The Effect of Subcellular Architecture on Osteogenic Differentiation
Open Access
- Author:
- Hale, Anastasia
- Millennium Scholars Program:
- Biology (BIOL)
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisor:
- Justin Lee Brown, Thesis Supervisor
- Keywords:
- Biomedical Engineering
Osteogenic Differentiation
YAP1
nanofibers
tissue engineering - Abstract:
- YAP1 is a transcription factor that has been found to localize in the nucleus of subconfluent cells and also has known roles in the Hippo pathway, which controls the growth of organs and suppresses tumor formation through the regulation of cellular proliferation. Recently, studies have investigated the function of YAP1 in osteogenesis, showing that a nanofiber surface can influence the cell signaling pathway and the localization of transcription factors, such as YAP1, specifically through the FAK/RhoA pathway. In order to investigate this phenomenon, I seeded human mesenchymal stem cells (hMSCs) on both electrospun nanofibers and flat PMMA coated surfaces. I used immunofluorescence to quantify relative YAP1 localization in the nucleus and cytoplasm, the number of focal adhesions (measured through vinculin), and FAK phosphorylation. I also stained the actin cytoskeleton and the nucleus in order to measure cell area, circularity, and symmetry. Additionally, I quantified alkaline phosphatase (ALP) to measure relative levels of osteogenic differentiation. Result show that nanofibrous architecture increases nuclear YAP1 localization and focal adhesion size compared to control flat surfaces. Additionally, the nanofiber surface promotes growth of cells with a decreased area and increased symmetry. Cells grown on nanofibrous surfaces also show greater expression of ALP, a marker of osteogenic differentiation. Ultimately, it can be concluded that nanofiber surfaces as compared to flat controls promote osteogenic differentiation through the FAK/RhoA/YAP1 pathway due to increased nuclear localization of YAP1, greater focal adhesion size, and enhanced expression of ALP in cells grown on nanofibrous materials. Understanding the FAK/RhoA/YAP1 pathway through modification of substrate architecture may aid in the development and optimization of biomaterials for tissue regeneration.