Demonstration of macromolecular inhibitors of calcification and nucleational factors in fluid from calcifying sites in cartilage.

An extracellular fluid phase (C(f1)), aspirated by micropuncture techniques from the hypertrophic cell zone of calcifying epiphyseal certilage, has been characterized in a calcifying system in vitro in respect to the behavior of sedimenting and supernatant fractions after high speed ultracentrifugation. To perform these tests on the starting samples of 20 nl of C(f1), macroscopic analytical methods were scaled down for the identification of relevant organic components, including hexuronic acid and proteinpolysaccharides (PPL). The mineral accretion system was designed to simulate physiologic conditions in the calcifying cartilage septa of normal rats, and the mineral used for seeding was an immature calcium phosphate similar to native cartilage mineral. Normal C(f1) or its dilutions in synthetic lymph up to 1:4 completely prevented mineral accretion in vitro. The inhibitory action was localized to the sedimented fractions after ultracentrifugation and could be destroyed by incubation with trypsin or hyaluronidase. The sediment of C(f1) contained 2 mg of hexuronic acid per ml of C(f1) and gave a strong reaction of identification for a light fraction of PPL by fluorescent antibodies to rat PPL. PPL fractions were tested in the same mineral accretion systems as C(f1) and exhibited responses similar to those of C(f1). Also, there was evidence of a mineral phase in C(f1) of normal rats, in C(f1) of rats with healing rickets, but not in C(f1) of untreated rachitic rats. These results are interpreted to indicate that certain PPLs function as an inhibitor of crystal growth at extracellular sites premonitory to calcification. Evidence for a low density inhibitor of mineral accretion was found in normal serum but not in C(f1).