Air embolization was used to kill 4-week-old New Zealand white rabbits and 8-week-old C57BL/6 mice acquired from Beijing Weitong Lihua Company (Beijing, China). The PLA General Hospital's Independent Ethics Committee authorized all animal experimentation techniques. MFCs from mice and rabbits were isolated and cultured using a prior method[4, 5]. Simply put, the meniscus was taken under sterile circumstances, the lateral synovium and lateral 1/3 of the meniscus were removed, and the medial 2/3 of the meniscus was preserved. The tissue was cut and processed for 6 hours with 0.15 percent Type II collagenase. Following that, the cell suspension was centrifuged (1500r/min for 5 minutes) to collect the main MFCs. Finally, the separated cells were resuspended in Dulbecco's minimal essential medium (DMEM)/F12 (Corning, US) supplemented with 10% fetal bovine serum (FBS) and 100U/ml penicillin/streptomycin solution (Gibco, US) at 37°C with 5% CO2. Every 2–3 days, the culture media was replaced. RAW264.7 macrophage cell lines were grown at 37°C in humidified conditions with 5% CO2 in DMEM (Gibco, US) with 10% FBS and 100U/ml penicillin/streptomycin solution.
Effects of STS on cell viability
Effects of STS on cell viabilities were assessed by Cell Counting Kit-8 (CCK-8). RAW264.7 macrophages (1×104/well) and rabbit MFCs(5×103/well) were seeded in 96-well plates. Cells were treated with various concentrations of STS (0, 5, 10, 20, 40ug/ml) for 24 h. Afterward, 100µl complete culture medium containing 10 µl CCK-8 solution (Dojindo, Japan) was added to each well and incubated at 37℃ in darkness for 4 h. The absorbance at 450nm of each well was detected using a microplate reader (Beckman, USA).
Effects of STS on macrophage repolarization
RAW264.7 macrophages were primed with 100ng/ml LPS (Sigma, US) for 24 hours to exhibit the M1 phenotype in order to assess macrophage repolarization from M1 to M2. To achieve the M2 phenotype, 20 ng/mL interleukin (IL-4) (PeproTech, US) was utilized. Following that, the M1 macrophages were treated with varying dosages of STS for an additional 24 hours. The polarization transitions were studied using quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescent (IF) labeling.
RAW264.7 mouse macrophages conditioned medium (CM) collection and its effect on mouse MFCs.
For 24 hours, RAW264.7 macrophages were stimulated with LPS to show the M1 phenotype and IL-4 to show the M2 phenotype. For an additional 24 hours, the M1 macrophages were exposed to different doses of STS. The supernatants of macrophages were extracted and centrifuged at 1000g for 5 minutes before being stored at -80°C for future research. The CM from developed macrophages was diluted in serum-free media at a 1:1 ratio before being introduced to mouse MFCs for chondrocyte apoptosis investigation.
Effects of STS on IL-1β-induced inflammation, oxidative stress, apoptosis, and ECM degradation in rabbit MFCs.
Rabbit MFCs were treated with or without 10 ng/mL IL-1β and various concentrations of STS (10, 20ug/ml) for 24 h. The effects of STS were evaluated by qRT-PCR, ELISA, western blot, and IF staining.
Quantitative real‑time PCR (qRT-PCR)
TRIzol(Ambion, USA) was used to extract total RNA from rabbit MFCs and RAW264.7 macrophages. A spectrophotometer was used to determine the concentration and purity of the isolated RNA. The RNA was then reverse-transcribed, and the resulting cDNA was amplified using polymerase chain reaction (PCR). PCR experiments (per well: 1 uL CDNA, 0.5 uL forward and reverse primers, 10 uL 2RealStar Green Fast Mixture (with ROX), 8 uL ddH2O) were carried out using an SYBR Green real-time PCR kit (GenStar) and the ArchimedX6 qRT-PCR system. All studies were repeated at least three times, and the amplification signals from individual target genes were standardized to glyceraldehyde3-phosphate dehydrogenase (GAPDH) levels. The primer sequences were shown in Table S1.
For RAW264.7 macrophages, the levels of IL-1β and TNF-α were evaluated using the mouse IL-1β ELISA kit (mlbio, ml301814), mouse TNF-α ELISA kit (mlbio, ml002095-J). For rabbit MFCs, rabbit MFCs were treated with IL-1β (10ng/ml) in the presence or absence of different concentrations (10 and 20 µg/ml) of STS for 24h. The culture supernatants were harvested for the following measurement. The levels of IL-1β, TNF-α, and matrix metalloproteinase13(MMP-13) were evaluated using the rabbit IL-1β ELISA kit (Jianglai, E72059), rabbit TNF-α ELISA kit (Jianglai, E72292), and rabbit MMP-13 ELISA kit (Jianglai, E72146). The measurement was performed in line with the product's instruction booklet.
