A hydrogel scaffold that maintains viability and supports differentiation of dental pulp stem cells
Introduction
The focus on conservative strategies for treatment of diseased dental pulps has led Endodontics to advance in the fields of stem cell biology, genetics, and tissue engineering. The new field of Regenerative Endodontics has emerged around the premise that it might be possible to generate a new dental pulp to treat necrotic teeth [1], [2], [3], [4]. The discovery of dental pulp stem cells (DPSC) capable of differentiating into many cell types [5], [6] has provided a significant boost to this field. Dental pulp stem cells contain specific sub-populations of specific progenitor cells making them ideally suitable to the engineering of dental tissues [7]. Of specific interest for Regenerative Endodontics, these cells can differentiate into both functional, dentin-making odontoblasts [8], [9] and vascular endothelial cells [10], [11]. These features are critically important, since adequate vascularization is vital for the regeneration of the dentin–pulp complex.
Despite the excitement around the clinical application of dental pulp stem cells, there are still several challenges that have to be overcome before regenerative approaches can be used routinely in Endodontics. An area of considerable interest now is the interaction between dental pulp stem cells and three-dimensional (3D) scaffolds. Scaffolds provide cell adhesion and enables cell proliferation, mimicking the microenvironment observed in natural tissues and organs [12]. In the context of dental pulp tissue engineering, scaffolds should have a relatively fast setting time, and provide adequate root canal modeling and adaptation [2]. Notably, at the time that this manuscript was prepared there were no commercially available scaffolds developed for dental pulp tissue engineering [13]. Puramatrix™ is a self-assembling peptide hydrogel that has been tested in differentiated primary cells and stem cells [14], [15]. It supports the regeneration of functional bone in murine calvaria [16]. The hydrogel comprises a 16-mer peptide in aqueous solution, which instantly polymerizes forming a biodegradable scaffold when introduced to physiological salt conditions. This capacity renders it ideal for clinical situations requiring both biocompatibility and rapid matrix formation. Here, we hypothesized that DPSC cultured in Puramatrix™ will survive, proliferate and differentiate into odontoblasts.
Section snippets
Materials and methods
Dental pulp stem cells (DPSC) were kindly provided by Dr. Songtao Shi (University of Southern California) and cultured as previously described [6]. Briefly, cells from 4th to 8th passage were grown in α-MEM medium (Invitrogen, Grand Island, NY, USA), supplemented by 20% FBS and incubated at 37 °C in 5% CO2.
Puramatrix™ supports the proliferation and survival of DPSC
We observed that DPSC proliferate in Puramatrix™ when seeded at a density of 1–4 × 105 cells/ml. In contrast, at the excessively high cell density (8 × 105 cells/ml), DPSC cells were not able to proliferate and instead fell to around the same as the maximum 72-hour data for the other densities of cells (Fig. 1). DPSC survived in all concentrations of Puramatrix (Fig. 2). Indeed, no difference was observed in DPSC proliferation in the different concentrations of Puramatrix that were tested here (p =
Discussion
Three-dimensional scaffolds are required for cellular organization and interaction in engineered tissue [12]. However, the use of any such scaffold in the small and closed environments of the root canal provides a singular challenge. Some scaffolds described in the literature are very successful environments for cell growth in vitro but are unsuitable for the demands of clinical practice [4]. Uniquely, Puramatrix™ is a liquid that may be potentially poured into a pulp chamber and which
Conclusion
Under the experimental conditions described, dental pulp stem cells survived and proliferated in a self-assembling peptide hydrogel. This class of materials represents a promising new alternative of injectable scaffolds for dental pulp tissue engineering.
Acknowledgements
This work was funded by grant R01-DE021410 from the NIH/NIDCR (JEN) and by CAPES, Brazilian government (BNC).
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These authors contributed equally to this work.