Suits Jocelyne M T, Khan Aasma A, Waldman Stephen D
Department of Chemical Engineering, Queen's University, Kingston, Ontario, Canada.
J Tissue Eng Regen Med. 2008 Aug;2(6):340-6. doi: 10.1002/term.102.
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 microg/construct). The storage of intracellular glycogen in engineered cartilage can be elicited by culturing the constructs in elevated volumes of medium (>or=1 ml medium/million cells), which might help to ensure appropriate metabolic function after implantation.
软骨组织工程最近的重点是开发植入后能够存活的功能性组织。一个这样的决定因素是工程组织在植入后维持其代谢活性的能力。在体内，软骨细胞含有细胞内糖原储备以支持新陈代谢，而这些细胞在体外组织生长过程中是否能够储存糖原尚不清楚。因此，本研究的目的是确定在组织工程软骨中引发糖原储存的合适营养条件。将分离的牛关节软骨细胞接种到无支架的三维培养物中，并在不同的营养条件（葡萄糖浓度和培养基体积）下培养4周。然后评估工程组织的细胞内糖原储存、葡萄糖利用和细胞外基质（ECM）积累。葡萄糖浓度（5-10 mM）和培养基体积（1-4 ml）对软骨组织形成没有明显影响。然而，细胞消耗的葡萄糖与提供的培养基体积成比例增加。乳酸产生也受到类似影响，但与消耗的葡萄糖成正比，表明葡萄糖利用发生了变化。同样，在培养基体积增加的情况下，工程组织的细胞内糖原染色呈阳性，这也通过生化方法得到证实（1 ml，1±2；2 ml，13±4；4 ml，13±3 μg/构建体）。通过在增加体积的培养基（≥1 ml培养基/百万细胞）中培养构建体，可以引发工程软骨中细胞内糖原的储存，这可能有助于确保植入后适当的代谢功能。
