Histological Characterization of Chondrocyte Pellets

After 21 days, pellets were washed with PBS and fixed in 10% neutral buffered formalin followed by paraffin embedding. Cross sections (5 μm thick) were stained with Safranin O-Fast Green for sulfated glycosaminoglycans (sGAG) using standard protocols. Immunohistochemical analysis was performed using a Collagen Type II (Col II) antibody (Developmental Studies Hybridoma Bank), followed by a goat anti-mouse-HRP (Jackson ImmunoResearch). Slides were developed using the ImmPACT DAB Peroxidase (HRP) Substrate (Vector Laboratories) and counterstained with VECTOR Hematoxylin QS (Vector Laboratories) per manufacturer’s instructions. Slides were visualized and images taken using a Nikon Eclipse 90i microscope (Tokyo, Japan). To measure total sGAG content, pellets were washed with PBS and each dry pellet was frozen at −80°C until ready to be assayed. sGAG content per pellet was quantified via the dimethylmethylene blue (DMMB) assay as previously described (n=3 pellets/group).^{11 (link)}