Decellularisation of the Dental Pulp for Use as a Scaffold in Regenerative Endodontics

Background: Endodontic management of immature necrotic permanent teeth is currently undergoing a paradigm shift from the conventional apexification and apical plug techniques to biological based regenerative procedures. The shift towards a tissue engineered clinical based procedure is highly attractive. However, the ideal scaffold to support pulp-dentine complex regeneration is yet to be identified. Aim: The primary aim of this work was to develop, characterise and assess the biocompatibility of an acellular extracellular matrix scaffold developed through decellularisation of rat and human dental pulp tissues. The secondary aim was to assess the ability of the scaffold to support human dental pulp stem cells attachment, viability, and differentiation. Methods: Following ethical approval, rat and human dental pulps were retrieved and decellularised. The efficiency of the decellularised protocol was assessed using histology and immunohistochemistry staining methods, scanning electron microscopy, and DNA quantification assays. Contact and extract cytotoxicity assays were performed to determine the biocompatibility of the developed scaffold. Decellularised scaffolds were recellularised with human dental pulp stem cells and cell viability was assessed, for up to 14 days, in culture. Expression of odontoblastic markers and molecular proteins within the recellularised scaffold were also investigated. Quantitative data were analysed using Student’s t-test and one-way analysis of variance using GraphPad Prism(Version 6). Results: Assessment of decellularised scaffolds revealed an acellular matrix with preservation of the connective tissue architecture and composition. Acellular scaffolds were biocompatible with normal cell growth in direct contact with the acellular scaffold. No difference in cellular activity was found following incubation in acellular scaffold extracts (p> 0.05). Live/Dead® confocal imaging showed high majority of viable cells. Furthermore, the scaffold was able to support human dental pulp stem cells viability and attachment following recellularisation. Immunolabelling of dental pulp stem cells within the recellularised scaffold revealed a positive expression against several tested odontoblastic markers and molecular proteins. Conclusion: The decellularisation protocol used showed promising results following decellularisation of rat and human dental pulp tissues in terms of developing an acellular biological scaffold with preserved extracellular structural components required for tissue specific regeneration.