OA model establishment in rabbits
This study was approved by the Institutional Ethics Committee of Nanjing First Hospital, Nanjing Medical University (China). All experimental rabbits were provided by Qinglong Shan Animal Breeding Center (China) and were fed standard laboratory food and water and were maintained in a room at the appropriate temperature (25°C, 24 h dark and light cycle). Thirty male New Zealand white rabbits (2.5–3.0 kg) underwent anterior cruciate ligament transection (ACLT) of the right knee[19]. Nine rabbits in the control group underwent a sham operation that did not dissect the anterior cruciate ligament. After surgery, penicillin (200,000U) was delivered via intramuscular injection twice a day for three consecutive days.
Cell isolation and culture
Four weeks after surgery, rabbits cartilage stemming from OA model group and sham operation group were isolated from the surface of the femoral condyle joint and tibial plateau. The cartilages were minced aseptically and then digested with trypsin and type II collagenase (Gibco, Grand Island, NY, USA). After digestion, chondrocytes were seeded in T25 flasks (5 × 106) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin (50U/mL), 10% fetal bovine serum (FBS) and streptomycin and cultured at 37℃ under 5% CO2. At 80–90% confluence, the chondrocytes were subcultured in a six-well plate (2 × 106 cells/well) for use in experiments.
immunofluorescence
OA chondrocytes were identified by immunofluorescence staining of aggrecan (MA3-16888, Thermo Fisher, Shanghai, China), collagen II (Origene, MD, USA) and PPARγ (LifeSpan, East Providence, RI, USA) [20]. Chondrocytes were fixed with 4% paraformaldehyde for 10 min, and then blocked with 1% bovine serum (BSA) for 1 h at room temperature. The chondrocytes were labeled for 2 h at 37℃ with antibodies specific for aggrecan, collagen II and PPARγ at 2 µg/ml in 0.1% BSA. Chondrocytes were then incubated with FITC-conjugated secondary antibodies (Agrisera, Vännäs, Sweden; 1:200) for 1 h at 37℃. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI, Thermo, Shanghai, China) for 5 min at in the absence of light. Total cells were counted following nuclear DAPI counterstaining in ten random fields per culture dish to determine the number of positive cells.
In vitro UTMDSV intervention experiment
For optimization, the chondrocytes (2 × 106) were treated with US at different intensities (0 W/cm2, 0.3 W/cm2, 0.6 W/cm2, 1.0 W/cm2) and different concentrations simvastatin-loaded microbubbles (1 µM, 10 µM, and 100 µM). This preliminary experiment showed that 1 µM simvastatin-loaded microbubbles produced the best effect under the parameters of 1 MHz and 0.3 W/cm2 US irradiation for 30 s. Subsequently, the chondrocytes were treated as follows. (1) Control group - no intervention; (2) US group - US irradiation (1 MHz, 0.3 W/cm2) for 30 s; (3) UTMD group-naked microbubbles under 1 MHz,0.3 W/cm2 US for 30 s; (4) SV group − 1 µM simvastatin; (5) UTMDSV group − 1 µM simvastatin-loaded microbubbles under 1 MHz, 0.3 W/cm2 US for 30 s. All groups were treated once every 2 days for 4 weeks.
PPARγ inhibitor intervention
In the early experiments, we explored the effects of different concentrations of PPARγ inhibitor (T0070907, Selleckchem, Houston TX, USA) on extracellular matrix proteins in chondrocytes. Finally, 12.5 µM T0070907 was selected for use in subsequent experiments[21]. The chondrocytes were seeded in six-well plates (2 × 106 cells/well) and treated as follows. (1) Control group − 0.1% DMSO; (2) PPARγ inhibitor group - treated with 12.5µM T0070907 for 48 h at 37℃; (3) PPARγ inhibitor + UTMDSV group - treated with 12.5µM T0070907 and 1µM simvastatin-loaded microbubbles under US (1 MHz, 0.3W/cm2) for 30 s, and continued cultivation for 48 h at 37℃; (4) UTMDSV group - treated with 1 µM simvastatin-loaded microbubbles under US (1 MHz, 0.3W/cm2) for 30 s and continued cultivation for 48 h at 37℃.
Western blot analysis
Total protein was extracted using lysis buffer (Keygenbio, Nanjing, Jiangsu, China) [4]. After being lysed on ice for 20 min, the lysate was centrifuged (12,000 rpm for 15 min, 4℃) and the supernatant was collected. The protein concentration was analyzed using a bicinchoninic acid protein assay kit (Keygenbio), and the remaining protein was mixed with 5 × sodium-dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) buffer and heated at 100 °C for 15 min. Proteins were then separated by SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF) membranes. All membranes were blocked with 5% skimmed milk at room temperature for 1 h. Membranes were incubated overnight at 4 °C with the following primary detection antibodies: anti-aggrecan (Thermo Fisher, 1:1,000), anti-collagen II (Origene, 1:1,000), and anti-PPARγ (LifeSpan, 1:1,000). Membranes were subsequently washed three times with Tween-Tris buffered saline (TBST) and incubated with secondary detection antibodies (Keygenbio) at 37 °C for 2 h. After washing as described previously, the proteins were visualized using an enhanced chemiluminescence reagent and quantified using the Image 4000 system (Tanon 6600, Beijing, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Abcam, Cambridge, USA) was quantified as an endogenous control protein. Quantification of protein expression in WB is relative value, which is the ratio of target protein expression divided by GAPDH (target protein/ GAPDH).
