Date of Completion

2-27-2013

Embargo Period

2-16-2013

Advisors

Liisa T. Kuhn, Ph. D.,; John R. Agar, D.D.S. M.A.

Field of Study

Dental Science

Degree

Master of Dental Science

Open Access

Open Access

Abstract

There is growing interest in the development of an engineering process by which dentin stem/progenitor cells can predictably proliferate, differentiate and produce mineralization nodules in cell culture. The engineering of 3D scaffolds could then be used to aid in proliferation and differentiation of these progenitor cells to aid in regeneration of the pulp/dentin complex in vivo.

In the present study, the goal was to characterize the MDPC-23 cell line and its behavior during proliferation. The cells in culture reach high numbers upon reaching confluence. When cells are growing in multilayers, as will occur in planned dentin regeneration experiments, it is difficult to accurately establish the cell number. DNA content can be used to determine cell number, but the correlation between cell number and DNA content for MDPC-23 cells needs to be validated.

We designed three experiments to establish growth curves and cell doubling time for the MDPC-23 cell line and finally to correlate cell numbers to DNA content in the MDPC-23 cell line. In the initial experiment to characterize cell growth, MDPC-23 cells number was plotted over a time period of 11 days. In the second experiment MDPC-23 cell growth was monitored for 11, with cell numbers counted manually using a hemocytometer. The cells from each time point were also used to measure the DNA content using fluorescence. The third experiment had more refined methods using a TC10™ automatic cell counter and then plotting these numbers to obtain the MDPC-23 growth curve. The cells were also used for DNA content calculation using the same dilution factor of 80x for all DNA samples.

The cell doubling times for two MDPC-23 cell cultures in Experiment #1 were 1.02 days and 1.12 days. In Experiment #2 the MDPC-23 cell doubling time was 0.94 days and for Experiment #3 it was 0.90 days. The average cell doubling time for all three experiments was 0.996 +/- 0.095 days.

The cell numbers and DNA content in Experiment #2 were not closely correlated (correlation coefficient of R2 = 0.875). The cell numbers and DNA content for Experiment #3 were closely correlated with a correlation coefficient of R2 = 0.98.

The MDPC-23 cell line has a typical S-shaped growth curve. This study confirmed the null hypothesis that MDPC-23 cell numbers as determined by cell counting closely correlate to DNA content.

Major Advisor

A. Jon Goldberg, Ph.D

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