Heavy chain transfer by tumor necrosis factor-stimulated gene 6 to the bikunin proteoglycan.

We present data that hyaluronan (HA) polysaccharides, about 14-86 monosaccharides in length, are capable of accepting only a single heavy chain (HC) from inter-α-inhibitor via transfer by tumor necrosis factor-stimulated gene 6 (TSG-6) and that this transfer is irreversible. We propose that either the sulfate groups (or the sulfation pattern) at the reducing end of the chondroitin sulfate (CS) chain of bikunin, or the core protein itself, enables the bikunin proteoglycan (PG) to accept more than a single HC and permits TSG-6 to transfer these HCs from its relatively small CS chain to HA. To test these hypotheses, we investigated HC transfer to the intact CS chain of the bikunin PG, and to the free chain of bikunin. We observed that both the free CS chain and the intact bikunin PG were only able to accept a single HC from inter-α-inhibitor via transfer by TSG-6 and that HCs could be swapped from the bikunin PG and its free CS chain to HA. Furthermore, a significant portion of the bikunin PG was unable to accept a single heavy chain. We discuss explanations for these observations, including the intracellular assembly of inter-α-inhibitor. In summary, these data demonstrate that the sulfation of the CS chain of bikunin and/or its core protein promote HC transfer by TSG-6 to its relatively short CS chain, although they are insufficient to enable the CS chain of bikunin to accept more than one HC in the absence of other cofactors.

Proteomic, mechanical, and biochemical development of tissue-engineered neocartilage.

BACKGROUND: The self-assembling process of cartilage tissue engineering is a promising technique to heal cartilage defects, preventing osteoarthritic changes. Given that chondrocytes dedifferentiate when expanded, it is not known if cellular expansion affects the development of self-assembled neocartilage. The objective of this study was to use proteomic, mechanical, and biochemical analyses to quantitatively investigate the development of self-assembled neocartilage derived from passaged, rejuvenated costal chondrocytes. METHODS: Yucatan minipig costal chondrocytes were used to create self-assembled neocartilage constructs. After 1, 4, 7, 14, 28, 56, or 84 days of self-assembly, constructs were analyzed through a variety of histological, biomechanical, biochemical, and proteomic techniques. RESULTS: It was found that temporal trends in neocartilage formation are similar to those seen in native hyaline articular cartilage development. For example, between days 7 and 84 of culture, tensile Young's modulus increased 4.4-times, total collagen increased 2.7-times, DNA content decreased 69.3%, collagen type II increased 1.5-times, and aggrecan dropped 55.3%, mirroring trends shown in native knee cartilage. Importantly, collagen type X, which is associated with cartilage calcification, remained at low levels (≤ 0.05%) at all neocartilage developmental time points, similar to knee cartilage (< 0.01%) and unlike donor rib cartilage (0.98%). CONCLUSIONS: In this work, bottom-up proteomics, a powerful tool to interrogate tissue composition, was used for the first time to quantify and compare the proteome of a developing engineered tissue to a recipient tissue. Furthermore, it was shown that self-assembled, costal chondrocyte-derived neocartilage is suitable for a non-homologous approach in the knee.

Proteomic, mechanical, and biochemical characterization of cartilage development.

The objective of this work is to examine the development of porcine cartilage by analyzing its mechanical properties, biochemical content, and proteomics at different developmental stages. Cartilage from the knees of fetal, neonatal, juvenile, and mature pigs was analyzed using histology, mechanical testing, biochemical assays, fluorophore-assisted carbohydrate electrophoresis, and bottom-up proteomics. Mature cartilage has 2.2-times the collagen per dry weight of fetal cartilage, and fetal cartilage has 2.1-times and 17.9-times the glycosaminoglycan and DNA per dry weight of mature cartilage, respectively. Tensile and compressive properties peak in the juvenile stage, with a tensile modulus 4.7-times that of neonatal. Proteomics analysis reveals increases in collagen types II and III, while collagen types IX, XI, and XIV, and aggrecan decrease with age. For example, collagen types IX and XI decrease 9.4-times and 5.1-times, respectively from fetal to mature. Mechanical and biochemical measurements have their greatest developmental changes between the neonatal and juvenile stages, where mechanotransduction plays a major role. Bottom-up proteomics serves as a powerful tool for tissue characterization, showing results beyond those of routine biochemical analysis. For example, proteomic analysis shows significant drops in collagen types IX, XI, and XIV throughout development, which shows insight into the permanence of cartilage's matrix. Changes in overall glycosaminoglycan content compared to aggrecan and link protein indicate non-enzymatic degradation of aggrecan structures or hyaluronan in mature cartilage. In addition to tissue characterization, bottom-up proteomics techniques are critical in tissue engineering efforts toward repair or regeneration of cartilage in animal models. STATEMENT OF SIGNIFICANCE: In this study, the development of porcine articular cartilage is interrogated through biomechanical, biochemical, and proteomic techniques, to determine how mechanics and extracellular matrix composition change from fetal to mature cartilage. For the first time, a bottom-up proteomics approach is used to reveal a wide variety of protein changes through aging; for example, the collagen subtype composition of the cartilage increases in collagen types II and III, and decreases in collagen types IX, XI, and XIV. This analysis shows that bottom-up proteomics is a critical tool in tissue characterization, especially toward developing a deeper understanding of matrix composition and development in tissue engineering studies.