Bioartificial matrices to modulate epithelial morphogenesis

Enemchukwu, Nduka Obichukwu

Title:

Bioartificial matrices to modulate epithelial morphogenesis

dc.contributor.advisor	García, Andrés J.
dc.contributor.author	Enemchukwu, Nduka Obichukwu
dc.contributor.committeeMember	Nusrat, Asma
dc.contributor.committeeMember	Barker, Thomas H.
dc.contributor.committeeMember	Collard, David M.
dc.contributor.committeeMember	Dixon, J. Brandon
dc.contributor.department	Mechanical Engineering
dc.date.accessioned	2015-01-12T20:28:32Z
dc.date.available	2015-01-13T06:30:04Z
dc.date.created	2013-12
dc.date.issued	2013-09-20
dc.date.submitted	December 2013
dc.date.updated	2015-01-12T20:28:32Z
dc.description.abstract	Acute injury of major epithelial organ systems (kidney, liver, lung, etc.) is collectively a principal cause of death worldwide. Regenerative medicine promises to meet these human health challenges by harnessing intrinsic cellular processes to repair or replace damaged tissues. Epithelial morphogenesis is a hard-wired, multicellular differentiation program that dynamically integrates microenvironmental cues to coordinate cell fate processes including adhesion, migration, proliferation, and polarization. Thus, epithelial morphogenesis is an instructive mode of tissue assembly, maintenance, and repair. Three-dimensional epithelial cell cultures in natural basement membrane (BM) extracts produce hollow, spherical cyst structures and have indicated that the BM provides the critical cell adhesion ligands to facilitate cell survival, stimulate proliferation, and promote polarization and lumen formation. However, the utility of natural BMs for detailed studies is generally limited by lot-to-lot variations, uncontrolled cell adhesive interactions, or growth factor contamination. The goal of this thesis was to engineer bioartificial extracellular matrices (ECM) that would support and modulate epithelial cyst morphogenesis. We have engineered hydrogels, based on a multi-arm maleimide-terminated poly (ethylene glycol) (PEG-4MAL), that present cell adhesive molecules and enzymatic degradation substrates and promote polarized epithelial cyst differentiation in vitro. To investigate the influence of matrix physical and biochemical signals on cyst morphogenesis, we independently varied the polymer weight percentage (wt%), the density of a cell adhesion ligand (RGD), and crosslink degradation rates of the hydrogels. Then, we evaluated functional outcomes including Madin-Darby canine kidney (MDCK II) epithelial cell survival, proliferation, cyst polarization, and lumen formation. We found that cell proliferation, but not cell survival, was sensitive to the polymer wt%, which is related to elastic modulus and crosslink density. This result defined a working range of PEG-4MAL concentration (3.5% - 4.5%) that promotes robust proliferation. Analysis of mature cysts indicated that 4.0% and 4.5% gels produced cysts resembling those typically grown in type I collagen gels while 3.5% gels produced cysts with higher incidence of inverted polarity and multiple lumens. Perturbation of matrix degradability using a slow-degrading crosslink peptide or matrix metalloproteinase inhibitors showed that the rate of matrix degradation exerts major influence on cyst growth in PEG-4MAL gels. We employed 4.0% PEG-4MAL hydrogels with RGD ligand density ranging over 0 – 2000 uM to discover that (1) lumen formation was eliminated in the absence of RGD, (2) extent of lumen formation increased with increasing RGD concentration, and (3) cyst polarity was inverted below a threshold of integrin binding to RGD. Together, these results show that the biochemical and physical properties of the matrix, particularly integrin binding and matrix degradability, effectively modulate establishment of apico-basal polarity and lumen phenotypes in MDCK II epithelial cyst structures. Furthermore, these studies validate PEG-4MAL hydrogels as a powerful culture platform to enable detailed investigation of matrix-directed modulation of epithelial morphogenesis.
dc.description.degree	Ph.D.
dc.embargo.terms	2014-12-01
dc.format.mimetype	application/pdf
dc.identifier.uri	http://hdl.handle.net/1853/52938
dc.language.iso	en_US
dc.publisher	Georgia Institute of Technology
dc.subject	Biomaterials
dc.subject	Polyethylene glycol
dc.subject	Epithelial morphogenesis
dc.subject	Cyst
dc.subject	Extracellular matrix
dc.subject	Epithelial cells
dc.title	Bioartificial matrices to modulate epithelial morphogenesis
dc.type	Text
dc.type.genre	Dissertation
dspace.entity.type	Publication
local.contributor.advisor	García, Andrés J.
local.contributor.corporatename	George W. Woodruff School of Mechanical Engineering
local.contributor.corporatename	College of Engineering
relation.isAdvisorOfPublication	6236e450-228b-4532-8b5e-812316ac90f3
relation.isOrgUnitOfPublication	c01ff908-c25f-439b-bf10-a074ed886bb7
relation.isOrgUnitOfPublication	7c022d60-21d5-497c-b552-95e489a06569
thesis.degree.level	Doctoral