Regulation of proteoglycan production by varying glucose concentrations controls fiber formation in tissue engineered menisci.

Fibrillar collagens are highly prevalent in the extracellular matrix of all connective tissues and therefore commonly used as a biomaterial in tissue engineering applications. In the native environment, collagen fibers are arranged in a complex hierarchical structure that is often difficult to recreate in a tissue engineered construct. Small leucine rich proteoglycans as well as hyaluronan binding proteoglycans, aggrecan and versican, have been implicated in regulating fiber formation. In this study, we modified proteoglycan production in vitro by altering culture medium glucose concentrations (4500, 1000, 500, 250, and 125 mg/L), and evaluated its effect on the formation of collagen fibers inside tissue engineered meniscal constructs. Reduction of extracellular glucose resulted in a dose dependent decrease in total sulfated glycosaminoglycan (GAG) production, but minimal decreases of decorin and biglycan. However, fibromodulin doubled in production between 125 and 4500 mg/L glucose concentration. A peak in fiber formation was observed at 500 mg/L glucose concentration and corresponded with reductions in total GAG production. Fiber formation reduction at 125 and 250 mg/L glucose concentrations are likely due to changes in metabolic activity associated with a limited supply of glucose. These results point to proteoglycan production as a means to manipulate fiber architecture in tissue engineered constructs. STATEMENT OF SIGNIFICANCE: Fibrillar collagens are highly prevalent in the extracellular matrix of all connective tissues; however achieving appropriate assembly and organization of collagen fibers in engineered connective tissues is a persistent challenge. Proteoglycans have been implicated in regulating collagen fiber organization both in vivo and in vitro, however little is known about methods to control proteoglycan production and the subsequent fiber organization in tissue engineered menisci. Here, we show that media glucose content can be optimized to control proteoglycan production and collagen fiber assembly, with optimal collagen fiber assembly occurring at sub-physiologic levels of glucose.