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Biocompatible coatings are often used to improve fixation between implants and tissues. Polyelectrolyte multilayer (PEMU) coatings built layer by layer with alternating pairs of polyelectrolytes can be tuned to improve cell interactions with surfaces. In Chapter 2, we show that human mesenchymal stem cells (hMSCs) induced with bone differentiation medium (BDM) to become osteoblasts, biomineralize crosslinked PEMUs built with the polycation poly(allylamine hydrochloride) (PAH) and the polyanion poly(acrylic acid) (PAA). Degrees of hMSC osteoblast differentiation and surface biomineralization reflect differences in extracellular matrix (ECM) deposited and organized by the cells on the smooth PAH-terminated PEMUs (PAH-PEMUs) and microstructured PAA-terminated PEMUs (PAA-PEMUs). BDM-induced hMSCs expressed higher levels of the early osteoblast differentiation marker alkaline phosphatase (ALP) sooner and assembled a less fibrillar collagen I organization on PAA-PEMUs than on PAH-PEMUs. Although cells on both types of PEMUs eventually expressed similar levels of the later stage osteoblast differentiation markers bone sialoprotein (BSP), osteonectin, and osteopontin, the cells organized a more amorphous Collagen I and denser BSP localization on PAA-PEMUs than on PAH-PEMUs. These ECM properties correlated with greater biomineralization on the PAA-PEMUs than on PAH-PEMUs. To further investigate ECM effects on hMSC behavior and differentiation, we developed a novel model for deposition and decellularization of cell secreted ECM on tissue culture plastic (TCP) and PEMU surfaces. The microenvironment presented by extracellular matrix (ECM) influences cell adhesion, proliferation and differentiation. Chapter 3 describes development of a cold EDTA-PBS protocol to remove intact cells from ECM deposited by cultured human bone marrow mesenchymal stems cells (hMSCs), osteogenic hMSCs, and two smooth muscle cell (SMC) lines, with minimal ECM damage and contamination. The decellularized ECM deposited by uninduced hMSCs enhanced naïve hMSC proliferation, stemness, and cell motility. Decellularized ECM deposited by osteogenic hMSCs early in the differentiation process stimulated naïve hMSCs osteogenesis and substrate biomineralization in the absence of added differentiation factors, but this osteogenic induction potential was lost by later differentiation stages. Both smooth muscle cell line decellularized ECMs induced naïve hMSC myofibroblast differentiation, but each ECM type induced naïve hMSC development into distinct SMC phenotypes. Chapter 4 describes our characterization of decellularized ECMs deposited by deposited by several cell types on PAH-PAA PEMUs. ECMs deposited on PAA-terminated PEMUs by hMSCs that were induced to become osteogenic with dexamethasone-containing bone differentiation medium (BDM) for 3, 8, and 15 days were decellularized. The ECM deposited for 3 days induced osteogenesis of naïve hMSCs and substrate biomineralization in the absence of the dexamethasone differentiation factor. This osteogenic induction potential was lower for ECM deposited for 8 and 15 days. ECMs deposited by human hTERT-immortalized myometrial smooth muscle cells (myomSMCs) induced naïve hMSCs to become myoblastic with 'synthetic' smooth muscle cell phenotypic characteristics. Overall, this research demonstrates the biocompatibility of PAH-PAA PEMUs, especially for biomineralization by osteogenic hMSCs. The protocol for decellularization of ECM deposited by cells on surfaces including PEMUs described here provides a potentially useful method for constructing biomaterial-ECM scaffolds and highlights the importance of ECM microenvironments on stem cell behavior.
A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Includes bibliographical references.
Thomas C. S. Keller, III, Professor Directing Dissertation; Joseph B. Schlenoff, University Representative; David M. Gilbert, Committee Member; Wu-Min Deng, Committee Member; Laura R. Keller, Committee Member.
Florida State University
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