Date of Award

Spring 1-1-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemical & Biochemical Engineering

First Advisor

Kristi S. Anseth

Second Advisor

Leslie Leinwand

Third Advisor

Stephanie J. Bryant

Fourth Advisor

Andrew P. Goodwin

Fifth Advisor

Jennifer N. Cha

Abstract

This thesis presents the development of hydrogel platforms to study the fibroblast-to-myofibroblast transition in valvular interstitial cells (VICs). These systems were used to characterize the effects of extracellular matrix cues on VICs, as well as the synergies between mechanical and biochemical signals. First, the impact of culture platform on VIC phenotype was assessed by culturing VICs in peptide-functionalized poly(ethylene glycol) hydrogels (2D and 3D) and comparing them to those cultured on tissue culture polystyrene (TCPS). Expression of the myofibroblast marker α-smooth muscle actin (αSMA), as well as by a global analysis of the transcriptional profiles1 demonstrated that TCPS caused significant perturbations in gene expression from the native VIC phenotype. The dimensionality of the hydrogel (2D vs 3D) was particularly influential in the regulation of genes related to cell structure and motility, developmental processes, proliferation and differentiation, and transport; these findings motivated the use of 3D cultures for the following experiments.

The effect of matrix modulus, particularly matrix stiffening, on encapsulated VICs was investigated2. To vary the matrix modulus without dramatic changes in VIC morphology, a method was developed for in situ stiffening of cell-laden hydrogels using sequential gelation steps. In contrast with prior findings in 2D, increased stiffness resulted in lower levels of myofibroblast activation, and suggested that stiffness alone was not sufficient to cause pathological activation of VICs to the myofibroblast phenotype in 3D. To facilitate the investigation of additional stimuli in a physiologically-relevant context, a high-throughput technique to encapsulate VICs within 3D hydrogels was developed and used to study VIC response to dynamic changes in matricellular signals3. A thiol-ene photoclick reaction provided temporal control over the presentation of peptide ligands to study their effects on VIC morphology and myofibroblast properties. Collectively, these studies demonstrate the ability to study and direct VIC phenotype through the temporal presentation of mechanical and biochemical cues in 3D polymer matrices.

References

1. Mabry, K. M., Payne, S. Z. & Anseth, K. S. Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype. Submitted.

2. Mabry, K. M., Lawrence, R. L. & Anseth, K. S. Dynamic stiffening of poly(ethylene glycol)-based hydrogels to direct valvular interstitial cell phenotype in a three-dimensional environment. Biomaterials 49, 47–56 (2015).

3. Mabry, K. M., Schroeder, M. E., Payne, S. Z. & Anseth, K. S. Three-dimensional high-throughput cell encapsulation platform to

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