated was centrifuged at 1500 r/min for 5 min. After that, the supernatant was discarded, and cells were cultured with DS medium at 37℃ with 5% CO2. All the animals were euthanatized according to the standards of the Ethics Committee of Chongqing Medical University.
Twenty-five male New Zealand rabbits (11 months old, 3.0-3.5 kg) were obtained from Chongqing Medical University and housed in individual cages with a cycle of 12 h of light and 12 h of darkness at 20-25℃. To construct the KOA model, the anterior cruciate ligament was transected from the right knee of 20 rabbits under general anesthesia (3% pentobarbital, 1 mL/kg) by using ophthalmic scissors as previously described . The other 5 rabbits served as controls. After postoperative wk 1, rabbits were induced to move for 30 min daily for 5 d per wk for 7 wk to promote OA development.
After 4 wk of OA induction , 20 rabbits with KOA were randomly divided into 4 groups including KOA (n=5), KOA+PBNPs (120 μg/mL article injection once a week for 6 weeks, n=5), KOA+LIPUS (n=5) and KOA+PBNPs combined with LIPUS (n=5) groups. Rabbits in all groups were sacrificed by air embolization at 6 wk after each intervention. All experiments were approved by the Animal Management Rule of the Chinese Ministry of Health and the Chongqing Medical University Animal Ethics Committee.
The femoral condyle articular cartilages from knee joints were collected 6 wk after ACLT and observed by naked eyes. The criterion of cartilage injury grading was referred to as Outerbridge’s grading standard  (Table 1).
After general observation, the specimens were fixed with neutral formalin and processed for histopathologic examination. The samples were decalcified in ethylenediaminetetraacetic acid for 3 wk, embedded in paraffin, and sliced into 4 μm sections using a microtome for microscopic examination. Pathologic changes of knee joint cartilage specimens were observed under a microscope, including surface irregularity, crack formation, and decrease of Safranin O staining of articular cartilage. Modified Mankin scoring scale (Table 2) was used to evaluate fibrosis, matrix distribution, cartilage loss, and chondrocyte colonization. Microscopic evaluations were performed by two independent experts in a double-blind fashion.
LIPUS (Osteotron Ⅳ, ITO Physiotherapy&Rehabilitation) was applied to chondrocytes and rabbits with KOA. LIPUS (acoustic intensities of 30 mW/cm2, 45 mW/cm2 and 60 mW/cm2, duty ratio 40%, central frequency 1.5 MHz, repetition frequency 1 kHz and operating time 20 min per day) was applied to the chondrocytes after being cultured 24 h at the bottom of the culture dish.
As for the rabbits with KOA, LIPUS was administrated to the right knee as follows: acoustic intensities of 60 mW/cm2, a duty ratio of 20%, central frequency of 1.5 MHz, repetition frequency of 1 kHz, the irradiation time of 20 min per day, and 5 days per week for 6 wk. The process was standardized with a device that the rabbits were placed in a supine position with the knee angled approximately 120 at the flexion position. The ultrasound probe was attached to the skin of the medial femoral condyle, and the target tissue was cartilage of the medial femoral condyle.
Cell death assessment
The effects of PBNPs on cartilage cell death were determined by Cell Counting Kit-8 (CCK-8) colorimetric assay (Sigma, USA). Briefly, podocytes were seeded into 96-well plates at a density of 104 cells per well and cultured in complete RPMI-1640 culture medium for 24 h. Then, the cells were treated with PBNPs at a concentration of 0, 30, 60, 120, 150, and 180 μg/mL respectively for 24 h and 120 μg/mL for 1, 2, 3, 4, 5, 6 and 7 days. CCK8 was added to each well and the cells were incubated at 37°C with 5% CO2 for 2 h. Absorbance was quantified at 450 nm using a multi-well fluorescent plate reader (Thermo Scientific Varioskan Flash, Thermo Fisher Scientific, USA). The rate of cell death was calculated.
