# Fibronectin-gelatin nanofilm coating improves dental pulp stem cell survival and differentiation in odontogenesis-mimicking organotypic 3D bilayered constructs

## TL;DR

A new nanofilm coating improves dental stem cell survival and differentiation in 3D models for studying rare oral diseases.

## Contribution

A fibronectin-gelatin nanofilm coating enhances human dental pulp stem cell viability and odontoblast differentiation in 3D constructs.

## Key findings

- FN/G coating improved hDPSC viability in thick constructs.
- Amelogenin was detected near odontoblast-like cells in the model.
- The model can be modified with CRISPR/Cas9 for patient-specific studies.

## Abstract

Healthcare professionals, researchers, patients and their families affected by rare diseases face many difficulties during diagnosis. A targeted diagnostic tool, using high-throughput sequencing (NGS) technologies, known as GenoDENT, used to unravel molecular diagnosis behind rare diseases with oral and dental manifestations, enabled the identification of more than 15% of variants of unknown significance (Class III - VUS), beside a high 70%–80% diagnostic rate in the analyzed patients’ cohort. VUS make diagnosis more difficult because they prevent precise correlation between genotype and phenotype. To overcome this issue, we are developing 3D in vitro models mimicking odontogenesis. Our first 3D models, made of odontoblast-like and ameloblast-like cells, were effective when using murine cells. They were stable over time and showed a good distinction between both used cell types. However, the formation of 3D models from human cells was less efficient, so we decided to couple the 3D model formation technique with the cell-accumulation method to produce stable 3D constructs. This process consists in covering the cells with a biomimetic artificial matrix made of fibronectin (FN) and gelatin (G). We found that FN/G coating improved viability of human dental pulp stem cells (hDPSC) in thick constructs and promoted odontoblast differentiation of hDPSC. In peripheral ameloblast-like cells, ameloblast-associated proteins such as amelogenin were detected in close contact with the odontoblast-like core. We believe that our model can be further modified to introduce patient-specific variations through gene-editing techniques like CRISPR/Cas9, for further development of new diagnostic tools applied to rare oro-dental diseases.