åTitle: Harnessing The Regenerative Potential of Purified Bovine Dental Pulp and Dentin Extracellular Matrices in a Chitosan/Alginate Hydrogel.

When a tooth is diseased or damaged through caries, bioactive molecules are liberated from the pulp and dentin as part of the natural response to injury and these are key molecules for stimulating stem cell responses for tissue repair. Incorporation of these extracellular matrix (ECM) derived molecules into a hydrogel model can mimic in vivo conditions to enable dentin-pulp complex regeneration. In this study, a chitosan/alginate (C/A) hydrogel was developed to sequester bovine ECM extracts. Human dental pulp cells (hDPCs) were cultured with these constructs and proliferation and cytotoxicity assays confirmed that these C/A hydrogels were bioactive. Sequential z-axis fluorescent imaging visualized hDPCs protruding into the hydrogel as it degraded. Alizarin red S staining showed hDPCs cultured with the hydrogels displayed increased calcium ion deposition, with dentin ECM stimulating the highest levels. Alkaline phosphatase activity was increased, as was expression of transforming growth factor-beta (TGF-β) as demonstrated using immunocytochemistry. Directional analysis following phase contrast kinetic image capture demonstrated that both dentin and pulp ECM molecules acted as chemoattractants for hDPCs. Data from this study demonstrated that purified ECM from dental pulp and dentin when delivered in a C/A hydrogel stimulated dental tissue repair processes in vitro. This article is protected by copyright. All rights reserved.