Date of Award

5-2011

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Bioengineering

Advisor

Dean, Delphine

Committee Member

Kennedy , Marian

Committee Member

Yao , Hai

Abstract

Dental pulp stem cells (DPSCs) have recently been described as a potential stem cell source for various regenerative medicine and tissue engineering applications. They appear to be multipotent, however more characterization is necessary to determine the true potential of these cells. An important aspect of using DPSCs, or any stem cell type, tissue engineering application is the microenvironment within the construct. The microenvironment could include construct mechanical properties, construct composition, and 3D dynamic conditions in vivo. This work aims to study those specific microenvironment effects on DPSCs. To determine the effects of mechanical properties of the substrate on DPSCs, they were seeded on polyacrylamide (PA) gels of different elastic moduli. These gels ranged from 3 kPa to 75 kPa and a glass coverslip was used as a control. They were also exposed to either standard stem cell media or an osteogenic differentiation media, to determine the potential of the DPSCs for osteogenic/odontogenic differentiation. The cultures were analyzed for morphological changes, osteopontin production, alkaline phosphatase (ALP) production, and mineralization. The results showed that the DPSCs adhered well to the PA gels for the first few days in culture, but by day 7, they were starting to detach from the PA gels and only remain viable in gel defects or along the edges. This selective growth was also reflected in the mineralization, which only occurred in areas of confluence for the cells on the PA gels. Interestingly, all cultures produced osteopontin and ALP, however by the end of the experiment, the cells cultured on glass had the highest ALP production. It appeared that without the addition of growth factors to induce other cell lineages, DPSCs defaulted to an osteogenic/odontogenic lineage.
To determine the effect of mineral composition, preliminary studies were done on bone marrow stromal cells (BMSCs) and 7F2 osteoblasts. These two cell types were exposed to either hydroxyapatite or fluoroapatite microparticles. The cells were analyzed for ALP production, collagen I production, osteocalcin production, and mineralization. The results showed that the BMSCs without any particles produced the most ALP from day 5 forward. This was reflected in the collagen I and osteocalcin production, as both the BMSCs and 7F2 osteoblasts had the highest production of both proteins in the control cultures without production. Interestingly, no mineralization was detected in any of the cultures except the control 7F2 osteoblasts at the final time point, day 14. More trials are necessary to achieve statistically significant results, however initial results indicate both particle types may be hindering normal osteogenic function. Finally, to determine the effects of both 3D and dynamic culture conditions on DPSCs, two culture systems were used. First, a parallel-plate flow chamber was designed to determine the effects of 2D fluid shear stress on the cells. Second, the 3DKUBETM from KIYATEC, Inc. was used for 3D dynamic culture. Both systems were evaluated for use with DPSCs and it was determined that the 3D dynamic system improved ALP production when compared with a 2D static system. Future work will determine if regeneration of a dentin-like structure can be achieved in a 3D dynamic system. In addition, the parallel plate flow chamber will be used to isolate the effects of uniaxial shear stress on the cells and compare them with the shear stresses created in 3D.

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