Chondrocyte Response to Cartilage Injury

描述（由申请人提供）：已知关节的急性创伤性损伤会增加继发性骨关节炎的风险，但尚不清楚这一过程是如何发生的。这种可导致骨关节炎风险增加的离散事件的存在激发了人们对开发体外创伤性关节损伤模型的兴趣。虽然动物和人类研究主要集中在完整关节的损伤上，但体外模型主要集中在了解损伤性机械压迫对关节软骨本身的影响。尽管体外模型不能解决所有损伤反应发生的事件，但它们可以量化特定事件和机制，包括明确的软骨加载方案的影响。除了描述损伤性压迫对基质和软骨细胞的影响外，还可以收集证据来描述软骨细胞如何以及为什么做出反应。本建议的具体目的是：(1)利用我们新开发的体外损伤模型，涉及在存在（或不存在）细胞因子（包括IL-1， tnf - α, IL-6）的情况下培养的机械损伤软骨，量化损伤和细胞因子对软骨细胞介导的基质转换的协同作用；(2)扩展和利用我们新开发的体外损伤模型，包括在关节囊组织存在（或不存在）的情况下培养的正常和机械损伤软骨，量化损伤和共培养对软骨细胞介导的分解代谢和合成代谢过程的协同作用；(3)量化损伤与细胞因子或人关节囊组织共培养对人膝关节和踝关节软骨软骨介导的分解代谢和合成代谢过程的协同效应；(4)利用原子力显微镜和高分辨率力谱等分子生物物理方法，定量测定损伤牛和人软骨中软骨细胞合成的基质蛋白聚糖和胶原的分子结构和分子力学功能；(5)确定机械损伤分级水平对富含聚集蛋白ECM分子力学和电力学相关的CS和KS-GAGs基因表达、信号通路和翻译后修饰的影响。

DESCRIPTION (provided by applicant): Acute traumatic injury to a joint is known to increase the risk for the development of secondary osteoarthritis, but it is unclear how this process occurs. The existence of such a discrete event that can lead to an increased risk of osteoarthritis has spurred interest in developing in vitro models of traumatic joint injury. Whereas animal and human studies have focused on injuries to the intact joint, in vitro models have focused on understanding the effect of the injurious mechanical compression primarily on the articular cartilage itself. Although in vitro models cannot address all the events that occur in response to injury, they allow quantification of specific events and mechanisms involving the effects of well-defined loading regimens on cartilage. In addition to describing the effects of injurious compression on the matrix and the chondrocytes, evidence can be gathered to describe how and why the chondrocytes respond. The Specific Aims of this proposal are: (1) Utilize our newly developed in vitro injury model involving mechanically injured cartilage incubated in the presence (and absence) of cytokines (including IL-1, TNF-alpha, IL-6) to quantify the synergistic effects of injury and cytokines on chondrocyte-mediated matrix turnover; (2) Extend and utilize our newly developed in vitro injury model involving normal and mechanically injured cartilage incubated in the presence (and absence) of joint capsule tissue to quantify the synergistic effects of injury and co-culture on chondrocyte mediated catabolic and anabolic processes; (3) Quantify the synergistic effects of injury and coculture with cytokines or human joint capsule tissue on chondrocyte-mediated catabolic and anabolic processes in human knee versus ankle cartilages; (4) Quantify the molecular structure and molecular mechanical function of matrix proteoglycans and collagens synthesized by chondrocytes and lost to the medium from injured bovine and human cartilages, using molecular biophysical methods including atomic force microscopy and high resolution force spectroscopy; and (5) Determine the effects of graded levels of mechanical injury on gene expression, signaling pathways, and post-translational modifications of CS and KS-GAGs relevant to the molecular mechanics and electromechanics of aggrecan-rich ECM.