Scaffold based tissue reconstruction inherently limits regenerative capacity due to inflammatory response and limited cell migration. In contrast, scaffold-free methods promise formation of functional tissues with both reduced adverse host reactions and enhanced integration. Cell-sheet engineering is a well-known bottom-up tissue engineering approach that allows the release of intact cell sheet from a temperature responsive polymer such as poly-N-isopropylacrylamide (pNIPAAm). pNIPAAm is an ideal template for culturing cell sheets because it undergoes a sharp volume-phase transition owing to the hydrophilic and hydrophobic interaction around its lower critical solution temperature (LCST) of 32°C, a temperature close to physiological temperature. Compared to enzymatic digestion via trypsinization, pNIPAAm provides a non-destructive approach for tissue harvest which retains its basal surface extracellular matrix and preserves cell-to-cell junctions thereby creating an intact monolayer of cell sheet suitable for tissue transplantation.
The overall thrust of this dissertation is to gain a comprehensive understanding of how tissue precursors are formed, harvested and printed from interactions with shape-changing pNIPAAm hydrogel. A simple geometrical microbeam pattern of pNIPAAm structures covalently bound on glass substrates for culturing mouse embryonic fibroblast and skeletal myoblast cell lines is presented. In order to characterize the cell-surface interactions, three main investigations were conducted: 1) the mechanism of cell detachment; 2) the feasibility of micro-contact printing tissue precursors onto target surfaces; and 3) the assembly of these tissues into three-dimensional (3D) constructs.
Detachment of cells from the shape-changing hydrogel was found to correlate with the lateral swelling of the microbeams, which is induced by thermal activation, hydration and shape distortion of the patterns. The mechanism of cell detachment was primarily driven by strain, which occurred almost instantaneously above a critical strain of 25%. This shape-changing pNIPAAm construct allows water penetration from the periphery and beneath the attached cells, providing rapid hydration and detachment within seconds. Cell cultured microbeams were used as stamps for micro-contact printing of tissue precursors and their viability, metabolic activity, local and global organization were evaluated after printing. The formation and printing of intact tissues from the shape-changing hydrogel suggests that the geometric patterning of pNIPAAm directs spatial organization through physical guidance cues while preserving cell functioning. Tissue precursors were sequentially assembled into parallel and perpendicular configurations to demonstrate the feasibility of constructing dense tissues with different organizations such as interconnected cell lines that could induce vascularization to solve perfusion issues in regenerative therapies. The novel approach presented in this dissertation establishes an efficient method for harvesting and printing of tissue precursors that may be applicable for the modular, bottom up construction of complex tissues for organ models and regenerative therapies.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-6829 |
| Date | 01 January 2015 |
| Creators | Akintewe, Olukemi O. |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
| Rights | default |
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