Fig. 5

HC-HA complex formation was increased in OA joints. A Heavy chain-hyaluronic acid (HC-HA) complex formation was analyzed by western blot. Paired synovial fluid samples were run both with and without hyaluronidase (HAase) pre-digestion to release the HA-bound HC. Sample mixes (12 μL with the equivalent of 1 μL of undiluted SF) were loaded per lane. Lane “–” = endogenous HC; lane “+” = endogenous HC + HA-bound HC. B HC-HA relative absorbance unit (a.u.) = ∆(HC/Pre-IαI) = [HC (Lane “+”)/Pre-IαI]–[HC (Lane “–”)/Pre-IαI], used to compare healthy and OA joints (n = 25 healthy and n = 61 OA). ****P < 0.0001. Data are displayed as box-and-whisker plots representing the first and third quartiles, median, and spread of the HC-HA complex levels for healthy and OA samples. C The induction of HC-HA complex formation in synovial membrane tissues was evaluated by immunostaining. Paraffin-embedded sections were stained with HA binding protein (green), the Dako IαI antibody (red, to detect HC-HA complex), and DAPI (blue). Increased HC-HA was detected in the intimal and subintimal regions of OA synovial membrane tissue and was prominent within OA synovial tissue vasculature and lymphatics. D HC-HA was observed within chondrocyte lacunae of superficial zone chondrocytes in OA but not healthy articular cartilage