Meniscus fibrochondrocytes mechano-hypoxia transduction

半月板纤维软骨细胞(MFC)被包裹在富含胶原和蛋白多糖的细胞外基质(ECM)中，它们合成并存在于膝关节内机械活跃和低氧压(低氧；1-7%O2)的微环境中。但对于MFC如何同时接收和破译机械负荷和低氧刺激，目前还没有基本的理解。*本提案旨在确定和描述MFC在MFC合成的ECM中如何同时响应机械刺激和低氧刺激。这一研究项目的发现将创造关于MFC细胞生理学的新知识，并创建一个与生理学相关的模型系统来研究MFC对机械载荷和缺氧的联合反应。我们已经证明转录因子--缺氧诱导因子-1(HIF-1)在MFC对低氧(5%O2)的反应中起重要作用。此外，低氧可增强MFC的基质形成表型。其他研究人员已报道，体外培养后，低氧增加了ECM植入关节软骨细胞的力学特性。为此，低氧信号在机械活动膝关节内MFC的生理学中可能起着至关重要的作用。然而，机械负荷和低氧对MFC基质形成表型的协同作用尚未在转录和翻译水平上被探索或理解。*我们将通过结合组织工程策略、转录组图谱和使用通路聚焦的基因表达PCR阵列技术，研究MFC合成基质中机械信号通路和缺氧信号通路的同时相互作用。以途径为中心的聚合酶链式反应阵列将使我们能够缩小范围并识别在机械负荷和低氧联合作用下同时调节的基因。我们将结合我们新收购的加拿大创新基金会(CFI)资助的TA Instruments机械加载生物反应器和BiSpherix XVIVO X3缺氧工作站的功能，探索嵌入由MFC体外培养形成的组织工程化ECM中的MFC机械负载和缺氧相结合的分子途径。实验后的分析将包括转录分析、定量聚合酶链式反应、蛋白质阵列、免疫荧光、组织学和生化分析。计划中的实验最终将通过确定联合机械负荷和缺氧对MFC的转录和翻译结果，在细胞生理学和自然科学中创造新的知识，并可能被证明对机械生物学领域具有重要意义。

Meniscus fibrochondrocytes (MFCs) are embedded in a collagen rich and proteoglycan constitutive extracellular matrix (ECM), which they synthesize and exist within a mechanically active and low oxygen tension (hypoxia; 1-7% O2) microenvironment within the knee. But there is no fundamental understanding on how MFCs simultaneously receive and decipher mechanical loads and hypoxic stimuli. ******This proposal aims to identify and characterize how MFCs simultaneously respond to mechanical and hypoxic stimuli within MFCs synthesized ECM. The findings of this research program will create new knowledge on MFC cell physiology as well as create a physiologically relevant model system to study the response MFCs to combined mechanical loads and hypoxia. ******We have demonstrated that the transcription factor, hypoxia inducible factor 1 (HIF-1), plays a significant role in the response of MFCs to hypoxia (5%O2). Moreover, hypoxia enhanced the matrix-forming phenotype of MFCs. Other investigators have reported that hypoxia increased the mechanical characteristics of ECM embedding articular chondrocytes after in vitro culture. To this end, hypoxia signaling may be of critical importance in the physiology of MFCs within a mechanically active knee. However, the concerted interplay of mechanical loads and hypoxia on MFCs' matrix-forming phenotype is yet to be explored or understood at both the transcriptional and translational level. ******We will investigate the simultaneous interplay of mechanical and hypoxia signaling pathways in MFCs embedded in MFCs synthesized matrix through the combination of tissue engineering strategies, transcriptome profiling and use of pathway-focused gene expression PCR array technology. Pathway-focused PCR array will enable us to narrow down and identify genes that are simultaneously modulated under combined mechanical loads and hypoxia. We will combine the capabilities of our newly acquired Canadian Foundation for Innovation (CFI) funded TA Instruments mechanical loading bioreactor and a Biospherix Xvivo X3 hypoxia workstation to explore the molecular pathways associated with combined of mechanical load and hypoxia in MFCs embedded in tissue engineered ECM formed from in vitro culture of MFCs. Post-experiment analyses will include transcriptomic analysis, qPCR, protein arrays, immunofluorescence, histology and biochemical assays. The planned experiments will ultimately create new knowledge in cell physiology and natural sciences by determining the transcriptional and translational outcome of combined mechanical loads and hypoxia on MFCs, and may prove significant to the fields of mechanobiology.