Materials and reagents
Gelatin from porcine skin (type A) was purchased from Sigma-Aldrich (St. Louis, MO) and used as received. Amine-terminated G5 PAMAM dendrimers were purchased from Dendritech (Midland, MI). Sodium periodate, dicyclohexylcarbodiimide, and N-hydroxysuccinimide were purchased from Macklin Reagent Co. (Shanghai, China). Adipic dihydrazide and Lipofectamine 3000 were obtained from Invitrogen (Carlsbad, California). MiR-140 and Cyanine5 (CY5)-miR-140 were designed and synthesized by GenePharma (Shanghai, China). RNase A and RNase inhibitor were purchased from Beyotime Biotechnology (Shanghai, China). All other chemicals were used as received without further purification.
Preparation and characterization of the G5-AHP/miR-140 complexes
G5-AHP dendrimers were synthesized as previously reported37. The purified product of G5-AHP was freeze-dried and characterized by 1H NMR in D2O (Avance III, Bruker 600 MHz, Germany). The complexes of G5-AHP/miR-140 were prepared by mixing G5-AHP and miR-140 at different N/P ratios. The N/P ratio was calculated according to the cationic groups (N number) on the G5-AHP surface to anionic phosphate groups (P number) of miR-140. All the G5-AHP/miR-140 complexes were incubated for 10 min at room temperature followed by characterization and activity evaluation. The morphology of the G5-AHP/miR-140 complex was observed by TEM (Hitachi, HT7700), and their particle size and zeta potential at N/P ratios of 0.5:1, 1:1, 2:1, 4:1, and 8:1 were determined on a Malvern Zetasizer Nano ZS90 (Malvern, UK) at 25 °C. The binding capacity of miR-140 and the G5-AHP dendrimer was estimated by agarose gel (Biowest, Spain) electrophoresis. G5-AHP/miR-140 complexes (N/P ratios of 0:1, 0.25:1, 0.5:1, 1:1, 2:1, and 4:1) were electrophoresed on a 2% (w/v) agarose gel at 100 V for 20 min. The miR-140 in the gels was stained with ethidium bromide and visualized under UV illumination (Tanon-2500, China). For the RNase A protection assay, G5-AHP/miR-140 complexes at an N/P ratio of 1:1 were incubated with RNase A at final concentrations of 5, 10, and 15 ng/µl. Afterwards, RNase inhibitors were used to eliminate the effect of RNase A. Then, the solutions were further analyzed using agarose gel electrophoresis as described above.
Fabrication of hydrogel MS and MS@G5-AHP/miR-140
Methacrylic-modified gelatin (GelMA) was synthesized as previously reported45. In brief, methacrylic acid was added dropwise to the gelatin solution (dissolved in PBS), and the reaction solution was stirred for 1 h at 50 °C. After that, 5× dilution PBS was added, and the mixture was extensively dialyzed against distilled water for 1 week at 40 °C. The purified GelMA porous foam was prepared by the freeze-drying method and stored at –80 °C. GelMA MSs were prepared using a modified microfluidic flow-focusing device46,47. Briefly, GelMA (5 wt%) and photoinitiator (0.5 wt%) were dissolved in PBS as the aqueous phase, and paraffin oil containing 5 wt% Span80 was used as the oil phase. They were injected into inlets of the microfluidic device from syringes. The flow rates of both the aqueous phase and oil phase were controlled by a syringe pump (Lead Fluid, China). The generated emulsion droplets were irradiated for 10 min by UV light (254 nm). The MSs were collected into 50 ml centrifuge tubes, suspended for 10 min to remove the paraffin oil as much as possible, and then washed with acetone and PBS three to five times to remove the paraffin oil and other additives. The GelMA MSs were then freeze-dried to absorb G5-AHP/miR-140 solutions, forming the MS@G5-AHP/miR-140 microgels.
