Hyaluronan Synthases in Normal and Regenerating Joint Cartilage

Abstract

Repair of full thickness joint cartilage defects is within reach of routine clinical practice. The quality of regenerating hyaline cartilage, however, is difficult to assess. Synthesis of an extracellular matrix with high hyaluronan content is crucial for its metabolic and functional properties. We studied hyaluronan synthase (HAS) expression in knee joints of adult sheep as a novel cellular marker for chondrocyte function. Six house-bred Merino sheep (age 4–6 years) underwent two-stage surgery of their femoral condyles for autologous chondrocyte transplantation (ACT). First, cells were isolated from biopsies and expanded in vitro for 2 weeks using standard culture techniques. In a second session, three defects were made and either left untreated, covered with periosteal flap alone, or in combination with chondrocyte suspensions injected under the flaps. After 16 weeks, biopsies were taken from the operated knees at the defect sites and from the untreated condyle. Specimens were processed for safranin O and electron microscopy, and for immunofluorescence using three different polyclonal anti-HAS antibodies recognizing one or all of the three known mammalian HAS. Control and regenerating tissues were compared regarding their morphology and the expression of HAS, in relation to collagens type I and II, and adult cartilage proteoglycan core protein. In comparison with untreated defects or with periosteal flap alone, ACT provided a neocartilage with better-differentiated morphology. In healthy joint cartilage, about 50% of the chondrocytes expressed HAS, independent of antibody. Following ACT, a higher density of chondrocytes was observed, of which more than 75% expressed HAS, whereas the regenerates without ACT showed a lower density of HAS-expressing cells. We propose to use HAS immunofluorescence as an additional marker of matrix synthesis by chondrocytes and joint cartilage regeneration.