Ab49945

For immunohistochemical evaluation, primary antibody, rabbit anti-col II (abcam, ab34712), rabbit anti-col I (abcam, ab34710) and mouse anti-col X (abcam, ab49945) were used in the present study. The goat anti-rabbit IgG H&L (HRP) (abcam, ab6721) was used as secondary antibody. The sections were first incubated with 0.4% pepsin (Roche) at 37°C for 1 h for antigen retrieval. The 3% H₂O₂ solution was used to block the endogenous peroxidase, and 1% BSA (Sigma) was applied to block nonspecific protein binding. The sections were first incubated with primary antibody overnight at 4°C, followed by the incubation with secondary antibody for 1 h at room temperature. Then, the color was developed by using the DAB substrate system.

The paraffin sections were baked at 65 °C overnight. Slides were then deparaffinized and rehydrated. Dako endogenous blocking reagent (S2003, Dako, Carpinteria, CA, USA) was then used to quench endogenous peroxidase for 15 min. Non-specific binding sites were blocked with 1:10 normal horse/goat serum (S-2000, Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. Primary antibodies: 1:400 dilution of MMP13 (ab39012, Abcam, Cambridge, UK), 1:1 000 dilution of ColX (ab49945, Abcam, Cambridge, UK); 1:400 dilution of Admts4 (ABT178, Millipore, Billerica, MA), and 1:500 dilution of Adamts5 (ab41037 Abcam, Cambridge, UK) were added, and the slides were incubated at 4 °C overnight. For IHC assays, the secondary biotinylated goat anti-mouse antibody (BA-9200, Vector Laboratories) at the dilution of 1:200 was added for 30 min on the second day, followed by incubation with 1:250 streptavidin (21130, Pierce, Rockford, IL, USA) for 30 min. Positive staining was detected by Romulin AEC Chromagen (Biocare Medical RAEC810L, Concord, CA, USA). For IF staining, an appropriate secondary antibody conjugated to a fluorescence probe was added, incubated at room temperature for 1 h, rinsed in PBS, and mounted in an anti-fading mounting media (Vector Laboratories, Burlingame, CA). Results were obtained using an Olympus BX43 upright microscope (Olympus Optical, Tokyo, Japan).

After micro-CT scanning, samples were then dehydrated in graded alcohol, decalcified. Before sectioning, samples were embedded in wax. Finally, samples were sectioned for H&E, safranin O, and IHC staining ab34712 (Abcam) for collagen II and ab49945 (Abcam) for collagen X according to the vendor’s protocol.

BM-MSC seeded constructs (Day 28) were analysed for DNA and sGAG content. Prior to performing assays, constructs were enzymatically digested with papain (125 ​μg/ml - Sigma) in a buffer containing 100 ​mM Sodium Phosphate (Sigma) with 5 ​mM Na₂EDTA (Sigma) at pH 6.5 as previously described [42 (link)]. sGAG quantification was performed using a DMMB assay as described previously. Quantification of dsDNA in the digested constructs was performed using a Quant-iT Pico Green dsDNA kit (Invitrogen) according to the manufactures protocol. Calcium content of constructs was determined by a calcium liquid colormetric assay as per the kit manufactures instructions (Sentinel Diagnostics). For histological analysis, samples were fixed with 4% paraformaldehyde (Sigma). Samples were dehydrated and wax embedded. Embedded constructs were then sectioned at a thickness of 5 ​μm using a microtome. Sections were stained with 1% Alcian blue to examine sulfated glycosaminoglycan (sGAG) and picrosirius red to examine collagen deposition. To identify the specific collagen types present in the constructs, immunohistochemistry was performed to detect collagen type I (Abcam ab90395 1:400), collagen type II (Santa Cruz-sc52658 1:400), and collagen type X (Abcam ab49945 1:200) as previously described [28 ].