Date of Award

1-1-2013

Document Type

Open Access Dissertation

Department

Chemistry and Biochemistry

Sub-Department

Chemistry

First Advisor

Qian Wang

Abstract

This research focused on developing and using plant virus based scaffolds to understand substrate control over cell alignment and differentiation. The first part of this work centered on development of virus based patterns that were then used to align and elongate aortic smooth muscle cells (SMCs). Virus patterns were generated in capillary tubes via a simple drying method. Three experimental parameters were used to control pattern formation: (1) protein concentration, (2) salt concentration, and (3) hydrophobicity of the pre-deposition surface. By controlling these parameters several aspects of the final virus patterns were controlled. First, virus orientation was controlled. Patterns were made that had rod like virus particles oriented either parallel or perpendicular to the long axis of the capillary tube. Second, patterns were formed that either had a monolayer or multilayer (5 layers) of virus. Finally, virus coverage was controlled. Stripe patterns were formed with different widths of virus stripes and different widths of spacing between virus stripes. Different pattern formations were generated by (1) interfacial assembly of tobacco mosaic virus (TMV) at the air/liquid interface and (2) a pinning-depinning process. These patterns were then used to investigate SMC morphology, elongation, alignment, and differentiation. Our data indicates that the virus based stripe patterns could control SMC morphology and induce the elongation and alignment of SMCs. However, no effect on SMC differentiation was observed.

The second part of this work focused on the use of genetically mutated viruses as substrates to support and improve the differentiation of mesenchymal stem cells (MSCs). The virus surfaces were created by coating high protein binding plates with genetically mutated viruses. These tobacco mosaic virus mutants display selected peptide fragments reported to bind integrin receptors or selected sequences derived from other integrin binding matrix proteins. Differentiation of MSCs into osteoblasts was monitored for 21 days. The differentiation was monitored through alkaline phosphatase activity, calcium quantification, and ELISA quantification of osteopontin and osteocalcin, two important biomarkers for osteogenesis. Experimental evidences generated by cytochemical staining and ELISA showed that certain mutant substrates supported differentiation of MSCs. Furthermore, substrates made of mutant viruses with multivalent RGD displays decreased the time to onset of mineralization.

The third part of this work combined a method for creating ordered virus patterns with the use of mutant TMV particles to create substrates that could both align and differentiate neuroblast and myoblast cells. Specifically, a flow assembly method was used to align either native or mutant TMV particles. The experimental parameters used to control virus alignment and density included: flow rate, virus concentration, salt concentration, and charge of the pre-deposition surface. These aligned virus patterns were then used as substrates to control cell growth. It was found that virus density and alignment played significant roles in the final orientation and differentiation of myoblasts and neuroblasts.

Collectively the research presented in this dissertation used the unique qualities of virus particles to create cell substrates that can align different cell types or modulate the differentiation of different cell types. These substrates may be used in the future to gain more insight into substrate based control over cell alignment and differentiation.

Rights

© 2013, Elizabeth Balizan

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