Demonstration of Macromolecular Inhibitor(s) of Calcification and Nucleational Factor(s) in Fluid from Calcifying Sites in Cartilage

ABSTRACT An extracellular fluid phase (Cf 1), as-pirated by micropuncture techniques from the hyper-trophic cell zone of calcifying epiphyseal cartilage, has been characterized in a calcifying system in vitro in re-spect to the behavior of sedimenting and supernatant fractions after high speed ultracentrifugation. To per-form these tests on the starting samples of 20 nI of Cf 1, 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 physio-logic conditions in the calcifying cartilage septa of nor-mal rats, and the mineral used for seeding was an im-mature calcium phosphate similar to native cartilage mineral. Normal Cf1 or its dilutions in synthetic lymph up to 1: 4 completely prevented mineral accretion in vitro. The inhibitory action was localized to the sedi-mented fractions after ultracentrifugation and could be destroyed by incubation with trypsin or hyaluronidase. The sediment of Cf1 contained 2 mg of hexuronic acid per ml of Cfi 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 Cfi and exhibited responses similar to those of Cf 1. Also, there was evidence of a mineral phase in Cf of normal rats, in Cf of rats with healing rickets, but not in Cf of untreated rachitic rats. These results are interpreted to indicate that certain PPLs func-tion as an inhibitor of crystal growth at extracellular sites premonitory to calcification. Evidence for a low density inhibitor of mineral accretion was found in nor-mal serum but not in Cfl. Received for publication 27 June 1968 and in revised for