Physical characterization test of MS@G5-AHP/miR-140
(I) BM: the particle size and morphology of MSs and MS@G5-AHP/miR-140 in the swollen state were measured by BM (LSM800, Zeiss, Germany).
(II) SEM: MSs and MS@G5-AHP/miR-140 were frozen at –80 °C and freeze-dried. The sample was gold-sputter coated for 30 s before being investigated under SEM (FEI USA).
(III) LSCM: FITC-conjugated GelMA was used to obtain fluorescent dye-labeled GelMA MSs. FITC-MSs and G5-AHP/CY5-miR-140 were colocalized using LSCM.
Chondrocyte culture and tensile strain loading
Chondrocytes were acquired from the knee articular cartilage of mice as previously reported48. Briefly, articular cartilage tissues were cut into small pieces (<1 mm3) and digested with 0.25% trypsin and 0.2% type II collagenase. The cells were obtained and cultured with DMEM/F12 media containing 10% fetal bovine serum (FBS) and antibiotics. Once the cells reached 80% confluence, they were subjected to cyclic tensile strain with a 0.5 Hz sinusoidal curve at 10% elongation for 24 h using a Flexcell1 FX-5000™ Tension System (Flexcell International Corporation, Burlington, NC).
In vitro cell transfection
Chondrocytes were seeded in 24-well plates and cultured for 24 h to reach 80% confluence. The G5-AHP/miR-140 complexes mixed with 0.33 µg CY5-miR-140 were diluted in 200 µl of DMEM/F12 medium and incubated at room temperature for 10 min. Afterwards, the cells were incubated with the complex solution for 6 h and then further cultured for 48 h with DMEM/F12 medium containing 10% FBS and antibiotics. The expression of CY5- miR-140 in the cells was observed by LSCM (LSM800, Zeiss, Germany) and accurately analyzed by flow cytometry (BD FACSCalibur, San Jose). Cells transfected with Lipofectamine 3000 were used as positive controls.
Quantification of mRNA and qRT-PCR
Total RNA from chondrocytes was extracted using TRIzol reagent (Invitrogen, Carlsbad, California) as previously reported48. One microgram of total RNA was used to synthesize cDNA using a RevertAid First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). The amplification of cDNA was performed by the SYBR Premix Ex Tag Kit (TaKaRa, Dalian, China). The primer sequences used in this study are shown in Supplementary Table S1.
Western blotting analysis
Western blot analysis was accomplished as previously reported49. Briefly, proteins were fractionated by SDS-PAGE (7.5–12.5% polyacrylamide gels) and transferred onto PVDF membranes (Millipore, Bedford, MA). The membranes were incubated with primary antibodies overnight at 4 °C. The horseradish peroxidase-conjugated secondary antibody (1:5000) was used to react with the corresponding primary antibody for 1 h. Antibody reactivity was observed using the Enhanced Chemiluminescence Western blot System (Amersham Biosciences). Image Processing and Analysis in Java (ImageJ) software was used to quantitatively evaluate the gray values of each band.
Immunofluorescence staining
The chondrocytes were fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton X100 for 15 min. Subsequently, the cells were incubated with mouse antiCOL2 antibody (1:100 dilution), anti-MMP13 antibody (1:100 dilution), and anti-ADAMTS5 antibody (1:100 dilution) at 4 °C overnight. The cells were washed with PBS three times and then incubated with appropriate Alexa Fluor-coupled secondary antibodies (Invitrogen, USA, 1:400) for 1 h. The cell nuclei were stained with 4’,6diamidino2phenylindole dilactate (Beyotime Biotechnology, China) at room temperature for 15 min. Finally, Alexa Fluor 594 phalloidin (Life Tech, USA) was used to label actin. The images were obtained by LSCM (LSM800, Zeiss, Germany).
