Mouse Anti newt Collagen type XII Hybridoma [NU3]

Background: For tendon tissue engineering, tenocyte-seeded scaffolds are a promising approach. Under conventional 2D culture however, tenocytes show rapid senescene and phenotype loss. We hypothesized that phenotype loss could be counteracted by simulated microgravity conditions. Methods: Human tenocytes were exposed to microgravity for 9 days on a Random Positioning Machine (RPM). Formation of 3D-structures (spheroids) was observed under light microscopy, gene expression was measured by real-time PCR. Cells under conventional 2D-culture served as control group. Results: Simulated microgravity reached a value of as low as 0.003g. Spheroid formation was observed after 4 days, and spheroids showed stable existance to the end of the observation period. After 9 days, spheroids showed a significantly higher gene expression of collagen 1 (Col1A1) compared to adherent cells under microgravity (4.4x, p=0.04) and compared to the control group (5.6x, p=0.02). Gene expression of collagen 3 (COL3A1) was significantly increased in spheroids compared to the control group (2.3x, p=0.03). Gene expressions of the extracellular matrix genes Tenascin C und Fibronectin (TNC and FN) were increased in adherent cells under microgravity compared to the 1g-control group, not reaching statistical significance (p=0.1 and p=0.3). For the gene expression of vimentin, no significant alteration was observed both in the adherent cells and in the spheroids compared to the 1g control group. Gene expression of the tenocyte-specific transcription factor scleraxis (SCX) was significantly increased in spheroids compared to the control group (3.7x, p=0.03). Conclusion: Simulated microgravity could counteract tenocyte senescence in vitro and serve as a promising model for scaffold-free 3D cell culturing and tissue engineering.

Cardiac fibrosis is characterized by excessive extracellular matrix deposition that contributes to compromised cardiac function and potentially heart failure. Cardiac pressure overload resulting from trans-aortic constriction in mice leads to cardiac fibrosis and increased Wnt/beta-catenin signaling in cardiac fibroblasts. Here, we conditionally induce beta-catenin loss of function in resident cardiac fibroblasts using Tcf21(MerCreMer) or in activated cardiac fibroblasts using periostin (Postn)(MerCreMer). We show that beta-catenin loss of function in cardiac fibroblasts after trans-aortic constriction significantly preserves cardiac function, and reduces interstitial fibrosis but does not alter the numbers of activated or differentiated cardiac fibroblasts in vivo. However, beta-catenin is specifically required in resident cardiac fibroblasts for fibrotic excessive extracellular matrix gene expression and binds Col3a1 and Postn gene sequences in cultured cardiac fibroblasts after induction of Wnt signaling. Moreover, cardiomyocyte hypertrophy is blunted with cardiac fibroblast-specific loss of beta-catenin after trans-aortic constriction in vivo. Thus, Wnt/beta-catenin signaling in resident cardiac fibroblasts is required for excessive extracellular matrix gene expression and collagen deposition after trans-aortic constriction.