JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH ARTICLE J Tissue Eng Regen Med 2008; 2: 340–346. Published online 8 July 2008 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/term.102 Glycogen storage in tissue-engineered cartilage Jocelyne M. T. Suits 1,3 , Aasma A. Khan 1,3 and Stephen D. Waldman 1,2,3 * 1 Department of Chemical Engineering, Queen’s University, Kingston, Ontario, Canada 2 Department of Mechanical and Materials Engineering, Queen’s University, Kingston, Ontario, Canada 3 Human Mobility Research Centre, Kingston General Hospital, Kingston, Ontario, Canada Abstract Recent focus in cartilage tissue engineering has been to develop functional tissue that can survive after implantation. One such determinant is the ability of the engineered tissue to be able to sustain its metabolic activity post-implantation. In vivo, chondrocytes contain stores of intracellular glycogen to support metabolism and it is unknown whether these cells can store glycogen during tissue growth in vitro. Thus, the purpose of this study was to determine the appropriate nutrient conditions to elicit glycogen storage in tissue-engineered cartilage. Isolated bovine articular chondrocytes were seeded in scaffold-free, 3D culture and grown under different nutrient conditions (glucose concentrations and media volumes) for 4 weeks. Intracellular glycogen storage, glucose utilization and extracellular matrix (ECM) accumulation of the engineered tissues were then evaluated. Glucose concentration (5 – 10 mM) and media volume (1 – 4 ml) had no apparent effect on cartilaginous tissue formation. However, glucose consumption by the cells increased in proportion to the volume of medium provided. Lactate production was similarly affected but in direct proportion to the glucose consumed, indicating a change in glucose utilization. Similarly, under elevated medium volume, engineered tissues stained positive for intracellular glycogen, which was also confirmed biochemically (1 ml, 1 ± 2; 2 ml, 13 ± 4; 4 ml, 13 ± 3 μg/construct). The storage of intracellular glycogen in engineered cartilage can be elicited by culturing the constructs in elevated volumes of medium (≥1 ml medium/million cells), which might help to ensure appropriate metabolic function after implantation. Copyright  2008 John Wiley & Sons, Ltd. Received 28 January 2008; Revised 24 April 2008; Accepted 22 May 2008 Keywords cartilage; tissue engineering; chondrocytes; glycogen; glucose utilization; extracellular matrix 1. Introduction Articular chondrocytes exist in a near-hypoxic environ- ment (Mankin et al., 1994), as demonstrated by the relatively low oxygen tensions within the cartilage: 5 – 7% O 2 at the articulating surface to <1% O 2 in the deep zone (Silver 1975; Zhou et al., 2004). For this reason, chon- drocytes primarily rely upon the anaerobic metabolism of glucose (glycolysis) to sustain their metabolic activ- ity (Lane et al., 1977; Lee and Urban 1997; Darling and Athanasiou 2003). During glycolysis, available glucose is rapidly converted to pyruvate, generating ATP. As very *Correspondence to: Stephen D. Waldman, Department of Chemical Engineering, Department Mechanical and Materials Engineering, McLaughlin Hall, Room 205, Queen’s University, Kingston, Ontario, Canada K7L 3N6. E-mail: waldman@me.queensu.ca little of the pyruvate enters the citric acid cycle (aerobic pathway), the majority of the pyruvate is then converted to lactic acid by lactate dehydrogenase and NADH (Miller et al., 1989). Since the yield of ATP from glycolysis is relative low (compared to aerobic metabolism), sufficient quantities of glucose are to sustain metabolic activity as well as to synthesize the sugar-containing macro- molecules of the cartilaginous extracellular matrix (ECM), e.g. glycosaminoglycans and glycoproteins (Windhaber et al., 2003). Synovial fluid is a primary source of nutrition for articular cartilage (Lane et al., 1977; Otte, 1991) which is secreted by the cells in the inner synovial membrane (both type A and type B cells; Mankin et al., 1994). Although synovial fluid contains glucose concentrations similar to that of serum (difference <0.5mM; Krachler and Domej, 2001), the available glucose is rapidly depleted within the upper regions of the tissue (Marcus, 1973). Copyright  2008 John Wiley & Sons, Ltd.