Collagen II and IX are deposited in the pericellular matrix. (a–f) Alginate beads were embedded in paraffin after five weeks of culture and sections stained with an antibody against collagen IX. Images a–c are duplicates of images d–f, except that cell borders are outlined with a white line in the latter series. a and d show chondrocytes expressing mutant COMP, b and e cultures overexpressing wild-type COMP, c and f untransduced cultures. Bars indicate 10 μm. (g) Collagen II secretion was assessed by pulse-chase experiments in cells expressing mutant (Mu) and wild-type (Wt) COMP. Numbers below the gels indicate the hours of chase. The positions of the molecular weight standards are indicated on the left, of collagen II to the right. The arrows mark origin and front of the separating gel. (h) The distribution of collagen II in the culture supernatant (Su), the citrate soluble alginate matrix (Ci), and the citrate resistant pellet (Pe) was evaluated after five weeks of alginate culture. The lanes showing the collagen II content of the citrate resistant pellets were exposed for a longer time than the other lanes. The position of cross-linked and pepsin-digested collagen II is indicated on the right side. The lower band observed exclusively in the supernatants represents the α2-chain of collagen I, produced at the outer surface of alginate beads (30, 32). Densitometric analysis revealed no significant variation in the proportion of collagen I to collagen II (32) between control and tranfected cultures.