Generation and Application of HERS Spheroids in Tooth Root Bioengineering
Objectives: The construction of biological root and its supportive tissue is the challenge for tooth replacement. Hertwig’s epithelial root sheath (HERS) plays important role in root formation while the number of the cells is limited. The three-dimensional (3D) culture is more likely to preserve the biological functions of the cells and expand the cells efficiently. Our aim is to find a new approach to expand HERS cells with vitality and regenerate dentin and periodontium by the combination of HERS spheroids and dental papilla cells (DPCs) Methods: HERS spheroids were generated via 3D-culture system with matrigel. They were characterized for their size, morphology, proliferation rate, immunophenotype, colony forming efficiency and multi-lineage differentiation potential by scanning electron microscopy, haematoxylin and eosin stain, cell counting kit 8, immunofluorescence, colony-forming assay and differentiation assays in comparison with two-dimensional cultured cells (2D-cells). The cementum, periodontal ligament and dentin formation were investigated by rat renal capsule implantation. Results: The HERS cells were arranged orderly in spheroids with diameters from 20μm to 60μm. There were much more cells in spheroids positive for stem cell markers and Ki67 proliferation antigen than the 2D-cells. The cells in spheroids could proliferate more quickly and retain more potential to conduct osteogenic/cementogenic, adipogenic and neurogenic differentiation than the 2D-cells. The spheroids could generate single cell clones while the 2D-cells could not. In vivo the spheroids transplants generated more cementum-like and periodontal ligament-like tissues than the 2D-cell transplants. DPCs/spheroids transplants generated more dentin-like tissues than the DPCs and DPCs/2D-cells grafts, indicating the potential role of HERS spheroids in promoting the differentiation of DPCs and dentin formation. Conclusions: The 3D cultured HERS spheroids are promising cell sources for periodontium and dentin regeneration and tooth root bioengineering.