Fischell Department of Bioengineering Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/6628

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Engineering Zonal Cartilage Through Utilization of a Mesenchymal Stem Cell Population
(2012) Coates, Emily Elizabeth; Fisher, John; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Articular cartilage has a limited ability to repair itself after damage due to injury or disease. Regenerative therapies using chondrocytes, the primary cartilage cell population, result in poor quality repair tissue and often cause further damage at the donor site. Furthermore, there are no current therapies which aim to regenerate the zonal organization and function of the tissue. In an effort to address both cell source limitations and zonal tissue regeneration the goal of the presented work was to utilize a mesenchymal stem cell (MSC) population to generate abundant numbers of chondrocytes with zonal phenotypes. To this end, zonal subpopulations of articular chondrocytes were isolated, characterized for differences in gene and protein expression, and exposed to scaffold environments designed to aid in phenotype retention. From these results, and reports in the literature, it was clear a major functional difference between zones was the production of a lubricating protein, proteoglycan 4 (PRG4), in the superficial zone only. Middle and deep zone cells were found to be phenotypically similar and distinct from superficial zone cells. It was further found that gene expression of PRG4 by superficial zone cells in alginate culture can be significantly enhanced by incorporation of matrix molecules hyaluronic acid (HA) and chondroitin sulfate (CS) to the scaffold environment. HA and CS also had favorable effects on MSC chondrogenesis by upregulating chondrogenic transcription factor Sox9 gene expression, and downregulating type I collagen (fibroblastic marker) gene expression. The potential of soluble signals derived from zonal (superficial or middle/deep) cartilage explants to drive MSC chondrogenesis was also investigated. Results show that signals derived from cartilage explants can induce chondrogenesis to varying degrees, with superficial zone explants inducing robust and sustained differentiation. This differentiation was found to be dependent on the proximity of the MSCs and tissue explants, implying that communication between MSCs and chondrocytes is necessary for chondrogenic induction. Coculture with superficial zone explants also upregulated MSC gene expression of PRG4. This research highlights the important functional differences between zonal chondrocyte populations and identifies MSCs as a progenitor population capable of differentiating into zone-specific chondrocytes.
TUBULAR PERFUSION SYSTEM BIOREACTOR FOR THE DYNAMIC CULTURE OF HUMAN MESENCHYMAL STEM CELLS
(2012) Yeatts, Andrew Bryan; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
In vitro culture techniques must be improved in order to increase the feasibility of cell based tissue engineering strategies. Limitations of current techniques are largely a result of the slow diffusion of molecules such as oxygen into the interior of three dimensional scaffolds in static culture. In order to enhance nutrient transport we have developed a novel bioreactor, the tubular perfusion system (TPS), to culture human mesenchymal stem cells (hMSCs) in three dimensional scaffolds. In our design, hMSCs are cultured on scaffolds tightly packed in a tubular growth chamber. Media is perfused by a peristaltic pump through the growth chamber and around the tightly packed scaffolds. In the first part of the work hMSCs are encapsulated in alginate scaffolds and results demonstrate bioreactor culture enhances late osteoblastic differentiation of hMSCs. An investigation into shear stress in the system revealed that osteogenic markers increase with increasing shear stress and that the differentiation of hMSCs is dependent on cell radial position within scaffolds. In order to enhance the ability to implant these constructs in vivo, a method to create an aggregated cell containing construct in vitro in a bioreactor system was developed. In this part of the work hMSCs are cultured in individual alginate beads in the TPS bioreactor and the beads are aggregated to form one large construct. Following this the TPS bioreactor was investigated to culture synthetic poly-L-lactic acid scaffolds which were fabricated using supercritical carbon dioxide gel drying. In addition to investigating the effects of perfusion on hMSC growth in these scaffolds, the effect of microporosity was investigated. In the final part of the work, a study was completed to determine how TPS culture influenced in vivo bone regeneration. Here alginate beads as well as synthetic PLGA/PCL constructs were used as scaffolds. Results revealed the efficacy of using the tubular perfusion system for bone tissue engineering and demonstrated increased bone formation as a result of hMSC implantation in both alginate and PLGA/PCL scaffolds. These studies highlighted the need for bioreactor culture in vitro as well as scaffolds to support in vivo tissue interaction.