Mechanically Tuned Sandwich Based 3D Hydrogels for Investigating the Fibroblast to Myofibroblast Transition
Date of Award
Master of Science in Biomedical Sciences
Abigail Hielscher, PhD
Xinyu Wang, PhD
Harold Komiskey, PhD, JD
Richard White, PhD
The breast cancer microenvironment is characterized by having an abnormal extracellular matrix (ECM), a protein-rich, non-cellular entity that provides structural support to the tissue. The abnormal ECM in the breast cancer microenvironment may be a result of fibroblast to myofibroblast transition. These myofibroblasts can promote growth and metastasis of the breast tumor by depositing additional ECM and making a stiffer stroma. The purpose of this study is to establish whether human mammary fibroblasts (HMFs) transition to activated myofibroblasts following culture in a mechanically tuned 3D gelatin based sandwich hydrogel. HMFs grown atop mechanically tuned gels served as controls (2D). To tune the mechanical stiffness, microbial transglutaminase (mTG), an enzyme that crosslinks lysine and glutamine residues, was used in concentrations of 30, 100, and 200 μg/ml to generate. a compliant, moderate, and stiff hydrogel, respectively. The HMFs were added to the compliant, moderate and stiff hydro gels and allowed 2 hours to adhere. To create the 3D hydrogel sandwich, a top compliant gel (30 μg/ml) was added. A western blot was performed on conditioned media of 3D hydrogels at day 2 and 4 to look at ECM protein expression for collagen I, collagen IV, Fibronectin, and Fibronectin ED-A. Results from these experiments were inconclusive. Next, we sought to determine if an increase in stiffness promoted TGF-β signaling. An ELISA was performed to quantify secreted TGF-β and results show that TGF-β levels increased between days 2 and 4 of culture for all tested stiffnesses in 2D and 3D gels. Comparing 2D and 3D results, TGF-β was decreased in 3D compared to 2D. Another ELISA was done on collected conditioned media for collagen and fibronectin. Here we found the results to show an increase of expression for both collagen I and fibronectin in the 2D model and at culture day 4. Last, confocal microscopy was used to determine whether increased mechanical stiffness promotes α-SMA co-localization with actin stress fibers. Findings from confocal microscopy show an increase in actin stress fibers in the 3D 30μg gels but a decrease in 100μg and 200μg gels. Overall these results do not support a robust myofibroblast phenotype with an increased mechanical stiffness. These findings illustrate that additional work will be needed in order to elucidate how mechanical stiffness in 3D supports a fibroblast to myofibroblast transit.
Byrd, Jana, "Mechanically Tuned Sandwich Based 3D Hydrogels for Investigating the Fibroblast to Myofibroblast Transition" (2018). PCOM Biomedical Studies Student Scholarship. 162.