Effect of Nutrient Availability on Tissue Formation
To determine the effect of nutrient availability on cartilaginous tissue formation, isolated articular chondrocytes were seeded in high-density 3D culture and maintained in varying amounts of media (1 – 8 mL or 0.5 – 4 mL/106 cells) under normoxic conditions for a period of 4 weeks. Increasing media availability appeared to significantly accelerate cartilaginous tissue deposition, as demonstrated by the changes in construct mass and ECM accumulation compared to 1 mL control (Figure 1a, Table 1). The observed response was non-monotonic with intermediate media volumes (4 mL or 2 mL/106 cells) eliciting the greatest effect on both GAG and collagen deposition (3.5-fold and 2.2-fold increase, respectively) without associated increases in cellularity (Figure 1a, Table 1). Histological assessment revealed similar trends with maximal tissue thickness (~ 0.5 mm) achieved under intermediate media volumes. Under all media volumes investigated, there were no observed changes in phenotype (Figure 1b) with tissue constructs staining positive for sulphated proteoglycans and collagen. Further analysis of collagen deposition (by immunohistochemical staining) indicated that the accumulated ECM was primarily collagen II, with only sporadic intracellular staining observed for collagen I. Finally, similar studies with rabbit and human chondrocytes (Figure S1) displayed strikingly similar trends suggesting this effect is conserved across different species.