Defense Date

2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Pathology

First Advisor

Shawn Holt

Abstract

To repair, replace, or regenerate damaged or diseased tissue has been a long-standing, albeit elusive, goal in medical research. Here, we characterize patient-derivable mesenchymal stem cell types, termed adipose-derived stem cells (ASCs). These cells, which can be derived from liposuction fat and lipoaspirate saline, are sources for patient-derivable extracellular matrix (ECM), fibrinogen (Fg) and adipose tissue extracellular matrix, and may prove useful for synthesizing new bone tissue analogues in vitro. Traditionally and rapidly isolated ASCs were thoroughly characterized as multipotent, having osteogenic, adipogenic, and chondrogenic differentiation potential, and they exhibited comparable proliferative lifespans. These ASCs also shared an indistinguishable immunophenotype when compared to bone marrow-derived mesenchymal stem cells, suggesting that these cells are an excellent source for bone following tissue engineering experimentation. In order to synthesize bone ex-vivo, electrospun scaffolds of fibrinogen (Fg), polydioxanone (PDO), and Fg:PDO blends were seeded with early passage ASCs, fibroblasts, or osteosarcoma cells and were maintained for 21 days in osteogenic or regular growth media. Constructs were analyzed both histologically and molecularly for evidence of osteoblastogenesis. Using SEM, the appearance of regular, mineralized-appearing structures were found in osteogenic-induced ASC seeded scaffolds beyond 14 days, only in the scaffolds containing Fg. Further, at 21 days of culture, Fg scaffolds with ASCs in osteogenic media became hard and brittle. Robust new collagen synthesis and matrix remodeling were observed on all Fg scaffolds, the levels of which were elevated over time. Pronounced mineralization was found throughout bone-induced ASC scaffolds, while control scaffolds (BJ foreskin fibroblasts) showed no mineral deposition (although they did demonstrate excellent cellularity). Analysis of gene expression (qRT-PCR) indicated that electrospun Fg supported osteoblastogenesis through the upregulation of alkaline phosphatase and osteocalcin gene expression. To confirm our gene expression results, osteogenic-induced ASCs on Fg scaffolds were also shown to secrete osteocalcin in the extracellular matrix, a key marker in osteoblastogenesis. Thus, electrospun Fg is an excellent material for ASC growth, proliferation, and osteogenic differentiation, providing an ideal system for furthering basic bone model-based research and for advancing regenerative medicine. In addition to establishing Fg as a source of scaffolding, we developed and characterized a novel method for isolating and subsequently electrospinning adipose tissue matrix. Because adipose ECM contains many primordial matrix proteins important for embryonic development and regeneration (such as laminin, type IV collagen, and fibronectin), adipose ECM may prove to be an autologous tissue engineering matrix and stem cell culture substrate. We show here that adipose tissue ECM can, in fact, be electrospun into a nanofiberous mesh, histologically shown to contain connective tissue, collagens, elastic fibers/elastin, proteoglycans, and glycoproteins in the newly synthesized matrix. We also show that this novel electrospun adipose tissue scaffold is capable of supporting stem cell growth. Taken together, experiments using ASCs cultured on extracellular matrices of electrospun Fg or adipose ECM present an excellent framework for future advances in regenerative medicine therapeutics and research.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

February 2010

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