Meniscus fibrochondrocytes mechano-hypoxia transduction

半月板纤维软骨细胞（mfc）嵌入富含胶原蛋白和蛋白聚糖的细胞外基质（ECM）中，它们在膝关节内机械活性和低氧张力（缺氧；1-7% O2）的微环境中合成并存在。但对于mfc如何同时接受和解读机械负荷和缺氧刺激，目前还没有基本的认识。******本提案旨在确定和表征mfc如何同时对mfc合成ECM中的机械和缺氧刺激做出反应。本研究项目的发现将为MFC细胞生理学创造新的知识，并创建一个生理学相关的模型系统来研究MFC对联合机械负荷和缺氧的反应。******我们已经证明转录因子缺氧诱导因子1 （HIF-1）在mfc对缺氧（5%O2）的反应中起着重要作用。此外，缺氧增强了mfc的基质形成表型。其他研究者报道缺氧增加体外培养后ECM包埋关节软骨细胞的力学特性。为此，缺氧信号在机械活动膝关节内mfc的生理学中可能是至关重要的。然而，在转录和翻译水平上，机械负荷和缺氧对mfc基质形成表型的协同相互作用尚未被探索或理解。******我们将通过结合组织工程策略、转录组分析和通路聚焦基因表达PCR阵列技术，研究嵌入mfc合成基质中的mfc中机械和缺氧信号通路的同时相互作用。途径聚焦PCR阵列将使我们能够缩小和识别在机械负荷和缺氧联合作用下同时调节的基因。我们将结合我们新获得的加拿大创新基金会（CFI）资助的TA Instruments机械负载生物反应器和Biospherix Xvivo X3缺氧工作站的能力，探索mfc在体外培养形成的组织工程ECM中嵌入机械负载和缺氧相关的分子途径。实验后分析将包括转录组分析、qPCR、蛋白质阵列、免疫荧光、组织学和生化分析。计划中的实验将最终通过确定联合机械负荷和缺氧对mfc的转录和翻译结果来创造细胞生理学和自然科学的新知识，并可能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.