Flow cytometric analysis
The treated cells were collected by centrifugation at 1000 r/min for 3 min, washed twice with ice-cold PBS, gently resuspended in 500 μL 1×Annexin V binding buffer containing 5 μL Annexin V-FITC and 3 μL of PI before being incubated at room temperature in the dark for 10 min. The percentage of apoptotic cells was analyzed by flow cytometry (BD FACSCalibur, Becton-Dickinson, USA).
Cell apoptosis was determined using the One-Step TdT-mediated dUTP Nick-End Labeling (TUNEL) Apoptosis Assay Kit (Beyotime, China). Pretreated cells in 12-well plates were washed twice with PBS, incubated with 50 μL TUNEL testing solution at 37℃ for 1 h in the dark, and then washed with PBS 3 times. After the membrane was sealed with anti-fluorescence quenching solution, the cells were observed at an excitation wavelength of 550 nm and the emission wavelength of 570 nm using a fluorescence microscope (Zeiss Fluorescence Microscope, Germany).
The pretreated cells in 12-well plates were incubated with 2’, 7-dichlorofluorescein diacetate (DCFH-DA) (10 μmol/L) at 37°C for 30 min and washed with PBS. The cell fluorescence was observed by using a fluorescence microscope (Zeiss Fluorescence Microscope, Germany) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.
Western blot analysis
Proteins extracted from cartilage cells were collected after treatment. The cells were lysed with RIPA (Beyotime, China) and PMSF, sonicated (noise-isolating tamber, Ningbo Scientz Biotechnology Co., Ltd.) for 12 s (20% power, 1-s pulse on, and 2-s pulse off), and centrifuged at 12,000 g for 15 min at 4℃. Protein concentrations were quantified with the BCA Protein Assay Kit (Beyotime, China). An equal volume of protein samples was loaded per lane and electrophoretically transferred onto PVDF membranes (Millipore, USA). The membranes were blocked in 5% skimmed milk for 1.5 h at room temperature and incubated with different primary antibodies: rabbit anti-cleaved caspase-3 (CST, USA), rabbit anti-Bcl2 (1:1000, CST, USA), rabbit anti-Bax (1:1000, CST, USA), rabbit anti-p-PI3K (1:1000, CST, USA), rabbit anti-PI3K (1:1000, CST, USA), rabbit anti-p-Akt (1:1000, CST, USA), rabbit anti-Akt (1:1000, CST, USA), rabbit anti-p-mTOR (1:1000, CST, USA), rabbit anti-mTOR (1:1000, CST, USA), rabbit anti-IL-1β (1:1000, Proteintech, USA), rabbit anti-MMP3 (1:1000, CST, USA), rabbit anti-MMP13 (1:1000, CST, USA), rabbit anti-p-JINK (1:1000, CST, USA), rabbit anti-JINK (1:1000, CST, USA), rabbit anti-p-c-Jun (1:1000, CST, USA), rabbit anti-c-Jun (1:1000, CST, USA), and β-actin ( 1:5000, Sungene Biotech, China) overnight at 4°C. After being washed with TBS-T, the membranes were incubated either with goat anti-mouse IgG (H+L) horseradish peroxidase (1:8000, MultiSciences, China) or goat anti-rabbit IgG (H+L) horseradish peroxidase (1:8000, MultiSciences, China) for 1 h at room temperature and incubated with ECL reagent (Advansta, USA). Protein blot images were captured using an ECL chemiluminescence system (GE Healthcare, Piscataway, NJ, USA) and quantified with Quantity One software.
Data were presented as mean±SD from 3 independent experiments. Analyses were carried out by using GraphPad Prism Software version 6.00 (San Diego, CA). The data complied with normal distribution and homogeneity of variance. Statistical comparisons between the two groups were analyzed using the two-tailed unpaired Student’s t-test. Differences among multiple groups were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey’s t-test. The value of P less than 0.05 was considered statistically significant.