Grant: Discovery Grants Program - Individual

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.