Spheroid formation and stemness preservation of human periodontal ligament cells on chitosan films

Abstract

Objective: The therapeutic potential of periodontal ligament cells for periodontal regeneration gradually decreases when cultured as a monolayer in vitro. Three-dimensional cell culture models provide an alternative to traditional monolayer cell culture. This study aimed to comparatively evaluate the influence of spheroid culture on periodontal ligament cells. Materials and Methods: Chitosan films were used to culture three-dimensional periodontal ligament cell spheroids. The proliferation, self-renewal, and osteogenic capacity of periodontal ligament cells derived from spheroids were evaluated and compared with cells cultured on a monolayer. Results: Viable spheroids of periodontal ligament cells were formed on chitosan films. Compared to monolayer cell culture, periodontal ligament cells exhibited decreased proliferation upon spheroid formation. In contrast, their expression of genes related to self-renewal was significantly higher comparison with cells cultured in a monolayer. Moreover, the formation of periodontal ligament cell spheroids increased their colony-forming unit ability and osteogenic differentiation capacity. Conclusion: The results demonstrate the successful use of chitosan films for the culture of periodontal ligament cell spheroids. Compared to cells cultured in monolayer, periodontal ligament cells in spheroids did not proliferate, but exhibited higher self-renewal gene expression, colony-forming unit and osteogenic capacity.