Model for tooth development in vitro

The ultimate goal of Tissue Engineering is the regeneration of whole organs or functional organ units. Recent approaches rely on the usage of adult tissue-specific cells in three-dimensional culture. It is now accepted that "Developmental Engineering", the recapitulation of critical initial steps of organogenesis in vitro, is more expedient. Identification of key players and basic mechanisms behind organ formation is one of the major challenges for tissue engineering. Developmental biology addresses these questions and much knowledge has been accumulated on the mechanisms behind organogenesis by studying animal models. However, animal models evince species-related limitations and arise ethical issues. Functional in vitro models emulating the physiological processes during human organ formation and homeostasis are requested and invaluable for future research. Here, a developmentally inspired approach is pursued to reproduce fundamental steps of human tooth organogenesis in vitro. The presented model comprises the usage of human dental pulp cells from adult donors that were expanded in monolayer before culture under non-adherent conditions to accomplish a 3D self-organized mesenchymal condensation. The cells reproducibly formed uniform organoids of 500 μm in diameter, resembling the size of a human mesenchymal tooth germ. Comprehensive gene expression analysis by qPCR and Next Generation Sequencing revealed a dramatic remodelling of the signalosome in the DPCs. The cells instantly responded to the changed culture conditions and subsequent cell-cell interaction with significantly modulated gene expression regarding cytoskeletal rearrangement. The suppression of RhoA activity was exhibited, a central molecule in actin-mediated mechanotransduction leading to cell shape change and subsequent induction of tissue-specific cell fate. The central role of actin-mediated signalling was evidenced by a functional assay, in which the actin polymerization and thereby cytoskeletal signalling was inhibited. No cell-cell contacts or condensation was observed in the DPCs upon administration of the inhibitor. Furthermore, central signalling pathways of mesenchymal condensation have been shown to be upregulated within the first six hours of the culture model. Among them are the TGFß- and Activin, the FGF, the Wnt, and the Notch pathway. The bivalent results from transcriptional analysis, regarding the activation status of the MAPK pathway, were unraveled by a MAPK reporter assay, which evidenced an immediate-early MAPK activity within minutes of condensation. A Collagen-binding eGFP-fused protein (CNA-eGFP) was applied to the DPC condensations to track Collagen protein secretion, which was visible after 4 hours. From protein analyses by immunohistological staining, after 4 days of condensation, the ECM components Tenascin, Fibronectin, Collagen type I and type IV were present in the DPC condensates. Long-term cultures of DPC condensates (4 weeks) showed odontogenic differentiation by upregulated expression of Dentin sialophosphoprotein, TGFß1, Activin, and BMP7. Inductive abilities of the self-organized dental mesenchyme were evidenced in co-culture with epithelial cells, that exhibited invagination and cytodifferentiation. The presented model of tooth development fulfills all demands on a functional human organogenesis model. It recapitulates the initial step of mesenchymal condensation and subsequent odontogenic differentiation and is capable of directing reciprocal differentiation induction of combined epithelium. Therefore, it represents a valuable model to study basic developmental mechanisms and is in the pipeline to find application in regenerative therapies.

Author dc:creator

• Rosowski, Jennifer

Advisor dc:contributor.advisor

• Lauster, Roland

Licence dc:rights.uri

• https://creativecommons.org/licenses/by/4.0/

Language dc:language.iso

en

Identifier URI

http://dx.doi.org/10.14279/depositonce-8142

OAI identifier oai:identifier

oai:depositonce.tu-berlin.de:11303/9032