Disparate response of articular- and auricular-derived chondrocytes to oxygen tension

Abstract

Purpose/aim: To determine the effect of reduced (5%) oxygen tension on chondrogenesis of auricular-derived chondrocytes. Currently, many cell and tissue culture experiments are performed at 20% oxygen with 5% carbon dioxide. Few cells in the body are subjected to this supra-physiological oxygen tension. Chondrocytes and their mesenchymal progenitors are widely reported to have greater chondrogenic expression when cultured at low, more physiological, oxygen tension (1-7%). Although generally accepted, there is still some controversy, and different culture methods, species, and outcome metrics cloud the field. These results are, however, articular chondrocyte biased and have not been reported for auricular-derived chondrocytes.

Materials and methods: Auricular and articular chondrocytes were isolated from skeletally mature New Zealand White rabbits, expanded in culture and differentiated in high density cultures with serum-free chondrogenic media. Cartilage tissue derived from aggregate cultures or from the tissue engineered sheets were assessed for biomechanical, glycosaminoglycan, collagen, collagen cross-links, and lysyl oxidase activity and expression.

Results: Our studies show increased proliferation rates for both auricular and articular chondrocytes at low (5%) O2 versus standard (20%) O2. In our scaffold-free chondrogenic cultures, low O2 was found to increase articular chondrocyte accumulation of glycosaminoglycan, but not cross-linked type II collagen, or total collagen. Conversely, auricular chondrocytes accumulated less glycosaminoglycan, cross-linked type II collagen and total collagen under low oxygen tension.

Conclusions: This study highlights the dramatic difference in response to low O2 of chondrocytes isolated from different anatomical sites. Low O2 is beneficial for articular-derived chondrogenesis but detrimental for auricular-derived chondrogenesis.

Keywords: Articular chondrocytes; auricular chondrocytes; cartilage tissue engineering; chondrogenesis; collagen cross-linking; oxygen tension.

Figure 1

Comparison of proliferation rates under different oxygen tensions. Low oxygen tension increased the rate of proliferation for both auricular and articular chondrocytes. This increase was greater for articular chondrocytes (16% vs. 3%). Auricular chondrocytes proliferated 2-fold faster than articular chondrocytes at 20% O₂ and 1.8-fold faster at 5% O₂ (p < 0.0001). Symbols indicate the mean for an experiment, and lines connect experiments. Bars show mean ± S.D.

Figure 2

Aggregate wet weights from chondrocytes grown and differentiated at 5% and 20% O₂. Wet weight was not significantly different between aggregates cultured at either oxygen tension for auricular chondrocytes but was significantly enhanced by low oxygen tension for articular chondrocytes. Auricular chondrocytes produce aggregates that are significantly heavier regardless of oxygen tension (p < 0.0001). Symbols indicate the mean for an experiment, and lines connect experiments. Bars show mean ± S.D.

Figure 3

GAG accumulation in aggregates. Auricular chondrocyte aggregates cultured under low oxygen tension accumulated significantly less GAG than those cultured at standard O₂. Articular chondrocytes displayed the opposite trend and accumulated significantly more GAG at low O₂. Auricular chondrocyte aggregates grown at standard O₂ had significantly more GAG than articular chondrocyte aggregates (p < 0.0001). Symbols indicate the mean of an experiment, and lines connect the experiment. Bars show mean ± S.D.

Figure 4

Collagen accumulation in aggregates. Auricular chondrocyte aggregates cultured under low oxygen tension had significantly less collagen content than those cultured at standard O₂. No consistent effect on collagen content was evident for articular chondrocytes. Symbols indicate the mean from an experiment, and lines connect the experiment. Bars show mean ± S.D.

