Huser C A M, Peacock M, Davies M E
Comparative Orthopaedics Research Group, University of Cambridge, UK.
Osteoarthritis Cartilage. 2006 Oct;14(10):1002-10. doi: 10.1016/j.joca.2006.03.012. Epub 2006 May 12.
Chondrocyte death, a notable feature of osteoarthritis, may play a role in the initiation of cartilage degeneration. The present study was aimed at uncovering the nature and involvement of cell death in the initiation of cartilage degeneration induced by mechanical trauma.
Articular cartilage discs obtained from healthy skeletally mature horses were subjected to a single-impact load (500 g from 50 mm) using a simple drop-tower device and cultured in vitro for 48 h. Chondrocyte death was examined using two independent methods: transmission electron microscopy and caspase-3 activity assay. To elucidate the signalling mechanisms involved in impact-induced cell death measured by terminal deoxynucleotidyl transferase-deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL), cartilage discs were incubated with specific caspase-3, -8 and -9 inhibitors prior to impact. Additionally, weight gain and glycosaminoglycan (GAG) release, markers of cartilage degeneration were monitored.
After 48 h, ultrastructural evidence of apoptosis was observed. Caspase-3 was activated after 12h of culture post-impact. When quantified by TUNEL, impact trauma induced death in 52.6% of superficial chondrocytes after 48 h in culture, compared to 4.2% in unimpacted controls. Specific caspases-3 and -9 inhibitors significantly reduced impact-induced apoptosis to 24.3% and 14.7%, respectively. Caspase-8 inhibition had no effect on chondrocyte death (60.3%). Impact-induced GAG release into the medium was significantly reduced by inhibition of cell death, but weight gain remained unaffected by caspase inhibition.
These results suggest that impact trauma-induced chondrocyte death is predominantly due to caspase-9-dependent apoptosis and is linked to cartilage degeneration.
软骨细胞死亡是骨关节炎的一个显著特征，可能在软骨退变的起始过程中发挥作用。本研究旨在揭示机械创伤诱导的软骨退变起始过程中细胞死亡的本质及参与情况。
从健康骨骼成熟的马获取关节软骨盘，使用简单的落塔装置施加单次冲击负荷（从50毫米高度落下500克重物），并在体外培养48小时。使用两种独立方法检测软骨细胞死亡：透射电子显微镜和半胱天冬酶-3活性测定。为阐明通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）检测的冲击诱导细胞死亡所涉及的信号传导机制，在冲击前将软骨盘与特异性半胱天冬酶-3、-8和-9抑制剂孵育。此外，监测体重增加和糖胺聚糖（GAG）释放，这是软骨退变的标志物。
48小时后，观察到凋亡的超微结构证据。冲击后培养12小时，半胱天冬酶-3被激活。通过TUNEL定量分析，培养48小时后，冲击创伤导致52.6%的表层软骨细胞死亡，而未受冲击的对照组为4.2%。特异性半胱天冬酶-3和-9抑制剂分别将冲击诱导的凋亡显著降低至24.3%和14.7%。半胱天冬酶-8抑制对软骨细胞死亡无影响（60.3%）。抑制细胞死亡可显著减少冲击诱导的GAG释放到培养基中，但半胱天冬酶抑制对体重增加无影响。
这些结果表明，冲击创伤诱导的软骨细胞死亡主要是由于半胱天冬酶-9依赖性凋亡，并且与软骨退变有关。
