Heywood Hannah K, Lee David A
School of Engineering and Materials Science, Queen Mary University of London, London, UK.
J Cell Physiol. 2010 Jan;222(1):248-53. doi: 10.1002/jcp.21946.
Autologous chondrocyte implantation requires a phase of in vitro cell expansion, achieved by monolayer culture under atmospheric oxygen levels. Chondrocytes reside under low oxygen conditions in situ and exhibit a glycolytic metabolism. However, oxidative phosphorylation rises progressively during culture, with concomitant reactive oxygen species production. We determine if the high oxygen environment in vitro provides the transformation stimulus. Articular chondrocytes were cultured in monolayer for up to 14 days under 2%, 5%, or 20% oxygen. Expansion under 2% and 5% oxygen reduced the rate at which the cells developed an oxidative phenotype compared to 20% oxygen. However, at 40 +/- 4 fmol cell(-1) h(-1) the oxygen consumption by chondrocytes expanded under 2% oxygen for 14 days was still 14 times the value observed for freshly isolated cells. Seventy-five to 78% of the increased oxygen consumption was accounted for by oxidative phosphorylation (oligomycin sensitive). Expansion under low oxygen also reduced cellular proliferation and 8-hydroxyguanosine release, a marker of oxidative DNA damage. However, these parameters remained elevated compared to freshly isolated cells. Thus, expansion under physiological oxygen levels reduces, but does not abolish, the induction of an oxidative energy metabolism. We conclude that simply transferring chondrocytes to low oxygen is not sufficient to either maintain or re-establish a normal energy metabolism. Furthermore, a hydrophobic polystyrene culture surface which promotes rounded cell morphology had no effect on the development of an oxidative metabolism. Although the shift towards an oxidative energy metabolism is often accompanied by morphological changes, this study does not support the hypothesis that it is driven by them.
自体软骨细胞移植需要一个体外细胞扩增阶段，这通过在大气氧水平下的单层培养来实现。软骨细胞在原位处于低氧条件下，并表现出糖酵解代谢。然而，在培养过程中氧化磷酸化会逐渐增加，同时产生活性氧。我们确定体外的高氧环境是否提供了转化刺激。将关节软骨细胞在2%、5%或20%氧气条件下进行单层培养长达14天。与20%氧气条件相比，在2%和5%氧气条件下扩增可降低细胞形成氧化表型的速率。然而，在2%氧气条件下扩增14天的软骨细胞耗氧量为40±4飞摩尔细胞⁻¹ 小时⁻¹，仍是新鲜分离细胞观察值的14倍。增加的耗氧量中有75%至78%是由氧化磷酸化（对寡霉素敏感）引起的。在低氧条件下扩增还减少了细胞增殖和8-羟基鸟苷释放，8-羟基鸟苷是氧化DNA损伤的标志物。然而，与新鲜分离的细胞相比，这些参数仍然升高。因此，在生理氧水平下扩增可减少但不能消除氧化能量代谢的诱导。我们得出结论，仅仅将软骨细胞转移到低氧环境不足以维持或重新建立正常的能量代谢。此外，促进细胞呈圆形形态的疏水性聚苯乙烯培养表面对氧化代谢的发展没有影响。尽管向氧化能量代谢的转变通常伴随着形态变化，但本研究不支持其由形态变化驱动这一假设。
