Dental pulp cells for tissue engineering. – PulpCell

The overall objective of this project is to develop innovative bioengineering approaches, tissue equivalents consisting in pulp cells seeded in 3D matrices, able to functionally regenerate the injured pulp. We believe that pulp cells can be a useful and convenient material for tooth bioengineering and this project may have a major impact for treating dental defects. In addition, pulp cells may also be important more generally for the bioengineering of craniofacial bones thanks to their embryological origin and to their multipotent properties, and we also propose to develop a tissue equivalent in this context. We will combine innovative in vitro and in vivo approaches, and carry them through several steps. Human teeth are frequently extracted during orthodontic treatment plans and the pulp (a reservoir of mesenchymal stem cells) can be easily collected.
First, we will isolate human pulp cell sub-populations enriched in cells displaying mesenchymal stem cell properties or endothelial progenitor properties, on the basis of their expression of cell surface markers. We will assess their ability to promote angiogenesis especially under hypoxic conditions (mimicking the future grafting site). In that purpose, 3D co-cultures of pulp cells and endothelial cells will mimic the in vivo recruitment of vascular cells from radicular pulp capillaries. In this model, pulp cells should promote vascularization and reinforce the neo-formed tubes. In vivo Doppler or Micro-CT (after contrast agent injection) data obtained through implantation of tissue equivalents in rat ectopic site will strengthen the exploration of angiogenic properties of pulp cells.
Second, tissue equivalent will be implanted in the emptied pulp chamber of rat molar (partial pulpectomy model). A major issue in tissue engineering is to determine the fate of implanted cells: will they stay in the implanted tooth and participate to the repair process, will they degenerate, or disseminate in the organism? To answer these questions, we will use, for the first time to our knowledge, investigative imaging methods (MRI and Scintigraphy) to track labeled pulp cells implanted in the rat pulp or to follow-up neoangiogenesis. These observations are challenging because the tooth organ is the most mineralized tissue of the organism, but our data using these imaging methods are promising. In vivo experiments will be carried out with pulp equivalents or with prevascularized tissue equivalents (seeded with both mesenchymal and endothelial pulp cells), which is an innovative approach that may enhance the vascularization of the implanted scaffold.
Third, the formation of a functional pulp tissue using the rat molar model will be explored. The nature of the repair process will be characterized through the analysis of the following criteria: 1) neovascularization and neoneurogenesis of the implanted tissue, 2) presence of newly differentiated odontoblast-like cells on the existing dentinal wall in the pulp chamber, 3) mineralized dentin formation by these newly differentiated cells. In parallel, similar experiments will be carried out to assess the potential of pulp cells to generate craniofacial bone. In this context, pulp cells will be seeded in dense collagen scaffolds and implanted in a critical-sized calvarial bone defect model.
Finally, similar experiments will be conducted in the dental pulp of the mini-pig, which is a model closer to humans, to get one step closer to the transfer to human clinic.
The PulpCell project requires knowledge in the following fields: biology of mineralized tissues, soft tissue interface, pulp cells, dental preclinical models, tissue engineering, imaging methods, hypoxia, and vascularization. The three partners are French experts in these complementary research areas.