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), aspirated by micropuncture techniques from the hypertrophic cell zone of calcifying epiphyseal cartilage, 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 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 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 Cf1 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 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 Cf1. 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 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 Cfl.

Evidence for a role of proteinpolysaccharides in regulation of mineral phase separation in calcifying cartilage

“…Earlier findings (1,2) important to the current report were (a) hypertrophic cell cartilage and extracellular fluid of normal rats or its dilutions in synthetic lymph up to 1: 4 completely prevented mineral growth in vitro, (b) the inhibitory action on mineral growth was confined to the sediment after ultracentrifugation and was abolished by prior incubation with trypsin or hyaluronidase, (c) the sediment of this extracellular cartilage fluid contained about 2 mg of hexuronate per ml of cartilage fluid and yielded a positive reaction of identification for proteinpolysaccharide with fluorescent antibodies to rat PPL-C,2 and (d) there was evidence of a mineral phase and/or organic nucleational agent for mineral production in hypertrophic cell cartilage fluid of normal rats and rats with healing rickets but not in cartilage fluid of untreated rachitic rats. These results were interpreted to indicate that certain PPC functioned as inhibitors of crystal growth at extracellular sites premonitory to calcification (2).…”

“…No effect of rickets on behavior of untreated or enzymically degraded fluids from uncalcified cartilages was observed (Table IV). In an additional experiment, centrifugation of the hypertrophic cell cartilage sediments after hyaluronidase treatment at 3000 g for 10 min was used to synthesize a "native fluid" mineral phase as previously reported (2). When this crude mineral sediment was resuspended at a level of 0.02 mg of phosphate per ml, in fresh cartilage fluid, there resulted an R1 + R2, SR partition not detectably different from that caused by MIV mineral phase (Table IV).…”

“…These results were interpreted to indicate that certain PPC functioned as inhibitors of crystal growth at extracellular sites premonitory to calcification (2).…”

“…Recently, an extracellular fluid phase was demonstrated by micropuncture techniques in the hypertrophic cell zone of calcifying epiphyseal cartilage1 and was characterized in respect to some parameters of calcification (1,2). Starting samples of only 20 nl of cartilage fluid compelled miniaturization of several classical methods for the separation and identification of relevant parameters including nucleotide, hemoglobin, protein, Ca, phosphate, hexuronate, and an immunofluorescent antibody method for whole proteinpolysaccharide complexes (PPC) identification (1)(2)(3). Partition by ultracentrifugation and enzymic treatment of protein-containing fractions as well as development of a microscopic system for measurement of mineral formation permitted study of factors in this cartilage fluid which initiate or inhibit mineral growth in vitro (1,2).…”

“…The Rl, R2, and SR2 subfractions of PPL-C from cartilages and from CPC precipitates of aspirated cartilage fluids were analyzed for protein and hexuronate content by methods of Lowry, Rosebrough, Farr, and Randall (7)' and Dische (8), respectively, as previously miniaturized (1,2). Ca was measured by a chlorophosphanazo dye method and phosphate by the method of Kuttner and Cohen (9), both also miniaturized (1-3).…”