The growth and morphology of fibroblasts cultured on a physically and chemically modified surface was investigated. The need to understand cellular relationships with surface topography and chemistry is essential in the fields of biomedical engineering and biotechnology. It is well documented that mammalian cell behavior senses and responds to the surrounding micro- and nano- scale environment, but the research defining the chemistry, surface architecture, and material properties for control of this behavior is still in its infancy. The cell response to a substrate is complex, involving membrane proteins, extracellular matrix (ECM), cytoskeletal rearrangement, and changes in gene expression. Conventional cell culture is carried out on two-dimensional (2-D) cell culture platforms, such as polystyrene (PS) or glass, and forces cell behavior to adapt and adhere to an unnatural, planar environment. The biological behavior of these cells is used as a starting point for drug screening, implant design, and metabolic processes, but this is a misrepresentation of cells in their native environment. This discrepancy may be hampering biological research or initiating experimental efforts that are invalid. This body of work seeks to address these issues and contains established protocols for inexpensive, pseudo three-dimensional (3-D) culture scaffolds. The research described offers a multi-disciplinary approach for fabrication of biomaterials to achieve user defined or in vivo cell behavior using human fibroblasts.
To provide insight into the design of alternative cell culture templates we have analyzed cell-surface interactions and characterized the surface properties. The substrates fabricated utilized micro-roughened surface topography with 2 – 6 µm wide features and surface chemistry as a method for controlling cell behavior. Surface roughness was templated onto polydimethylsiloxane (PDMS) and PS. The fabricated polymer surfaces have been characterized by atomic force microscopy (AFM), contact angle goniometry, fluorescence microscopy, and infrared (IR) spectroscopy. Initial studies of the textured surface yielded a super-hydrophobic surface with a 154° contact angle and high surface adhesion that was investigated using surface free energy calculations. This was followed by modification of the micro-roughness with self-assembled monolayers (SAMs), proteins, or thin films of polymer for use as a culture platform for cells. Cell behavior on the modified polymers was compared and analyzed against unmodified surfaces and tissue culture PS dishes. Cell morphology on rough PDMS surface was altered by the surface topography decreasing the average cell area to 1760 µm2 compared to an average cell area of 3410 µm2 on smooth PDMS. Gene expression changes were also noted with a 2.3 fold increase in the matrix metalloproteinase, MMP14, in cells on the rough surface compared to cells cultured on Petri dishes. Surface roughness was also combined with other surface modification methods for cell culture, including cell alignment and cell sheet engineering. 50 µm wide lines of fibronectin (FN) patterned on the rough PDMS induced cell directionality while still maintaining a pseudo 3-D culture system creating the first cell culture surface of its kind. The micro-roughness was also templated onto PS and chemically modified with a thermo-responsive polymer. This novel surface produced confluent cell sheets that detached from the surface when cooled below 32°C. Cell sheets cultured on the modified PS surfaces had an increase in FN fibril formation stimulated by the surface roughness when compared to cell sheets detached from a smooth, control surface.
The minor alterations to surface topology were proven to be effective in modifying cell biochemical response compared to cells cultured on flat substrates. Differences in surface topography and chemistry stimulated changes in cell adhesion, cytoskeletal arrangement, ECM composition, and gene expression. These cell properties were used as markers for comparison to native cell systems and other reports of 3 D culture scaffolds. The mechanism of altering cell response is discussed in each chapter with respect to the specific type of surface used and compared to cell response and behavior on planar culture systems. New fabrication procedures are described that include the incorporation of other surface modification techniques such as SAMs, surface patterning, and thermo-responsive polymer grafting with surface roughness for original cell culture platforms to mimic an in vivo environment. The research presented here demonstrates that micro- and nano- changes to surface topography have large impacts on the cell-surface relationship which have important implications for research and medical applications involving adherent cells.
| Identifer | oai:union.ndltd.org:wpi.edu/oai:digitalcommons.wpi.edu:etd-dissertations-1295 |
| Date | 30 May 2014 |
| Creators | Stanton, Morgan M |
| Contributors | Christopher R. Lambert, Advisor, Arne Gericke, Department Head, James P. Dittami, Committee Member, John C. MacDonald, Committee Member, Bulent Mutus, Committee Member |
| Publisher | Digital WPI |
| Source Sets | Worcester Polytechnic Institute |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Doctoral Dissertations (All Dissertations, All Years) |
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