Optimization of histoprocessing methods to detect glycosaminoglycan, collagen type II, and collagen type I in decalcified rabbit osteochondral sections

摘要

Rabbit models frequently are used as a small animal cartilage repair model, where hyaline articular cartilage repair can be identified by the presence of type II collagen, the absence of type I collagen, and an abundant glycosaminoglycan component (GAG). Staining methods for these three extracellular matrix components should therefore function in the same sample after histologic processing. The goal of the current study was to optimize histoprocessing techniques to maximally retain GAG, while still enabling the immunodetection of both collagen type II and collagen type I in decalcified rabbit osteochondral sections. We compared four different fixation methods and two different decalcification procedures in both paraffin and cryostat sections using a histological grading scale for five tissue characteristics, including chondrocyte morphology, bone marrow structure, cartilage GAG staining with Safranin O, cartilage immunostaining for type II collagen, and bone matrix immunostaining for type I collagen. We found that addition of ruthenium hexaammine trichloride to the fixation solution provided the best retention of chondrocyte morphology. However, ruthenium hexaammine trichloride interfered with immunodetection of type II collagen, an effect not observed for any of the other fixation conditions. Additionally, paraffin embedding was found to suppress immunodetection of type I collagen in bone matrix compared with cryosectioning. Nonetheless, bone marrow structure was better preserved in paraffin sections. Taken together, our results show that fixation in formalin or para-formaldehyde, followed by decalcification in acid with trace fixative, and then cryosectioning permitted 1) adequate preservation of chondrocyte morphology, 2) Safranin O staining of GAG in cartilage matrix, 3) immunostaining of type II collagen in cartilage matrix, and 4) immunostaining of type I collagen in bone matrix.