Date of Award

12-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Advisor

Burg, Karen JL

Committee Member

LaBerge , Martine

Committee Member

Dreau , Didier

Committee Member

Webb , Ken

Abstract

It is estimated that this year more than 200,000 women in the United States will be diagnosed with breast cancer. Treatment for most occurrences of breast cancer will often include surgical removal of the tumorigenic tissue, resulting in a soft tissue defect within the subcutaneous tissue of the skin. Post-surgical reconstruction methods are often sought by patients to restore the aesthetic function of the breast via cellular or non-cellular methods; however, because of complications associated with currently used methods for breast reconstruction, researchers are investigating tissue engineering methods to produce viable autologous adipose tissue for breast reconstruction.
Previous research in our laboratory has focused on the development of an injectable composite system for breast reconstruction that uses injectable microcarrier beads as support scaffolds for cellular growth. In vitro and in vivo studies of this system have shown that the polymeric microcarriers not only support cellular attachment, but also facilitate proliferation and differentiation of adipose cells, and yield biocompatible tissue response in an in vivo host. Successful clinical implementation of this system, like many other adipose tissue engineering methods, has been limited by an inability to engineer adipose tissue of sustained volume long-term. It is hypothesized that more optimal in vitro culturing methods for adipose cells could yield populations of fat cells capable of differentiating to adipocytes and retaining their capacity for lipid-filing over time. The goal of this work, therefore, was to evaluate specific factors that influence adipose cell differentiation in an attempt to create a more successful injectable composite system for breast reconstruction. While adipogenesis is influenced by many factors, for this work, we investigated two specific areas: 1) the influence of adipose cell-mammary epithelial cell interactions and 2) the influence of fatty acids on adipose cell differentiation in vitro.
A series of conditioned media studies were first performed to determine how adipogenesis of primary adipose cells from bovine, murine, and human tissue samples was influenced by mammary epithelial cell-conditioned media. Depending on the source of conditioned media and the species of adipose cell, adipose cell differentiation was either positively or negatively influenced by the conditioned media as evidenced by increased or decreased triglyceride production and lipid accumulation in the two-dimensional cultures. Characterization of the components of the conditioned media samples showed that mammary epithelial cell-conditioned media contains measurable quantities of factors that influence adipogenesis.
To investigate the influence of fatty acids on adipose cell differentiation, a series of studies were performed to identify an optimal concentration of fatty acid and an optimal culture time for supplementing adipose cells with a fatty acid that would stimulate fatty acid uptake and subsequent lipid filling of the cells. Exogenous fatty acids in cell culture medium were found to influence adipogenesis. Microcarrier beads containing fatty acids were then manufactured and used in three-dimensional cell culture experiments to determine if microcarrier beads could be used for fatty acid delivery while simultaneously supporting adipose cell growth and differentiation.
Results of the studies performed in this work suggest that fatty acids as well as specific factors produced by mammary epithelial cells will influence adipogenesis, suggesting that these factors may be used to enhance the in vitro culturing of adipose cells for the injectable composite system. Methods to more optimally culture the adipose cells in vitro could be used to identify strategies that could then reduce the concerns of tissue resorption and volume-reduction that have limited the success of breast tissue engineering strategies clinically. Ideally, the incorporation of fatty acids and/or cellular components into the injectable composite would allow immediate delivery of a cellular implant without the need for in vitro cultivation.

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