Effects of cations on cartilage structure: swelling of growth plate and degradation of proteoglycans induced by chelators of divalent cations.

Slices of fresh ovine and bovine epiphyseal cartilages swell following extraction in 0.05 M disodium ethylenediaminetetraacetate (EDTA) in Tris buffer, pH 5.8 and 7.4, at 4 degrees and 37 degrees. The swelling is strikingly visible to the unaided eye and is most pronounced in the growth plate region of the epiphysis. Other chelators--ethyleneglycol-bis (beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), and citrate buffer--also induce swelling. Swelling is associated with increased degradation of proteoglycans (PG) especially at pH 5.8, however, collagen seems to be unaffected. These effects are prevented by the addition of certain divalent cations (Ca, Mg, Zn) to the extraction media. At higher concentrations, the monovalent cation sodium also prevents swelling. It is concluded that divalent cations are required to maintain structure and function of cartilage. Freezing and thawing the cartilage did not prevent swelling or degradation, which suggests that these phenomena are not dependent on living chondrocytes. Although PG degradation and loss is markedly increased at 37 degrees as compared with 4 degrees, swelling is unaffected. It is concluded therefore that the degradative effects are enzymatic but the swelling is physicochemical. Other cartilages (nasal, manubrium) also swell and show histochemical evidence of PG degradation. These effects are minimal compared with the effects induced in the growth plate. It is inferred that growth plate contains more proteases than other cartilages and has properties that make it more susceptible to swelling. Swelling of the growth plate occurs even when the metaphysis is attached to it albeit to a lesser extent than when it is freed of underlying bone. A hypothesis is offered which attempts to link these phenomena with chondrocyte and matrical imbibition of water (swelling) in the zone of hypertrophy of the growth plate.

Loss of proteoglycans during decalcification of fresh metaphyses with disodium ethylenediaminetetraacetate (EDTA).

Recent immunofluorescent and histochemical data did not detect changes in the concentration of proteoglycans between noncalcified and calcified cartilage in fetal bovine growth plate or metaphyseal bone. These findings were constant, regardless of prior fixation before demineralization with disodium ethylenediaminetetraacetate (EDTA) or prior demineralization before fixation. Previous experience has shown that EDTA can extract proteoglycans from calcified cartilage. With this in mind, we determined the amount of proteoglycan extracted from calcified cartilage in metaphyseal bone and uncalcified growth plate cartilages during decalcification of unfixed fresh tissues with EDTA. To this end, fresh growth plate cartilages and metaphyses were decalcified at 5 degrees C for 48 hours in a buffered solution of EDTA to which several protease inhibitors were added. Under these conditions 20-25% of the total proteoglycan (measured as uronic acid and hexosamine) was extracted from mineralized cartilage but only about 1% from the uncalcified (growth plate) cartilages. Thus, histochemical and immunohistochemical studies appear to be insensitive measures of proteoglycan concentrations in histological sections of mineralized tissue and may not give quantitative information.

Changes in cartilage proteoglycans associated with calcification.

The purposes of these experiments were to study the biosynthetic and postbiosynthetic relationships between proteoglycans in noncalcified growth cartilage and calcified cartilage in metaphysis from the costochondral junctions of immature rabbits. Based on in vivo experiments in which 35 S-sodium sulfate was injected into rabbits, it is shown that proteoglycans from the hypertrophic region becomes part of the calcified cartilage matrix which is to be incorporated into the metaphysis. The proteoglycan aggregates in the growth apparatus undergo partial disaggregation and degradation. There is approximately a 25% decrease in aggregation from regions of the rib distal to the metaphyseal-growth plate junction (69%) to the region proximal to it (50%). In contrast, in their final state in calcified cartilage, the proteoglycans are more completely disaggregated and the proteoglycans subunits are smaller, as adjudged from gel chromatography. Control experiments indicate that although some artifactual disaggregation is produced by the extraction process, it is not of the same magnitude as that seen in the actual isolation experiments nor are the subunits reduced in size.

Studies on extractable and resistant proteoglycans from metaphyseal and cortical bone and cartilage.

Soluble proteoglycans (SPG) were extracted from bovine (BCC) and human (HCC) costal cartilages by the dissociative method using 4 M guanidinium chloride (GuHCl). Proteoglycans which are resistant to extraction (RPG) were obtained following collagenase digestion or hydroxylamine treatment of the cartilage residues. Similarly, SPG were extracted from bovine metaphyseal and cortical bone using EDTA. The RPG were extracted from the bones using hydroxylamine. Density gradient fractionation under dissociative conditions of cartilage SPG and RPG followed by chromatography on Sepharose 2B revealed that A1D1 RPG are smaller than the SPG. SPG reacted with either collagenase or hydroxylamine are also smaller than the parent SPG. A1D1 fractions obtained from BCC-SPG and RPG or from mixtures of SPG and acid-soluble collagen are free of hydroxyproline. Hydroxyproline is not completely separated from HCC-RPG. Density gradient fractionation of bone proteoglycans and Sepharose chromatography of the A1 and A1D1 fractions showed that those obtained from metaphysis are larger than those from cortical bone. This was attributed to the presence of calcified cartilage in metaphyseal bone. The A1D1 fractions of the metaphyseal proteoglycans seemed to undergo self-association since this fraction is larger than the A1 fraction from which it is derived. Cortical bone proteoglycans do not behave similarly. Density gradient purification under dissociative conditions failed to separate hydroxyproline from the proteoglycans obtained from bone. It is hypothesized that in bone proteoglycans and collagen might be linked.

Resistant proteoglycans in epiphyseal plate cartilage. Variations in their distribution in relationship to age and species.

The localizations of resistant proteoglycans (RPGs) in the epiphyseal plates of rats, dogs, and humans are similar. In the epiphysial plates from young rats, dogs, and humans, the RPGs form a stratum at the junction of the zones of resting and proliferating cells. Non-calcified cartilage RPGs are associated with cells which have the potential for proliferation or column organization. As the individuals age, RPGs are found in intercolumnar regions or at times are even absent. There is also a type of RPGs in calcified cartilage, including the calcified cartilage subjacent to the articular surface, in all species. In human epiphyseal plates, looser fibrillar RPGs change abruptly to a more condensed type in the zone of provisional calcification. Calcified cartilage RPGs stain more intensely with toluidine blue and may represent a different type of RPGs.