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Insulin, Ascorbate, and Glucose Have a Much Greater Influence Than Transferrin and Selenous Acid on the In Vitro Growth of Engineered Cartilage in Chondrogenic Media

Vunjak-Novakovic, Gordana; Cigan, Alexander D.; Nims, Robert J.; Albro, Michael B.; Esau, John D.; Dreyer, Marissa P.; Hung, Clark T.; Ateshian, Gerard A.

The primary goal of this study was to characterize the response of chondrocyte-seeded agarose constructs to varying concentrations of several key nutrients in a chondrogenic medium, within the overall context of optimizing the key nutrients and the placement of nutrient channels for successful growth of cartilage tissue constructs large enough to be clinically relevant in the treatment of osteoarthritis (OA). To this end, chondrocyte-agarose constructs (phi4x2.34 mm, 30x106 cells/mL) were subjected to varying supplementation levels of insulin (0× to 30× relative to standard supplementation), transferrin (0x to 30x), selenous acid (0x to 10x), ascorbate (0x to 30x), and glucose (0x to 3x). The quality of resulting engineered tissue constructs was evaluated by their compressive modulus (E-Y), tensile modulus (E+Y), hydraulic permeability (k), and content of sulfated glycosaminoglycans (sGAG) and collagen (COL); DNA content was also quantified. Three control groups from two separate castings of constructs (1x concentrations of all medium constituents) were used. After 42 days of culture, values in each of these controls were, respectively, E-Y=518 plus or minus 78, 401 plus or minus 113, 236 plus or minus 67 kPa; E+Y=1420 plus or minus 430, 1140 plus or minus 490, 1240 plus or minus 280 kPa; k=2.3 plus or minus 0.8x10-3, 5.4 plus or minus 7.0x10-3, 3.3 plus or minus 1.3x10-3 mm4/N times s; sGAG=7.8 plus or minus 0.3, 6.3 plus or minus 0.4, 4.1 plus or minus 0.5%/ww; COL=1.3 plus or minus 0.2, 1.1 plus or minus 0.3, 1.4 plus or minus 0.4%/ww; and DNA=11.5 plus or minus 2.2, 12.1 plus or minus 0.6, 5.2 plus or minus 2.8 μg/disk. The presence of insulin and ascorbate was essential, but their concentrations may drop as low as 0.3x without detrimental effects on any of the measured properties; excessive supplementation of ascorbate (up to 30x) was detrimental to E-Y, and 30x insulin was detrimental to both E+Y and E-Y. The presence of glucose was similarly essential, and matrix elaboration was significantly dependent on its concentration (p less than 10-6), with loss of functional properties, composition, and cellularity observed at less than or equal to 0.3x; excessive glucose supplementation (up to 3x) showed no detrimental effects. In contrast, transferrin and selenous acid had no influence on matrix elaboration. These findings suggest that adequate distributions of insulin, ascorbate, and glucose, but not necessarily of transferrin and selenous acid, must be ensured within large engineered cartilage constructs to produce a viable substitute for joint tissue lost due to OA.

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Also Published In

Title
Tissue Engineering Part A
DOI
https://doi.org/10.1089/ten.tea.2012.0596

More About This Work

Academic Units
Biomedical Engineering
Publisher
Mary Ann Liebert
Published Here
January 31, 2014