Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO 80309, USA.
Osteoarthritis Cartilage. 2012 Nov;20(11):1326-35. doi: 10.1016/j.joca.2012.06.015. Epub 2012 Jul 10.
The objectives for this study were to determine whether radical initiated photopolymerizations typically employed for cell encapsulations lead to oxidative stress incurred by chondrocytes and whether the development of a pericellular matrix (PCM) decreases this oxidative stress and has longer-term benefits on chondrocyte function.
Freshly isolated bovine chondrocytes were encapsulated in poly(ethylene glycol) (PEG) hydrogels devoid of a PCM or with a PCM, confirmed by immunocytochemistry (IC), and cultured for up to 2 weeks. Reactive oxygen species (ROS) production and damage to cell membrane by lipid peroxidation were accomplished using carboxy-2,7-difluorodihydrofluorescein diacetate (carboxy-H(2)DFFDA) and by malondialdehyde (MDA) content, respectively. Gene expression and proteoglycan synthesis were analyzed using reverse transcription (RT)-quantitative PCR (qPCR) and (35)SO(4) incorporation, respectively.
The photopolymerization reaction, which alone generates radicals and extracellular ROS, led to oxidative stress in chondrocytes evidenced by increased intracellular ROS and lipid peroxidation. The presence of a PCM decreased intracellular ROS and abrogated membrane lipid peroxidation, improved aggrecan, collagen II and collagen VI expression, and enhanced proteoglycan synthesis.
The development of the PCM prior to photoencapsulation in PEG hydrogels reduces oxidative stress and improves chondrocyte anabolic activity. Our data suggest this reduction occurs by decreased ROS diffusion into the cell and decreased membrane damage. Our findings suggest that minimizing oxidative stress, such as through the presence of a PCM, may have long-term beneficial effects on tissue elaboration when employing photopolymerizations to encapsulate chondrocytes for cartilage tissue engineering applications.
本研究旨在确定用于细胞包封的自由基引发光聚合是否会导致软骨细胞产生氧化应激，以及细胞外基质 (PCM) 的形成是否会降低这种氧化应激，并对软骨细胞功能产生长期益处。
将新鲜分离的牛软骨细胞包封在缺乏 PCM 或具有 PCM 的聚乙二醇 (PEG) 水凝胶中，通过免疫细胞化学 (IC) 进行确认，并培养长达 2 周。使用羧基-2,7-二氟二氢荧光素二乙酸酯 (carboxy-H(2)DFFDA) 和丙二醛 (MDA) 含量分别测定活性氧 (ROS) 产生和细胞膜脂质过氧化损伤。使用逆转录 (RT)-定量 PCR (qPCR) 和 (35)SO(4)掺入分别分析基因表达和蛋白聚糖合成。
光聚合反应本身会产生自由基和细胞外 ROS，导致软骨细胞发生氧化应激，表现为细胞内 ROS 和脂质过氧化增加。PCM 的存在降低了细胞内 ROS 和膜脂质过氧化，改善了聚集蛋白聚糖、胶原 II 和胶原 VI 的表达，并增强了蛋白聚糖的合成。
在 PEG 水凝胶中进行光包封之前形成 PCM 可减少氧化应激并提高软骨细胞合成代谢活性。我们的数据表明，这种减少是通过减少 ROS 向细胞内扩散和减少膜损伤来实现的。我们的研究结果表明，通过减少氧化应激（例如通过存在 PCM），当使用光聚合来包封软骨细胞用于软骨组织工程应用时，可能对组织生成具有长期有益的影响。
