Background

Periodontal disease is a prevalent issue and a major cause of tooth loss. Although the periodontal ligament (PDL) is known to contain resident stem cell populations, it is poorly understood. Therefore, harvesting PDL cell populations and maintaining their characteristics without in vitro culture will provide insight to guide regenerative therapies.

Periodontal ligament stem cells (PDLSCs) are capable of differentiating into osteo-/cemento-lineage cells in vitro and in vivo (Seo et al., 2004; Zhao et al., 2021). Cementoblasts are cells responsible for the attachment of PDL fibres to the tooth. Little is known about the differentiation pathways within the PDL that lead to cementoblast formation due to difficulties in isolating this cell population (Matthews et al., 2016).

Previously, human periodontal ligament cells were isolated to evaluate their regenerative potential, which involved separation of the PDL from tooth roots and subsequent in vitro culture (Park et al., 2011). A protocol was established to isolate rat PDL cells (Kaneda et al., 2006), demonstrating that dissociation of PDL tissue using 2 g/ml collagenase and 0.25% trypsin for 110 min allows all attached PDL cells to be harvested. Separation of PDL tissue from mouse teeth is more difficult due to their small size. We used collagenase P for 30-45 min and found that mouse PDL cells can be collected effectively. We developed a protocol to collect sufficient PDL cells from mouse molars for single-cell transcriptomics analysis (Zhao et al., 2021).