Lubricin

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).

Decalcified bone sections were deparaffinized with xylene, and endogenous peroxidase activity was quenched by treatment with 3% H₂O₂ for 15 min, followed by antigen retrieval by trypsinization for 10 min. Sections were then blocked with normal goat serum for 30 min and incubated at 4 °C overnight with primary antibody followed by the appropriate biotinylated secondary antibody and horseradish peroxidase-conjugated streptavidin-biotin staining. Immunoreactivity was visualized with a 3,3′-diaminobenzidine tetrahydrochloride kit (ZSGB-BIO, Beijing, China) followed by counterstaining with Methyl green. Primary antibodies against the following proteins were used: collagen II (1:400; Chondrex, Redmond, WA, USA), FGFR3 (1:200; Santa Cruz Biotechnology, Dallas, Texas, USA), Osteocalcin (1:200; Santa Cruz Biotechnology), PCNA (1:200; Epitomics, Burlingame, CA, USA), Collagen X (1:200; Abcam, Cambridge, MA, USA), Aggrecan (1:200; Millipore, Billerica, MA, USA), MMP13 (1:200; Proteintech, Chicago, IL, USA), ADANTS5(1:200; Abcam), lubricin (1:600; Abcam), IHH (1:100; Abcam) and RUNX2 (1:200; Santa Cruz Biotechnology). The number of immunoreactive cells in three central regions of condylar cartilage section was counted by using Image-Pro Plus 5.1.