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.