Dental pulp stem cell adhesion and differentiation on a novel therapeutic biomaterial

Jacob C. Scherba1,2, Kyle H. Vining1,2, Adam D. Celiz1,3, David J. Mooney1,2.
1. Wyss Institute for Biologically Inspired Engineering. 2. Harvard John A. Paulson School of Engineering and
Applied Sciences. 3. University of Nottingham.
Introduction: Over 1 billion dental fillings are performed annually in the United States according to the U.S. Food and Drug Administration. Current dental decay treatments, however, can lead to pulp injury that may necessitate costly and painful procedures, like root canals and extractions. Using a high-throughput microarray, we identified AT03 as a leading candidate to facilitate dental pulp stem cell (DPSC) adhesion. Subsequently, we scaled to a bulk biomaterial that can be cured in situ via UV irradiation. In this study, we seek to understand the mechanism of DPSC adhesion to our material and investigate its ability to allow for differentiation of DPSCs into odontoblasts. Understanding these processes is the basis for enabling dentin regeneration in teeth, and it informs our investigation of the therapeutic capability of our material in vivo. The mechanistic understanding of the behavior of these cells on our material, if applied properly, has considerable implications for routine therapeutic dentistry.

Materials and Methods: To investigate the mechanism of adhesion, RNA was isolated from adhered cells on AT03 and Bis-GMA (current industry standard) and tissue culture plastic (TCP) controls at 8 and 48 hours. F- actin and immunostaining were performed to illustrate cell morphology and focal adhesion as well as proliferative activity. For differentiation, RNA was collected from adhered DPSCs at 21 days. RT qPCR was performed to explore relative levels of odontoblastic and osteogenic differentiation.

Results and Discussion: Phase microscopic images qualitatively determined that human DPSCs adhered more strongly to our AT03 polymer than to Bis-GMA. In addition, cell counts were performed after 48 hours to determine the number of viable cells. There were significantly fewer viable DPSCs on Bis-GMA compared to both TCP and our AT03 polymer (p < 0.0005). Gene expression analysis showed that AT03 supports expression of osteogenic differentiation markers after 21 days in culture.

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Figure 1. Adhesion of DPSCs is illustrated in the phase microscope images to the left, characterized by the distinct elongated morphology of the cell. In addition, a significant reduction in viable cell count is exhibited between Bis-GMA and both AT03 and TCP. Data is presented + SD.

Conclusions: Human DPSCs adhere more strongly to our AT03 polymer than to the industry standard polymer, Bis-GMA, and there are more viable cells present on our material over time. The fact that our material allows for the growth of viable DPSCs could radically alter therapeutic dentistry. The facilitation of osteogenic differentiation is also promising because hard tissue mineralization plays a key role in dental tissue repair. Applied in the clinic, our material could allow for the regeneration of dentin in patients who might otherwise require expensive and painful procedures like extractions or root canals. We are currently investigating the behavior of our material in vivo using a rat dental pulp injury model.