Type x collagen

For histology, samples were fixed in 10% formalin for 24 hours, decalcified with Immunocal (Thermo Fisher Scientific), dehydrated in ethanol, embedded in paraffin, and sectioned to 5 μm. Sections were stained for (i) hematoxylin and eosin (H&E), (ii) Alcian Blue with Nuclear Fast Red, (iii) Picosirius Red, and (iv) Movat’s pentachrome. The use of Movat’s pentachrome to evaluate cartilage/bone after implantation is widely reported. The green color indicates the presence of both collagens (staining yellow) and glycosaminoglycans (staining blue by Alcian Blue, which is one of the components of Movat’s stain). All reagents were from Sigma-Aldrich, unless otherwise specified. Immunohistochemistry was performed using the following antibodies: type I collagen, type II collagen, type X collagen, lubricin, and osteopontin (Abcam, Cambridge, MA, USA). Sections were processed according to manufacturer’s instructions. Immunobinding was detected with biotinylated secondary antibodies using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Images were acquired using an Olympus FSX-100 microscope (Olympus, Center Valley, PA, USA).

The samples were fixed in 4% paraformaldehyde, followed by dehydrating with ethanol, clearing with xylene, embedding in paraffin wax and 5 μm sections were prepared. The sections were stained with safranin-O/fast green/iron hematoxylin staining for sulfated GAGs. Immunohistochemical (IHC) analyses for type I collagen, type II collagen, and type X collagen were performed on sections using primary antibodies (type I collagen; Abcam plc, UK, type II collagen; Kyowa Pharma Chemical, Japan, type X collagen; Abcam plc, UK) [1 (link)]. The deparaffinized sections were incubated in 3% H₂O₂ for 10 min to block endogenous peroxidase activity, followed by incubation in proteinase K (DAKO, Glostrup, Denmark) for 5 min for antigen retrieval, and then in 10% goat serum for 30 min to avoid non-specific binding of primary antibodies. Primary antibody was applied to each section and incubated for 1 h. Detection was then performed using Histofine® Simple Stain™ MAX PO (MULTI; Nichirei Bioscience, Tokyo, Japan) and Simple Stain™ DAB Solution (Nichirei Bioscience). All procedure was done at room temperature, and during each step, the sections were washed three times with 0.1% Tween 20 in PBS for 5 min. Immunostaining for type I collagen, type II collagen, and type X collagen were assessed with Aperio CS2 (Leica Microsystems, Wetzlar, Germany) based on positive stained area.