RAW264.7 macrophages and rabbit MFCs were fixed in paraformaldehyde with 0.1% Triton X-100 (Sigma, USA). To inhibit nonspecific binding, 10% goat serum was utilized. The cells were then treated with antibodies overnight. The cells were treated for 1 hour at room temperature with fluorophore-conjugated secondary antibodies (Alexa Fluor goat anti-mouse 488 or goat anti-rabbit 488, Abcam) at a 1:100 dilution to visualize the relevant subsets. Cells were then stained with 4,6-diamidino-2-phenylindole (DAPI) for 5 minutes. The stained slides were examined under a fluorescence microscope (Nikon, Tokyo). Image J (version 1.5.0, USA) was used to count the number of positive cells. The following antibodies are used: inos(Abcam). CD206(CST). ACAN(Novus). and collagen II (Novus).
Flow cytometry for mouse MFCs apoptosis analysis
Mouse MFCs (106 /well) in a 6-well plate were treated with collected RAW264.7 mouse macrophages conditioned medium for 3 days. Mouse MFCs apoptosis rates were analyzed with annexin V-FITC apoptosis detection kit (Beyotime) according to the manufacturer's instructions.
In the icebox, rabbit MFCs were lysed for 1.5 h with RIPA lysate containing 0.1 percent PMSF. The fractured samples were then spun at 12,000 RPM for 20 minutes at 4°C in a centrifuge. The supernatant is the complete protein solution. The bicinchoninic acid technique was used to determine protein concentration and compute loading volume. The separating gel and concentrating gel were then prepared, and electrophoresis commenced after loading samples in a certain order. After electrophoresis on the gel substrate, the gel was removed and transferred to the PVDF membrane. The membrane and the first antibody (1:1000) were incubated overnight at 4°C. The membrane was incubated with the second antibody (1:3000) at room temperature for 1 hour on the second day. Finally, the gray value is determined after the PVDF membrane has been exposed to a 200 uL ECL solution. The following antibodies are used: Interleukin-1 receptor-associated kinase 4(IRAK4, CST), TNFR-associated factor 6 (TRAF6, CST), p-IκB (Bioss), nuclear factor kappa-B (p-NFκB p65, Bioss), extracellular regulated protein kinases (p-ERK, Bioss), p-p38 (Bioss), c-Jun N-terminal kinase (p-JNK, Bioss), and β-actin (Abcam).
Fabrication of the scaffold
Fabrication of STS delivery MECM based hydrogel hybrid solutions
The method for preparing and identifying inner MECM and outer MECM are shown in the Supplementary Material. STS delivery MECM based hydrogel hybrid solutions were made by dissolving sodium alginate in 10ug/ml STS solution at 2% (w/v) containing 2% MECM particles.
Fabrication of STS delivery PCL-MECM based hydrogel hybrid scaffold
The rabbit meniscus was completely removed from the knee joint. Micro-ct was used to obtain imaging information of the native meniscus. Solidworks 2018 software was used to design a pure PCL scaffold model and export it in STL format. The scaffold was then fabricated by a 3D layer-by-layer fused deposition modeling (FDM) printer (PanoSpace BioPro) using PCL (Mn = 80000, Sigma) (Table S2). Inner MECM based hydrogel hybrid Solutions and outer MECM based hydrogel hybrid solutions of sustained-release STS were injected into the inner and outer sides of the PCL scaffold respectively. After that, the hybrid scaffold was cross-linked for 2 minutes using 102 mM CaCl2 ions.
STS releasing behavior of the hybrid scaffold
In order to measure the STS releasing behavior of the MECM based hydrogel, STS delivery MECM based hydrogel hybrid solutions were poured into a 3mm diameter mold, then crosslinked with 102 mM CaCl2 ions for 2 min. scaffolds were immersed in PBS at 37°C during the test period. At each time point (1 h, 3 h, 6 h, 12 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, and 48 h), 200 ul of supernatant was removed and replaced with an equal volume of fresh PBS. Then the absorbance of supernatants at 265 nm wavelength was determined by UV spectrophotometer and the concentration of STS in the supernatants was calculated according to the standard curve of STS.
