Primary cilia support cartilage regeneration after injury

Mice

All mice were maintained under C57BL/6J background in a specific pathogen-free (SPF) facility under a 12/12 h day/night illumination cycle. Gli1-CreER mice, R26RTdtomato mice, and Col1a1(2.3 kb)-GFP mice were used as described previously.39,53 All animals were bred according to the National Institutes of Health’s Guide for Care and Use of Laboratory Animals. All animal studies were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committees of Tongji University, and followed all ARRIVE recommendations (Animal Studies: Reporting of In Vivo Experiments) guidelines.

Growth plate injury model

As described previously, an injury model within the growth plate was established in male wild-type mice.8 Briefly, 4-week-old mice were performed under general anesthesia by isoflurane inhalation. Before the operation, the tibias were shaved, and disinfected by iodophor balls. The joint capsule between the femur and tibia was incised and opened with microsurgical scissors. A 30 g needle was passed through the hypertrophic zone of the proximal tibial growth plate. The control mice used in this study are sham-operated. The joint capsule and skin were closed with a 6–0 suture and a 4–0 suture (Jinhuan Medical, China).

Micro-CT analysis

Dissected tibias were fixed in 4% paraformaldehyde (PFA) for 24–48 h. The tibias analyzed by micro-CT 50 (Scanco Medical, Zurich, Switzerland) at a scan resolution of a 14 μm slice increment with a voltage of 70 kV and a current of 200 μA. One hundred slices of trabecular bone underneath the growth plate were reconstructed for statistical analysis. The parameters of trabecular bone volume/total volume (BV/TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), and trabecular bone space (Tb.Sp) were quantified according to the standard procedures.

Histological assessment

For histological analysis, tibias were decalcified in 10% EDTA (pH 7.4) at 4 °C for 3–4 weeks. After dehydration through a series of graded ethanol concentration, specimens were embedded in paraffin and cut into 5-μm-thick sections. Then sections were deparaffinized with xylene and rehydrated in a descending series of ethanol concentrations. Hematoxylin and eosin (H&E) (Sangon Biotech, Shanghai, China) staining was performed for morphological evaluation. The sections were stained with Safranin O/Fast Green (SO/FG) (Solarbio, Beijing, china) for extracellular matrix analysis in growth plate according to the manufacturer’s protocol.

Immunofluorescence and confocal imaging

For immunofluorescence staining, tibias were embedded in optimal cutting temperature compound (OCT) (Sakura, Taizhou, China) and sectioned at an 8 μm thickness after decalcification. Antigen retrieval was performed using hyaluronidase or 0.1% trypsin; sections were blocked in 5% FBS in PBS-T (0.1% Triton X-100 in PBS), The sections were incubated overnight at 4 °C with anti-type II collagen (COL II) (1:200, Boster Biological Technology, Wuhan, China), anti-aggrecan (ACAN) (1:200, Boster Biological Technology, Wuhan, China), anti-IHH (1:500, Proteintech Group, Wuhan, China), anti-acetylated α-tubulin (1:1 000, Sigma-Aldrich. St Louis, MO), anti-CD73 (1:200, Proteintech Group, Wuhan, China), anti-IFT140 (1:50, Proteintech Group, Wuhan, China) On the second day, the sections were incubated with the appropriate secondary antibody conjugated with fluorophore (1:1 000, Invitrogen, Carlsbad, California). Sections were subsequently stained with DAPI (Sigma-Aldrich. St Louis, MO). All fluorescence microscopy images were acquired using Nikon TI2-E + A1 R confocal microscope (Nikon, Japan) or Eclipse Ni-U microscope (Nikon, Japan). Imaris microscopy Image Analysis Software (Oxford instruments) was employed for counting cell numbers by using spot detection. In brief, DAPI+ cell and EdU+ cell numbers in growth plate were counted by each channel and then calculate the EdU+ cells of growth plate, and methods of quantification of Gli1+ cells and ciliation in growth plate were same. ImageJ software (US National Institutes of Health, United States) was used to quantify the fluorescence area of IHH and COL II within growth plate that followed by standard protocol.

Analysis of LCM-seq data

The counts matrix containing the number of counts for each gene and each sample was obtained from the GEO database (GSE113982).28 We thank Phillip T. Newton et al. for sharing the LCM-seq data of growth plate on GEO database. The analysis was performed using R version 4.1.3. Differential expression analysis was performed using the DEseq2 R package. Differentially expressed genes were collected for the heat map by using pheatmap package. Pathway analysis of differentially expressed genes was analyzed by using Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The ggplot2 package was used to visualize the expression prolife of ciliary genes.

Cell culture

Chondrogenic cell line ATDC5 was cultured as previously described.54 Cells were maintained at 37 °C and 5% CO2. DMEM (Gibco, Shanghai, China) with 5% FBS containing 1% Insulin-Transferrin-Selenium (ITS) supplement (Gibco, Shanghai, China) and vitamin C were gently added to induce chondrocyte differentiation for fourteen days. 4 mmol·L−1 chloral hydrate (Sigma-Aldrich. St Louis, MO) was added to cells to remove primary cilia every two days. 10 nmol·L−1 SAG (Selleck, Shanghai, China) was added to cells during chondrogenic differentiation. Cell pellets were fixed with 4% PFA for 15 minutes and stained with Alcian-Blue Staining Solution (Solarbio, Beijing, China) for 30 min to characterize the chondrogenic differentiation.

