Growth of Immature Articular Cartilage in Vitro: Correlated Variation in Tensile Biomechanical and Collagen Network Properties

Abstract

Articular cartilage biochemical composition and mechanical properties evolve during in utero and in vivo growth, with marked differences between fetus, newborn, and young adult. The objectives of this study were to test whether in vitro growth of bovine fetal and newborn calf articular cartilage explants resulted in changes in biochemical and tensile properties during up to 6 weeks of free-swelling culture in serum-supplemented medium. During this culture period, both fetal and calf cartilage grew markedly in size, increasing in dry and wet mass by 150-270%. This was due in part to increases in sulfated glycosaminoglycan (+248%), collagen (+96%), and pyridinoline cross-link (+133%). This was accompanied by an increase in water content so that the concentration of matrix components decreased, despite the overall net increase in mass. The ratio of pyridinoline cross-link to collagen remained low and characteristic of immature tissue. The equilibrium and dynamic tensile moduli and strength of both fetal and calf cartilage decreased during the culture period. The biochemical and biomechanical properties of the cartilage explants were correlated, such that the low values of modulus and strength were associated with low concentrations of collagen and pyridinoline. Thus, the tested culture conditions supported growth and maintenance cartilage in an immature state, but did not induce biomechanical or collagen network maturation.

Mechanisms of cartilage growth: Modulation of balance between proteoglycan and collagen in vitro using chondroitinase ABC

“…In the case of the first phenotype, incubation with 20% FBS resulted in expansive cartilage growth ( Figure 7B) characterized by a marked increase in tissue thickness and wet weight ( Figures 1A and B) and in the content of GAG and collagen ( Figures 2B and C). The result was a reduction in the concentrations of GAG, collagen, and Pyr (Figures 2B-D) and a concomitant diminution in tensile mechanical integrity (Figures 5 and 6), similar to previous findings on growth of immature cartilage in serum-supplemented medium (19)(20)(21). These findings suggest that a free-swelling, serum-supplemented culture environment induces remodeling and reorganization of the tissue matrix that facilitates a relatively loose and weak collagen network and allows tissue expansion.…”

“…Because the various zones of normal cartilage exhibit differences in biochemical composition and mechanical properties, the middle layer was also analyzed, and it displayed an initial state different from that of the superficial layer. The patellofemoral groove was used as the source of tissue, similar to tissue used in previous studies (15,19,21). Consequently, the biochemical and biomechanical properties of tissue samples at the time of explant were similar to those reported previously (15,21).…”

“…Articular cartilage was harvested from the patellofemoral groove of 3 newborn (1-3-week-old) bovine calves as described previously (21). Blocks, 9 mm ϫ 3 mm in area, were prepared using a sledge microtome either to include the intact articular surface (the superficial layer, ϳ0.4 mm thick) or to include the middle zone, starting at a distance of ϳ0.6 mm from the articular surface (the middle layer, 0.25 mm thick).…”

“…The residual cartilage and failed portions of the corresponding tensile strip (see below) were analyzed together to quantify the biochemical composition of the explants. Samples were lyophilized, weighed dry, and solubilized with proteinase K (21). Portions of the tissue digest were analyzed to quantify the content of DNA (32), GAG (33), and hydroxyproline (34).…”

“…In contrast to this type of in vivo growth and maturation, growth of cartilage explants in vitro in serum-supplemented medium results in a net deposition of proteoglycan that is greater than that of collagen, along with a decrease in mechanical integrity. For explants of immature cartilage, incubation in serum-supplemented medium results in an increase of tissue volume, maintenance of proteoglycan concentration, and a decrease in the concentrations of collagen and Pyr crosslinks (19)(20)(21). These changes in composition are associated with a decrease in tensile modulus and strength (21).…”