β-Catenin Signaling Leading to the Maintenance and Differentiation of MC3T3 Cells in Response to Electrospun Nanofibers

Open Access
Author:
Nagurney, Rebecca Ann
Area of Honors:
Bioengineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Justin Lee Brown, Thesis Supervisor
  • William O Hancock, Honors Advisor
Keywords:
  • Nanofibers
  • Electrospinning
  • PMMA
  • PolyHEMA
  • Polyvinyl Alcohol
  • Beta-Catenin
  • Osteogenesis
Abstract:
Current techniques to direct cells to certain lineages to grow tissue are very expensive and impractical for future applications. Because current techniques are expensive and impractical, scientists and engineers need to develop new techniques and methods that are affordable and can be scaled appropriately. The research for this thesis focused on directing MC3T3-E1 S4 cells to become bone cells by studying the translocation of beta-catenin. Research began by electrospinning polymer fibers of different diameters onto glass coverslips coated with PolyHEMA (PHEMA) and Polyvinyl Alcohol (PVA). The fiber mats produced were seeded with the cells and tested using immunofluorescence and western blotting techniques. The hypothesis of this study was that larger diameter fibers will have more nuclear beta catenin because the cells will tend to wrap around the fibers and form fewer cell-to-cell junctions, whereas smaller diameter fibers will have more cytoplasmic beta-catenin because they will spread on the fibers and reach out to make cell-to-cell junctions. Qualitative experimental results were obtained through immunofluorescence. The images showed beta-catenin along the cell membrane in adherens junctions on the flat surfaces and small nanofiber surfaces. For the large nanofiber surfaces, beta-catenin was seen along the cell membrane in adherens junctions and in the cell nucleus. Quantitative results were obtained through environmental scanning electron microscopy (ESEM) and western blotting. The fiber diameters measured using ESEM and ImageJ showed a significant statistical difference in diameter between the 22% PMMA (small) and 25% PMMA (large) fibers. This enforces the hypothesis that the difference in fiber diameter will have a different effect on cell fate. Western blots measured the intensity of beta-catenin and cadherin throughout the cells that were seeded on the flat and nanofiber surfaces. Trends show an increase in quantity of beta-catenin and cadherin on the nanofiber structures. The trend suggests that the fibers influenced beta-catenin to move throughout the cell and lead to transcriptional changes.