Cholesterol efflux analysis
The cholesterol efflux from chondrocytes was measured using a fluorometric cholesterol efflux assay kit (Sigma–Aldrich, St. Louis, MO, USA)[22]. Briefly, the chondrocytes were seeded in 96-well plates at a density of 2 × 104 cells/well and allowed to adhere overnight at 37 °C under 5% CO2. After adherence, cells were washed with FBS-free Roswell Park Memorial Institute (RPMI, Sigma–Aldrich) medium without FBS. The cells were then labeled with cholesterol and incubated for 16 h at 37 °C under 5% CO2. Subsequently, chondrocytes were incubated for 5 h (37 °C, 5% CO2) with 100µL of samples acting as cholesterol acceptors; only FBS-free RPMI was added to the blank control wells. The supernatants (medium) were then transferred to a fresh 96-well plate and the fluorescence was measured (Fm, Ex/Em = 482/515 nm) using a SpectraMax M3 microplate reader (Molecular Devices, San Jose, CA, USA). The adherent cells were solubilized by cell lysis buffer to measure the fluorescence (Fc, Ex/Em = 482/515 nm). The cholesterol efflux (%) was calculated according to the following formula: Cholesterol efflux (%) = Fm/(Fc + Fm) × 100% (Fm = fluorescence intensity of the medium; Fc = fluorescence intensity of the cell lysate).
Oil red O staining
The triglyceride level in chondrocytes was detected using an oil red O staining kit (Keygenbio). The chondrocytes in each group were seeded in a six-well plate (2 × 106 cells/well) and incubated for 24 h (37°C under 5% CO2). After washing with PBS, the chondrocytes were fixed in 10% neutral formaldehyde for 30 min, and then stained with oil red O staining solution at 60℃ for 10 min. Cells were exposed to decolorizing solutions A and B twice at room temperature. After re-staining with hematoxylin for 20–60 s, the cells were rinsed with decolorizing solution B and observed under a microscope (Olympus BX43, Tokyo, Japan). Isopropanol solution dissolved cells in each group (500 ul/well), and then transferred to a 96-well plate to detect absorbance at 490 nm using a TECAN Infinite M200PRO microplate reader (Thermo).
In vivo intra-articular injection experiment
To optimize the in vivo experiment, we evaluated different ultrasound intensities (1 W/cm2, 2 W/cm2) and periods (2 min, 5 min) of intervention in the articular cavity of the right knee of the experimental rabbit. The rupture of simvastatin microbubbles was evaluated using ultrasound images, and the optimal ultrasound parameters (frequency of 1 MHz, intensity of 2 W/cm2 for 5 min) were identified. The OA model rabbits were randomly divided into four groups using random number table method (n = 6/group) based on the following treatments: OA group, UTMD group, SV group, and UTMDSV group. The sham operation rabbits were used as a control group (n = 6). The treatments were delivered by injection into the right knee joint cavity as follows: UTMD group - naked microbubbles under US irradiation (1 MHz, 2 W/cm2) for 5 min; SV group − 0.1 mg/mL simvastatin; UTMDSV group − 0.1 mg/mL simvastatin-loaded microbubbles under US irradiation (1 MHz, 2 W/cm2) for 5 min; control and OA model groups - equivalent volume of saline. Each group received 0.2 mL/kg. All groups were treated once every 7 days for 4 weeks and the rabbits were sacrificed by air embolism for histological and WB analyses after treatment.
T-CHO and HDL-C analysis
Synovial fluid was collected from the knee joint of rabbits and placed at room temperature for 30 min before centrifugation at 4,000r for 5 min. The TC and HDL-C level were determined using oxidase-peroxidase coupling (COD-PAP) test kit (JianCheng bioengineering, Nanjing, China). The absorbance was measured at 510 nm and 546 nm using a TECAN Infinite M200PRO microplate reader (Thermo).
Histological analysis
Histological analysis of articular cartilage was performed for all groups[23]. The femoral condyles and tibial plateau of the right knees were fixed in 10% formaldehyde for 3d. And then were sectioned along the sagittal plane, embedded in paraffin and sectioned after decalcification. Sections were stained with fresh Weigert iron hematoxylin for 5 min, and then washed in running tap water. After soaking for 15 s in acid alcohol differentiation solution (1% hydrochloric acid, 70% alcohol), slides were washed with distilled water for 10 min before immersion in fast green stain for 5 min. Next, the sections were washed briefly with 1% acetic acid for 15 s to remove residual fast green stain. Subsequently, sections were stained with safranin-O for 5 min, dehydrated with 95% alcohol, cleared with xylene and finally sealed in optical resin. The following characteristics of the sections prepared from each group were graded according to Mankin’s scoring principles: the tide-line integrity, the number and structure of chondrocytes, and dye intensity. The severity of tissue lesions was graded and standardized as a scoring criterion, with higher scores reflecting greater severity of the lesion. The sections were evaluated independently by two observers using a blinded method and average scores were calculated as the result.
Statistical analysis
The vivo animal studies were randomized, except for the in vitro cell experiments. No samples or animals were excluded from the analysis except rabbits who died early in the process of experiment (30 rabbits in vivo experiments and 9 rabbits in vitro experiments, no animals were excluded). All in vivo data were expressed as the mean and each data point represents an individual rabbit. All in vitro data are expressed as mean with 95% CI. The data displayed normal variance. The independent t-test and one-way analysis of variance (ANOVA) were used to identify the significance of the difference followed by Least significant difference (LSD). All statistical analysis was performed using SPSS software (version 21.0, IBM, USA). P< 0.05 were considered to indicate statistical significance.