Decellularised dental pulp tissue as a potential biological scaffold for endodontic tissue regeneration

Background: There is an unmet need for regenerating functional dental pulp, particularly in non-vital immature permanent teeth where dental pulp necrosis arrests further radicular maturation, putting young patients at risk of tooth loss. Despite the progress achieved over recent decades, dental pulp regeneration still faces challenges in promoting post-implantation vascularisation and inducing odontoblast-like cell differentiation.

Aim: The overall aim of the research presented in this thesis was to investigate the potential of a decellularised dental pulp matrix (DDP) of bovine origin to facilitate vascularisation and support dental pulp regeneration.

Methods: Bovine dental pulp tissues were retrieved and decellularised. The efficiency of the decellularisation method was evaluated using histological analysis, DNA quantification assay, immunohistochemical staining and scanning electron microscopy. Furthermore, the cytocompatibility of the developed DDPs were assessed using contact and extract cytotoxicity assays. DDPs were then recellularised with human dental pulp stem cells (hDPSCs) and analysed in vitro using scanning electron microscopy, fluorescent staining and confocal scanning laser microscopy, Live/Dead® cells assay, Quant-iT™ PicoGreen® dsDNA assay and histology. The effect of DDPs on gene expression of markers involved in angiogenesis and odontogenesis in hDPSCs was evaluated in vitro using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). A preliminary in vivo study was then conducted in which hDPSCs-seeded and unseeded DDPs were inserted in debrided human premolar root slices and implanted subcutaneously into immunodeficient mice. Samples were retrieved after 30 days and analysed using histological and immunohistochemical staining.

Results: Acellular dental pulp matrices retaining the native histoarchitecture, vasculature, essential extracellular matrix components and several growth factors were generated following bovine dental pulp decellularisation. The in vitro cytocompatibility evaluation of the developed DDPs revealed no apparent cytotoxic effect on the growth and morphology of the cells grown in direct contact with the DDPs, and on the viability of cells grown in an extract of DDPs. Upon recellularisation of the DDPs with hDPSCs, the in vitro analyses showed cell engraftment with progressive repopulation of the matrix and vasculature of the DDPs, and with enhanced gene expression of the markers involved in angiogenesis and odontogenesis. In vivo implantation of root slices with hDPSCs-seeded DDPs revealed apparent vascularisation enhancement, whilst those with unseeded DDPs showed host cell recruitment and infiltration.

Conclusions: The developed decellularised dental pulp matrix is a cytocompatible, pro-angiogenic and pro-odontogenic scaffold characterised by the retention of native histoarchitecture, vasculature, essential extracellular matrix components and angiogenic and odontogenic growth factors in the matrix following decellularisation. Seeding of hDPSCs onto the DDP led to progressive cell repopulation of the matrix and vasculature, enhanced expression of the markers involved in angiogenesis and odontogenesis in hDPSCs and improved in vivo vascularisation capacity of the DDP. Moreover, the DDP has a chemotactic activity in vivo, enabling host cells mobilisation and recruitment. The findings of this research suggest that a combination of DDP and hDPSCs could provide a promising vascularisation promoting strategy for dental pulp regeneration via cell transplantation. Furthermore, the use of DDP, in its acellular form, could provide a strategy for dental pulp regeneration via cell homing.