Collagen 10

The hydrogels were fixed in 4% paraformaldehyde for 24 h, embedded in OCT compound, and processed using standard procedures. For the immunofluorescence microscopy, 20-µm-thick histological sections were used, which were fixed for 10 min with 4% paraformaldehyde in PBS at room temperature, washed three times with 1 × PBS, and permeabilized with 0.5% Triton-X for 10 min. Sections were washed with PBS and blocked for 45 min in blocking buffer (5% BSA and 0.5% Tween-20 in 1× PBS) containing 10% normal goat serum at room temperature. For immunostaining, sections were incubated overnight at 4 °C with collagen 10 (1:200, Sigma), RUNX2 (1:200, Abcam, Cambridge, UK), chondroitin sulfate (1:100, Abcam), TGF-β2 (1:200, Abcam), and DKK1 (1:200, Abcam). The secondary antibodies were goat anti-rabbit Alexa Fluor 568 and goat anti-mouse Alexa Fluor 488 (Abcam), which were incubated for 1 h at room temperature. Images were acquired using the Cytation 3 Cell Imaging Multi-Mode Reader (Biotek Instruments, Inc., Winooski, VT, USA). An automated detection of the fluorescent intensities was used to analyze the expression.

Chondrogenic pellets were fixed in 4% formaldehyde (Sigma Aldrich) and embedded in paraffin. Sections (3 µm) were stained with Alcian blue 8GX and counterstained with hematoxylin (both from Sigma Aldrich), or stained with alizarin red-S alone. Immunohistochemical staining for collagen 2 (clone 6b3; MerckMillipore Saint-Quentin en Yveline, France) and collagen 10 (Sigma Aldrich) was carried out after antigen retrieval with boiling citrate buffer for two minutes (Dako, Trappes, France) and incubation with primary antibody (1/100) overnight at 4°C. Antibody detection was performed using a goat anti-mouse multiHRP (MerckMillipore) and histogreen (Eurobio-Abcys, Les Ulis, France), sections were counterstained with hematoxylin (Sigma Aldrich).