Sc 52658

The specimen sections were analyzed by immunohistochemical methods to determine the level of IL1β (Abcam, San Francisco, CA, USA, Ab-9787, 1:200), MMP13 (Abcam, ab75606, 1:100), type II collagen (Santa Cruz Biotechnology, CA, USA, Sc-52658, 1:100), SOX9 (Abcam, ab26416, 1:100), BMP2 (Abcam, ab6285, 1:100), BMP5 (ThermoFisher Scientific, Cleveland, OH, USA, PA5-97037, 1:100), and BMP6 (ThermoFisher Scientific, Cleveland, OH, USA, PA5-75427, 1:100). The harvested specimens were fixed in 4% PBS-buffered formaldehyde for 2 days and decalcified in PBS-buffered 10% EDTA solution for one month. Decalcified paraffin-embedded samples were cut into 5-μm-thick sections and transferred onto polylysine-coated slides. Sections of the specimens were immunostained with antibodies to identify the protein markers. The immunoreactivity of the specimens was demonstrated using a horseradish peroxidase (HRP)-3′-,3′-diaminobenzidine (DAB) cell and tissue staining kit (R&D Systems, Minneapolis, MN, USA). The immunolabeled positive cells were quantified from five areas in three sections of the same specimen using a Zeiss Axioskop 2 plus microscope (Carl Zeiss, Berlin, Germany). All images were captured using a Cool CCD camera (SNAP-Pro c.f. Digital kit; Media Cybernetics, Carlsbad, CA, USA), and data were analyzed using Image-Pro^® Plus image analysis software (Media Cybernetics, Carlsbad, CA, USA).

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 ].

For the cell immunofluorescence assay, cells were fixed in 4% PFA, permeabilized with 0.3% Triton X‐100 for 15 min, and blocked with 1% bovine serum albumiin (BSA) for 30 min. After that, cells were incubated with primary antibodies against COL2A1 (1:100, Santa Cruz, sc‐52658), SOX9 (1:100, Abcam, ab76997), and COL1 (1:200, Affinity, AF7001) overnight at 4 °C. Then, cells were washed and incubated with secondary antibodies Alexa Fluor 488 (1:1000, Invirogen, A21202) or Alexa Fluor 546 (1:500, Invirogen, A11035) for 2 h in the dark. After counterstaining with DAPI, images were taken by confocal microscopy (Olympus) and fluorescence was quantified by ImageJ software.