Figure 5

Lysyl oxidase expression and activity in aggregates. (A) Oxygen tension had no consistent effect on lysyl oxidase expression in auricular chondrocyte aggregates. LOX trended toward higher expression at low oxygen tension with articular chondrocyte aggregates. Auricular chondrocytes had significantly higher gene expression than articular chondrocytes of lysyl oxidase at each oxygen tension (p < 0.05). (B) In terms of activity, there was a significant decrease in auricular chondrocytes grown under low oxygen tension but no significant effect on the articular chondrocytes. Lysyl oxidase activity was significantly higher per cell in auricular chondrocytes over articular chondrocytes only at 20% oxygen tension (p < 0.05). Symbols indicate the mean from an experiment, and lines connect the experiment. Bars show mean ± S.D.

Figure 6

Collagen cross-link density in aggregates. Collagen cross-link density was significantly lower for auricular chondrocyte tissue grown at low oxygen tension, but no consistent change was seen for the articular chondrocytes. Auricular chondrocytes grown at standard O₂ had significantly more cross-linking (p < 0.01). Symbols indicate the mean from an experiment, and lines connect the experiment. Bars show mean ± S.D.

Figure 7

Histology and immunohistochemistry. Safranin-O/Fast green staining shows greater accumulation of GAG in auricular chondrocyte aggregates differentiated at 20% O₂ vs. 5% O₂; the reverse is true for the articular cartilage aggregates. Collagen type II staining (E–H) shows no apparent difference in the auricular chondrocyte aggregates but is clearly stronger in articular chondrocyte aggregates at 5% O₂ vs. 20% O₂. Collagen type X staining (I–L) is stronger in auricular chondrocyte aggregates at 20% O₂ vs. 5% O₂ and is more matrix associated. In articular chondrocyte aggregates, there is a slightly stronger staining at 5% O₂ vs. 20% O₂. Elastin staining (M–P) was weak across all samples with a slightly greater accumulation at 20% O₂ for the auricular chondrocyte aggregates only.

Figure 8

Higher magnification histology and immunohistochemistry. Safranin-O/Fast green staining shows greater accumulation of GAG in auricular chondrocyte aggregates differentiated at 20% O₂ vs. 5% O₂; the reverse is true for the articular cartilage aggregates (A–D). Collagen type II staining (E–H) shows no apparent difference in the auricular chondrocyte aggregates but is clearly stronger in articular chondrocyte aggregates at 5% O₂ vs. 20% O₂. Collagen type X staining (I–J) is stronger in auricular chondrocyte aggregates at 20% O₂ vs. 5% O₂ and is more matrix associated. In articular chondrocyte aggregates there is a slightly stronger staining at 5% O₂ vs. 20% O₂. Elastin staining (M–P) was weak across all samples with a greater accumulation at 20% O₂ for the auricular chondrocyte aggregates only.

Figure 9

Biomechanical testing of articular chondrocyte sheets. Articular chondrocyte derived, scaffold-free, tissue engineered sheets have greater mechanical stiffness when cultured at low (5%) oxygen tension than at standard (20%) O₂. Auricular sheets did not form testable constructs at low oxygen tension. Symbols indicate the mean from an experiment, and lines connect the experiment. Bars show mean ± S.D.

Figure 10

Type II and type XI collagen heterpolymer formation in aggregates. (A) Coomasie blue-stained SDS-PAGE gel of pepsin solubilized collagen showing α1(II), α1(XI), α2(XI) and α1(II) chains. (B) Western blot of samples equivalent to those in A and probed with anti-type II collagen antibody (1C10) confirmed type II collagen chains synthesized by auricular and articular chondrocytes under standard and low O₂ conditions. (C) Western blot of samples identical to those in B and probed with antibody 5890. This antibody specifically recognizes the N-telopeptide domain of α1(XI) collagen when cross- linked to chains of α1(II) and β1(II). (D) Western blot of samples identical to those electrophoresed in B (above) and probed with mAb 10F2. This antibody specifically recognizes the C- telopeptide domain of type II collagen when it is cross-linked to α1(II) collagen chains as we have shown before for murine cartilage (34). The antibody also detected the α1(XI) chain in articular chondrocyte matrix grown in 20% O₂ but not under 5% O₂.