An Analysis of the Collagen Synthesized by Chondrocyte Cultures Derived From the Chick Embryo Tibiotarsus

241 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1980.Chondrocyte cell cultures originating from the histologically distinct zones of the tibiotarsus synthesize chondroitin sulfate and type II collagen in primary and secondary cultures. The collagen is secreted into two extracellular collagen pools, the medium (CM) and matrix (Ma) fractions. The CM-collagen fraction contains procollagen and the intermediates in its metabolic maturation to {(alpha)1(II)}(,3) collagen. The Ma-collagen fraction contains mostly the pN-collagen and fully processed collagen. Small amounts of other bands that have a mobility slightly slower than the (alpha)1 chains are seen in this fraction after limited pepsin digestion. Under the cell culture conditions examined, the kinetics of collagen accumulation was found to be very similar for the cells derived from each of the distinct zones. The kinetic experiments also showed type II collagen to be synthesized on an equal weight basis with chondroitin sulfate.After separation of the denatured collagen by SDS-PAGE, a 60K {('3)H}-proline-labeled band was found in the medium of some of the chondrocyte cultures. Limited pepsin digestion cleaved the 60K band to a 45K band. The 60K band was shown to originate from triple helical collagen by its hydroxyproline content, its resistance to limited pepsinization, its ability to form SLS crystallites and its digestion with bacterial collagenase. The 60K collagen contained a cleavage site for human skin collagenase. The 60K collagen was not generated from the the type II collagen found in these cultures as shown by cyanogen bromide peptide mapping, partial chymotryptic peptide mapping, and digestion with an animal collagenase. The 60K collagen band may be formed by degradation of some other high molecular weight collagen precursor in these cultures or it may be a novel, undergraded biosynthetic product of the chondrocytes.In organ culture the 60K collagen was only present in the zones 3a and 3b. In primary cell culture, the 60K collagen appeared in zones 2a, 2b, 3a, and 3b often increasing in concentration in the latter zones. In secondary cultures, the 60K collagen was synthesized by zone 1 cells and to a small extent by the tarsus cells in addition to the cells from the other zones. From this evidence the 60K collagen does not appear to be a cell culture artifact. Instead, it appears to be a collagen marker of senescent chondrocytes.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD