Effect of oxygen tension on tissue-engineered human nasal septal chondrocytes

Abstract

Tissue-engineered nasal septal cartilage may provide a source of autologous tissue for repair of craniofacial defects. Although advances have been made in manipulating the chondrocyte culture environment for production of neocartilage, consensus on the best oxygen tension for in vitro growth of tissue-engineered cartilage has not been reached. The objective of this study was to determine whether in vitro oxygen tension influences chondrocyte expansion and redifferentiation. Proliferation of chondrocytes from 12 patients expanded in monolayer under hypoxic (5% or 10%) or normoxic (21%) oxygen tension was compared over 14 days of culture. The highest performing oxygen level was used for further expansion of the monolayer cultures. At confluency, chondrocytes were redifferentiated by encapsulation in alginate beads and cultured for 14 days under hypoxic (5 or 10%) or normoxic (21%) oxygen tension. Biochemical and histological properties were evaluated. Chondrocyte proliferation in monolayer and redifferentiation in alginate beads were supported by all oxygen tensions tested. Chondrocytes in monolayer culture had increased proliferation at normoxic oxygen tension (p = 0.06), as well as greater accumulation of glycosaminoglycan (GAG) during chondrocyte redifferentiation (p < 0.05). Chondrocytes released from beads cultured under all three oxygen levels showed robust accumulation of GAG and type II collagen with a lower degree of type I collagen immunoreactivity. Finally, formation of chondrocyte clusters was associated with decreasing oxygen tension (p < 0.05). Expansion of human septal chondrocytes in monolayer culture was greatest at normoxic oxygen tension. Both normoxic and hypoxic culture of human septal chondrocytes embedded in alginate beads supported robust extracellular matrix deposition. However, GAG accumulation was significantly enhanced under normoxic culture conditions. Chondrocyte cluster formation was associated with hypoxic oxygen tension.

Figure 1.

Light micrographs of human nasal septal chondrocytes cultured as monolayers for 8 days under (A) 5%, (B) 10%, or (C) 21% oxygen tension. Chondrocytes exhibit the elongated morphology typical of dedifferentiated chondrocytes.

Figure 2.

The effect of oxygen tension on chondrocyte proliferation. Human nasal septal chondrocytes from 12 patients were cultured as monolayers for 14 days under (diamond) 5, (square) 10, or (triangle) 21% oxygen tension. There was a trend toward increased cell proliferation when chondrocytes were cultured under normoxic (21%) culture conditions (p = 0.06; mean ± SD).

Figure 3.

The effect of oxygen tension on DNA and sulfated glycosaminoglycan (sGAG) accumulation during chondrocyte redifferentiation. After monolayer expansion under normoxic culture conditions, human nasal septal chondrocytes from 12 patients were redifferentiated by encapsulation and culture in alginate beads for 14 days under (diamond) 5, (square) 10, or (triangle) 21% oxygen tension. Over the 14-day culture period, chondrocyte DNA per bead did not vary significantly and remained similar under the three oxygen tensions tested. In contrast, the amount of sGAG per bead increased with increasing oxygen tension (p < 0.05; mean ± SD).

Figure 4.

The effect of oxygen tension on accumulation of collagen during chondrocyte redifferentiation. After 14 days of redifferentiation in alginate beads, the amount of total collagen per DNA was similar between the three oxygen tensions tested (p > 0.05; mean ± SD).

Figure 5.

Chondrocytes redifferentiated under hypoxic (5 and 10%) and normoxic (21%) oxygen tensions developed substantial extracellular matrix (ECM) after 14 days of redifferentiation by encapsulation and culture in alginate beads. Chondrocytes showed characteristics of differentiated chondrocytes including (A–C) robust accumulation of glycosaminoglycan (GAG) detected by Alcian blue histochemistry as well as (D–F) low levels of type I collagen immunohistochemical staining and (G–I) strong type II collagen immunohistochemistry) with minimal immunohistochemical staining (J–L) with a nonspecific IgG.

Figure 6.

Redifferentiation of human nasal septal chondrocytes in alginate beads under decreased oxygen tension resulted in an increased number of chondrocyte clusters. Linear regression analysis revealed a positive correlation between the number of chondrocyte cell clusters formed and decreasing oxygen tension. Regression line and corresponding R² and p values are shown.