Cell viability analysis
To assess the biocompatibility of the scaffolds, a Live Death@ Viability/Cytotoxicity Kit (BioVision, San Francisco, America) was employed. MFCs were added at a density of 5×105/ mL to STS delivery MECM-based hydrogel hybrid solutions before the fabrication of an MFCs-loaded hybrid scaffold. The MFCs-loaded hybrid scaffold was cultured for three days before being stained with live-dead staining according to the product manual. Then, we imaged at 488 and 552 nm with a Leica TCS-SP8 laser confocal microscope (Wetzlar, Germany).
In vivo assessment of the early host response and macrophage polarization of the hybrid scaffold.
Eight Sprague Dawley (SD) rats were used to evaluate the early host response and macrophage polarization of the hybrid scaffolds in vivo. Eight Sprague Dawley (SD) rats were randomly divided into two groups of four rats each. They were PCL-MECM based hydrogel hybrid scaffold (PCL-hydrogel) and STS delivery PCL-MECM based hydrogel hybrid scaffold (PCL-hydrogel-STS10). Following anesthesia, the hybrid scaffold is inserted into the subcutaneous tissue. The rats were killed one week after implantation. Remove the scaffold, along with the surrounding capsule and tissue. Frozen microtomes were used to cut the samples into 5um thick pieces after OCT embedding was completed. The levels of inflammation and macrophage polarization were assessed by hematoxylin and eosin (H&E), immunofluorescence staining (CD206, CST), and immunohistochemical staining (IL-1β, Abcam). For immunohistochemical staining, sections were treated with the first antibodies for an overnight period at 4°C. Sections were then incubated in the second antibody for 1 hour after being rinsed three times with PBS. After being sealed with neutral resins, the portions were exposed to DAB solution and photographed. Dark brown is considered a positive expression. Image J software (version 1.5.0, USA) was used for quantitative/semiquantitative analysis of the results.
In vivo repair
A total of twenty New Zealand white rabbits (male, five months old) were obtained from the Beijing Weitong Lihua Company (Beijing, China). At the PLA General Hospital, the study was approved by the Institutional Animal Care and Use Committee. Twenty rabbits were randomly divided into four groups of five rabbits each (two knees of each rabbit were used). They were the negative control group (negative control), PCL-MECM based hydrogel hybrid scaffold group (PCL-hydrogel-MFCs), STS delivery hybrid scaffold group (PCL-hydrogel-MFCs-STS10), and the positive control group (positive control).
The method for establishing critical-size medial meniscal defect was performed as described in our previous study[4, 45]. Briefly, To expose the internal structure of the knee joint, the patella is rotated laterally. The rabbits were then subjected to an entire medial meniscectomy, with the exception of 5% of the external rim, which was implanted with a scaffold and sutured to the residual rim. MFCs were added into hybrid scaffold at a density of 5×106/ mL in the PCL-Hydrogel-MFCs groups and PCL-Hydrogel-MFCS-STS10 groups. Culture in DMEM for 24h one day before transplantation. The rabbits in the negative control group had a fake procedure, whereas the rabbits in the positive control group merely had meniscectomy. Layer by layer, the wound was sutured. The rabbits were maintained in fixed, individual cages after surgery and were thoroughly monitored for infection and other problems. Air embolization killed rabbits three months later. Meniscus and cartilage samples from the knee were obtained for further examination.
After the animals were killed, the meniscus was fixed in 4% paraformaldehyde for 2 d, paraffin embedding, and cut at approximately 7 µm. Following the manufacturer's instructions, the sections were stained with H&E and toluidine blue (TB). The meniscal regeneration was then estimated using a semiquantitative histological grading method. Three independently trained researchers blindly analyzed all photos in terms of reparative tissue with bonding, the presence of fibrochondrocytes, and safranin O stainability using the Ishida scoring system. The articular surface was examined by fixing osteochondral samples from the femur and the tibia for 48 hours in 4 percent paraformaldehyde and then decalcifying for 7 weeks in EDTA solution. the samples were sliced into 7um slices and stained with H&E. An individual specimen's score was based on the mean value from all of its cartilage sections, according to Mankin scoring criteria.
All data were expressed as means ± standard deviation (SD) for a minimum of n = 3. SPSS 22.0 statistical software was used for the statistical analysis. Statistical significance was indicated by a p-value < 0.05. T-tests were used to compare the two groups. One-way ANOVA was used to compare the three groups.