Tissue tek

For histology and immunohistochemistry (IHC), unfixed chondrocyte‐laden alginate beads were covered in Tissue‐Tek (Thermo Fisher), snap frozen in liquid nitrogen, and cut in 14–20 μm sections at −20°C using a cryostat. Sections were mounted on Superfrost Plus Gold microscopic slides (Thermo Scientific) and stained with Alcian Blue (pH 1.0) for histological examination or processed for IHC. For IHC, sections were incubated for 2 h with monoclonal antibodies against type II collagen (II‐II6B3, Developmental Studies Hybridoma Bank) or type VI collagen (MAB3303, EMD Millipore). Following incubation, sections were washed in PBS and incubated for 30 min with EnVision (Dako). Finally, sections were incubated with AEC substrate for 10 min and visualized using microscopy. Postprocessing was undertaken using NIS Elements software (Nikon Instruments Europe B.V.).
Control cartilage samples derived from goat ears were embedded in paraffin using standard histological techniques, cut in 5 μm sections, and stained using Alcian Blue, Mayer's Hematoxylin‐Eosin, type II and type VI collagen to assess glycosaminoglycan distribution, morphology, and collagen distribution, respectively (Supporting Information Fig. S1).

Mice were intracardially perfused with PBS and 4% PFA, fixed with 4% PFA in PBS overnight at 4°C and then cryoprotected in 30% sucrose (Merck Millipore, 107687) in PBS for 48 h. Next, samples were frozen in Optimal Cutting Temperature (Tissue-Tek; Thermo Fisher Scientific, 23–730–571). Sagittal sections (30 µm) were obtained with a CM 1950 Ag Protect freezing microtome (Leica, Solms, Germany). Sections were incubated with the primary antibody 72 h at 4°C in a 0.1 N phosphate buffer containing 1% bovine serum albumin (Sigma-Aldrich, A9418) and 1% Triton X-100 (Sigma-Aldrich, X100). After washing with blocking solution, sections were incubated with donkey Alexa Fluor-conjugated secondary antibody overnight at 4°C (Thermo Fisher Scientific, A-21206 or AP180SA6MI). Finally, sections were washed and mounted with Prolong Gold Antifade (Thermo Fisher Scientific, P36930). Images were taken with a confocal LSM710 META microscope (Carl Zeiss AG, Oberkochen, Germany).