Type 2 collagen

The total protein from chondrocytes or CEP tissue was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China). Protein quantification was performed with the BCA Protein Quantitation Kit (Beyotime, China). Next, the protein was separated on an SDS-polyacrylamide gel before transferring into a PVDF membrane (Millipore, United States). The membrane was then blocked with skim milk, incubated overnight at 4°C with primary antibody, washed 3×, and incubated at room temperature for 2 h with secondary antibody. The antibodies used in this study were collagen type II (1:3,000; Abcam, United Kingdom), aggrecan (1:1,000; Abcam, United Kingdom), MMP-13 (1:3,000; Abcam, United Kingdom), CITED2 (1:500; Santa Cruz Biotechnology), GAPDH (1: 5,000; Abcam, United Kingdom), and goat anti-rabbit secondary antibody (1:5,000; Abcam, United Kingdom). The protein bands were quantified using Image J software and GADPH was used as a loading control.

Aggregates were fixed in 3.7% paraformaldehyde for 2 h at 4 °C in the refrigerator, washed with PBS, and soaked in 3% sucrose overnight at 4 °C in the refrigerator and then embedded in OCT and cryosectioned. The sections (8-μm thick) were stained for GAG with Safranin-O and Alcian Blue. Immunohistochemical staining for extracellular matrix molecules was carried out using the following antibodies: collagen type II (Iowa University), type X (Abcam), fibronectin (FN) (Abcam), N-cadherin (Abcam), Cav-1 (cell signaling), and β-catenin (Abcam). After washing the samples to remove mounting media, the sections were treated for enzymatic antigen retrieval for Col II and Col X. In the case of Cav-1 and FN trypsin antigen retrieval was done prior to staining, and for N-cadherin and β-catenin, visualization heat antigen retrieval was used. Negative controls were established for each antibody by omitting the primary antibodies and using isotype control. For immunofluorescent (IF) visualization after incubation with primary antibodies, samples were incubated with secondary antibodies labeled with Alexa Fluor 594 and 488 (Invitrogen) followed by DAPI nuclear staining.

Mice were sacrificed at 3 weeks of age, hindlimbs were dissected and fixed in either PFA (histology) or 95% ethanol 5% acetic acid (immunohistochemistry) for 48h in 4^°C. The limbs were then decalcified in 20% EDTA pH 7.4 over 2 weeks, wax embedded and cut into 6μm sections. Haematoxylin/eosin (H&E) staining was used to visualise the general morphology of the tissue, using the automated Thermo Shandon stainer.
Immunohistochemistry and BrdU labelling (measurement of cell proliferation) were performed as described previously [30 (link)]. Primary antibodies were used at a dilution of 1:500 (ER stress: BiP, GRP94 and CRELD2 from R&D Systems; PDIA6 from Abcam, p58IPK from Santa Cruz Biotechnology; ECM: type I collagen, type II collagen from Abcam; matrilin-3 from R&D Systems; type X collagen [68 (link)]). BrdU labelled cells were counted using the Watershed algorithm on the Fiji ImageJ platform (National Institutes of Health, Bethesda, Maryland, USA; [67 (link)]) and presented as percentage of total cells in the proliferative zone and On-Way ANOVA was used for statistical analysis of data.

For histology, samples were fixed in 10% formalin for 24 hours, decalcified with Immunocal (Thermo Fisher Scientific), dehydrated in ethanol, embedded in paraffin, and sectioned to 5 μm. Sections were stained for (i) hematoxylin and eosin (H&E), (ii) Alcian Blue with Nuclear Fast Red, (iii) Picosirius Red, and (iv) Movat’s pentachrome. The use of Movat’s pentachrome to evaluate cartilage/bone after implantation is widely reported. The green color indicates the presence of both collagens (staining yellow) and glycosaminoglycans (staining blue by Alcian Blue, which is one of the components of Movat’s stain). All reagents were from Sigma-Aldrich, unless otherwise specified. Immunohistochemistry was performed using the following antibodies: type I collagen, type II collagen, type X collagen, lubricin, and osteopontin (Abcam, Cambridge, MA, USA). Sections were processed according to manufacturer’s instructions. Immunobinding was detected with biotinylated secondary antibodies using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Images were acquired using an Olympus FSX-100 microscope (Olympus, Center Valley, PA, USA).

Formalin fixed hindlimbs were rinsed and decalcified with Cal-X (ThermoFisher) solution for a period of 14 days. Once decalcified, the samples were embedded in paraffin blocks for sectioning. Coronal sections measuring five microns were cut and stained with Hematoxylin & Eosin (H&E) and Safranin O for qualitative analysis and OARSI scoring. Picrosirius red staining was employed to visualize collagen intensity as previously described [40 (link)]. Immunohistochemistry (IHC) was employed to measure macrophage infiltration and activity at 14 days post-injury via CD68 (ThermoFisher) and iNOS (Abcam, Cambridge, UK), respectively. For Day 56 samples, IHC was performed to analyze Type II collagen (Abcam).