CCK-8 tests
Chondrocytes were seeded in 12-well plates at a cell density of 4 × 104 ml−1 and cocultured with G5-AHP/miR-140 and MS@G5-AHP/miR-140 using Transwell chambers for 1, 3, and 5 days. All experiments were performed in triplicate. The Cell Counting Kit8 assay (Dojindo Laboratories, Kumamoto, Japan) was applied to investigate the cytotoxicity of MS@G5-AHP/miR-140 on chondrocytes. Briefly, 100 µl of WST-1 cell proliferation reagent was added to each well followed by incubation for 2 h at 37 °C. Afterwards, the mixed medium was transferred to 96-well plates. The optical density value of the samples was measured on a microplate reader (Infinite F50, Tecan, Switzerland). The absorbance value of the control group cells was defined as 100% survival.
Live/dead staining
The cell biocompatibility of MS@G5-AHP/miR-140 was measured by a Live/Dead Cell kit (Life Tech, USA). The cells were stained with 500 µl of live/dead staining solution for 15 min and then observed using LSCM. Green fluorescence indicates viable cells with esterase activity, whereas red fluorescence shows dead cells. The live cell numbers were quantitatively analyzed using Image Processing and Analysis in Java (ImageJ) software.
In vitro MMP-responsive tests
(I) GelMA MSs were treated with or without 10 ng/ml MMPs, and the morphology of MSs at 1, 3, 5, and 7 days was assessed by BM.
(II) MS@G5-AHP/CY5-miR-140 was treated with or without 10 ng/ml MMPs. Briefly, 50 µl of the solution was transferred to 96well plates away from light after treatment for 1, 3, 5, 7, and 14 days. The fluorescence intensity of each sample was measured on a microplate reader (Infinite F50, Tecan, Switzerland). The CY5-miR-140 contents proportional to fluorescence intensity were calculated according to the standard curve established between CY5-miR-140 content and G5-AHP/CY5-miR-140 fluorescence intensity.
(III) Coculture of primary mouse chondrocytes with MS@G5-AHP/CY5-miR-140 using Transwell chambers was divided into two groups in triplicate. The Transwell + MMP group was treated with 10 ng/ml MMPs. The expression of CY5-miR-140 was visualized by LSCM after two days. The control group was treated only with G5-AHP/miR-140.
Animal experiments
DMM surgery was applied to generate an animal model of OA in male C57 mice (8 weeks old; n = 25)50. After anesthetization, a medial articular approach was used to expose the arthrosis, and then, the quadriceps was gently turned laterally to avoid damaging the patellar ligament. Afterwards, the medial meniscus was dissociated, and the medial meniscus ligament (MMTL) was transected in a manner that did not injure the articular cartilage. Finally, the medial capsular incision was sutured after restoring the quadriceps well and closing the skin. Sham operations involving exposure of the joint capsule without MMTL transection were performed as a nonoperated control. The model mice were randomly divided into the PBS, MS@G5-AHP/miR-NC, G5-AHP/miR-140, and MS@G5-AHP/miR-140 groups (n = 5 for each group) and intraarticularly injected once per fortnight with PBS, MS@G5-AHP/miR-NC, G5-AHP/miR-140, or MS@G5-AHP/miR-140. All animal experiments were performed according to the protocol approved by the Animal Research Committee of Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, China, which complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Radiological evaluation and histological staining analyses
Radiographical evaluation of mice with OA was conducted at 1 and 12 weeks after surgery, and digital plain radiographs of the morphology of mouse knees were acquired by an MX-20 Cabinet X-ray System (Faxitron, Tucson, AZ, USA). SkyScan1172 high-resolution micro-CT (Bruker, Kontich, Belgium) was applied to scan knee joints as previously described51. Reconstructed and three-dimensional modeled data were obtained for further analysis. The samples were then dehydrated and embedded in paraffin. Six-micrometer sections were obtained by cutting tissues longitudinally. HE staining, toluidine blue staining and safranin Ofast green staining were used to analyze histological changes and proteoglycan content as reported previously52.