Scratch-wound assay

ATDC5 cells were plated into a 24-well culture plate for 24 h. Add medium with 100 mmol·L−1 SAG for one day or 4 mmol·L−1 chloral hydrate for two days. The culture medium was then removed, and the inoculated cells’ surface was scratched with a 10 μL pipette tip and marked the scratch wound. Then they were gently washed with PBS to remove the floating cells and added culture media with 0.5% FBS. Photograph the scratches at 0 h and 12 h. The distance that the cells migrated to the wounded area during this time was measured by ImageJ software (US National Institutes of Health, United States).

EdU cell proliferation assay

EdU (5-ethynyl-2’-deoxyuridine, Beyotime, Shanghai, China) dissolved in PBS was administered to mice twice, at six and three hours before euthanization at the indicated days (500 µg for 4 w per injection). BeyoClick™ EdU-594 or 488 (Beyotime, Shanghai, China) was used to detect EdU in the paraffin section.

Pharmacological manipulation of Hedgehog signaling pathway

For Hedgehog agonist or antagonist experiments, SAG (Selleck, Shanghai, China) or LDE225 (Selleck, Shanghai, China) was reconstituted in DMSO as a stock solution (20 mg·mL-1) and kept in -20 °C until use. SAG or LDE225 was diluted in Sunflower oil (Macklin, Shanghai, China) and intraperitoneally administered at 25 µg/g b.w., once a day for seven days. (total seven doses).7

Quantitative real-time PCR assay

Growth plate was isolated under stereoscopic microscope by using microforceps to remove the articular cartilage and secondary ossification centers. The total RNA was isolated from a growth plate or ATDC5 cells with RNAiso Plus reagent (Life, Shanghai, China). cDNA was synthesized using a First Strand cDNA Synthesis Kit (TaKaRa, Beijing, China). Real-time PCR was conducted with a SYBR Green Master Mix (Yeasen, Shanghai, China). The expression levels of mRNAs were normalized to that of the housekeeping gene Gapdh. Gene-specific primer sequences are listed in Supplementary Table 1.

Flow cytometry

Proximal epiphyses of the tibia were manually dislodged, and attached soft tissues and woven bones were carefully removed using forceps. Then isolated the growth plate structure under stereoscopic microscope by using microforceps to remove the articular cartilage and secondary ossification centers. Dissected epiphyses were minced using microsurgical scissors and incubated with 3 mg·mL-1 collagenase I and 0.2% collagenase II (Sigma-Aldrich) in Hank’s Balanced Salt Solution (HBSS, Sigma-Aldrich) at 37 °C for 30 min. Cells were mechanically triturated using a vortex and filtered through a 70 μm cell strainer (Corning, NY, USA) into a 15 mL tube on ice to obtain a single-cell suspension. After washing, tissue remnants were incubated with collagenase at 37 °C for another 30 min, and cells were filtered into the same tube. Cells were pelleted and resuspended in FACS buffer (Hank’s buffer, 2%FBS, 5 mmol·L−1 EDTA) for flow cytometry. Following standard protocols, the dissociated cells were stained with the following antibodies (1:200, eBioscience, Shanghai, China) to identify the mSSCs, pre-BCSP, BCSP. Allophycocyanin (APC)-conjugated CD31 (390), CD45 (30F-11), Ter119 (TER-119), PE/Cy5-CD90.2 (53-2.1), phycoerythrin (PE)-conjugated CD51 (RMV-7), PE/Cy7-conjugated CD105 (MJ7/18), fluorescein isothiocyanate (FITC)-conjugated CD200 (OX90). Flow cytometry analysis was performed using a four-laser BD LSR Fortessa (Ex. 405/488/561/640 nm, BD, USA) and BD FACSDiva software. Acquired raw data were further analyzed on FlowJo version 10.0.7.

Senescence-associated β-galactosidase (SA-β-Gal) detection

The SA-β-gal assay was performed according to the manufacturer’s protocol (Beyotime, Shanghai, China). Briefly, tibias were embedded in OCT and sectioned at 8 μm thickness after decalcification. The sections were fixed for 15 min and washed with 1× PBS, and then incubated with staining solution overnight at 37 °C.

Growth plate micro-perforation surgery model

4-week-old male Col1a1(2.3 kb)-GFP mice were performed anesthesia and disinfection, and the surgery model was established as mentioned previously.7 Left tibias were operated, while right tibias were untreated and used as an internal control. All mice were randomly divided into administration of DMSO and SAG group. The joint capsule between the femur and tibia was incised and opened with microsurgical scissors. A hole was made in the intercondylar region of tibias using a 26-gauge needle and then remove a cylindrical area of the growth plat in a stepwise manner by endodontic K-files (#20, 25, 30, 35) (Dentsply, PA, USA).

Statistics

Statistical data are presented as the mean ± SD. Comparisons between the two groups were analyzed using a two-tailed, unpaired Student’s t-test. A one-way ANOVA test was used when the data involved multiple group comparisons. GraphPad PRISM v.8.0.1 was used for statistical analysis. P < 0.05 was considered statistically significant.

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