Adult equine bone marrow stromal cells produce a cartilage-like ECM mechanically superior to animal-matched adult chondrocytes.

Our objective was to evaluate the age-dependent mechanical phenotype of bone marrow stromal cell- (BMSC-) and chondrocyte-produced cartilage-like neo-tissue and to elucidate the matrix-associated mechanisms which generate this phenotype. Cells from both immature (2-4 month-old foals) and skeletally-mature (2-5 year-old adults) mixed-breed horses were isolated from animal-matched bone marrow and cartilage tissue, encapsulated in self-assembling-peptide hydrogels, and cultured with and without TGF-beta1 supplementation. BMSCs and chondrocytes from both donor ages were encapsulated with high viability. BMSCs from both ages produced neo-tissue with higher mechanical stiffness than that produced by either young or adult chondrocytes. Young, but not adult, chondrocytes proliferated in response to TGF-beta1 while BMSCs from both age groups proliferated with TGF-beta1. Young chondrocytes stimulated by TGF-beta1 accumulated ECM with 10-fold higher sulfated-glycosaminoglycan content than adult chondrocytes and 2-3-fold higher than BMSCs of either age. The opposite trend was observed for hydroxyproline content, with BMSCs accumulating 2-3-fold more than chondrocytes, independent of age. Size-exclusion chromatography of extracted proteoglycans showed that an aggrecan-like peak was the predominant sulfated proteoglycan for all cell types. Direct measurement of aggrecan core protein length and chondroitin sulfate chain length by single molecule atomic force microscopy imaging revealed that, independent of age, BMSCs produced longer core protein and longer chondroitin sulfate chains, and fewer short core protein molecules than chondrocytes, suggesting that the BMSC-produced aggrecan has a phenotype more characteristic of young tissue than chondrocyte-produced aggrecan. Aggrecan ultrastructure, ECM composition, and cellular proliferation combine to suggest a mechanism by which BMSCs produce a superior cartilage-like neo-tissue than either young or adult chondrocytes.

Exercise and injury increase chondroitin sulfate chain length and decrease hyaluronan chain length in synovial fluid.

OBJECTIVES: (1) To investigate the effects of exercise and osteochondral (OC) injury on synovial fluid (SF) chondroitin sulfate (CS) and hyaluronan (HA) concentration and chain length, (2) to compare SF and cartilage CS data from joints with OC fragmentation, and (3) to compare SF CS and HA profiles with those seen in serum from the same horses. METHODS: Serum and SF were obtained from (1) normal horses after 8 weeks rest, (2) the same horses after 9 months treadmill training, and (3) horses with OC injury from racing. Articular cartilage was also collected from group 3 horses. Concentrations and chain lengths of CS and HA were determined by gel chromatography and fluorophore-assisted carbohydrate electrophoresis. RESULTS: SF CS peak chain length in the OC injury group increased significantly (18.7kDa) when compared to rested horses (11.6kDa), with exercise producing an intermediate chain length (15.6kDa). Cartilage and serum from the OC injury group had the abnormally long CS chains seen in SF from these horses. Total SF HA was significantly lower in the OC injury group compared to the rested group. Both the OC injury group and the exercised group had significant decreases in SF HA chain length compared to the rested group. CONCLUSIONS: Chain length of SF CS was increased by exercise and OC injury. Exercise resulted in a modest increase, whereas OC injury caused a marked increase. In contrast to CS, SF HA chain length was decreased by OC injury, and to a lesser extent by exercise. Chain length analysis of SF CS and HA may provide a useful tool for evaluation of joint health.

Variations in matrix composition and GAG fine structure among scaffolds for cartilage tissue engineering.

OBJECTIVE: To compare matrix composition and glycosaminoglycan (GAG) fine structure among five scaffolds commonly used for in vitro chondrocyte culture and cartilage tissue engineering. DESIGN: Bovine articular chondrocytes were seeded into agarose, alginate, collagen I, fibrin and polyglycolic acid (PGA) constructs and cultured for 20 or 40 days. In addition to construct DNA and sulfated GAG (sGAG) contents, the delta-disaccharide compositions of the chondroitin/dermatan sulfate GAGs were determined for each scaffold group via fluorophore-assisted carbohydrate electrophoresis (FACE). RESULTS: Significant differences were found in cell proliferation and extracellular matrix accumulation among the five scaffold groups. Significant cell proliferation was observed for all scaffold types but occurred later (20-40 days) in PGA constructs compared to the other groups (0-20 days). By 40 days, agarose constructs had the highest sGAG to DNA ratio, while alginate and collagen I had the lowest levels. Quantitative differences in the Delta-disaccharide composition of the GAGs accumulated in the different scaffolds were also found, with the most striking variations in unsulfated and disulfated delta-disaccharides. Agarose constructs had the highest fraction of disulfated residues and the lowest fraction of unsulfated residues, with a 6-sulfated/4-sulfated disaccharide ratio most similar to that of native articular cartilage. CONCLUSIONS: The similarities and differences among scaffolds in proteoglycan accumulation and GAG composition suggest that the scaffold material directly or indirectly influences chondrocyte proteoglycan metabolism and may have an influence on the quality of tissue engineered cartilage.