Basement Membrane of Hertwig’s Epithelial Root Sheath Is Involved in Dental Follicle Cell Differentiation into Cementoblasts

The cementum is a mineralized tissue that covers the tooth root and is necessary for anchoring periodontal ligament fibers to the teeth. During root development, dental follicle (DF) cells differentiate into cementoblasts, periodontal ligament fibroblasts, and osteoblasts to form the cementum, periodontal ligament, and bone, respectively. However, the mechanisms underlying these cell fate decisions remain unclear. Using scanning electron microscopy, we observed a basement membrane (BM)-like structure between the forming root dentin on the periodontal ligament side and the Hertwig's epithelial root sheath (HERS), as well as similar BM structures on the dentin surface after HERS removal. We hypothesized that these HERS-derived BM structures are involved in the differentiation of DF cells into cementoblasts and investigated the effect of basement membrane components (BMCs) on the differentiation of DF cells. Using a cell line (MDF) derived from DF cells of the incisors, we studied adhesion to hydroxyapatite. The undifferentiated MDF cells showed no adhesion. However, when coated with the BMC secreted by the HERS cell line (HERS02T), established from the first lower molars of tdTomato mice, MDF cells demonstrated increased initial adhesion, alkaline phosphatase activity, and calcification ability on hydroxyapatite. Additionally, we performed a comprehensive proteomic analysis of BMC secreted by HERS02T cells and-based on total spectral counts-identified three major proteins: laminin-332, tenascin-C, and periostin. These protein components were coated onto apatite-coated dishes and tested for the induction of MDF differentiation. The results showed that the coating significantly upregulated the expression of the cementoblast differentiation markers <i>Sparcl1</i> and <i>Ibsp</i>. These results suggested that HERS may detach after BM formation on dentin surfaces. Subsequently, DF differentiate into cementoblasts using the BM when they migrate to the dentin surface.