Establishment and characterization of immortalized mouse ameloblast-like cell lines

Objectives: Enamel organ epithelium (EOE) gives rise to the major epithelial-derived cell types of tooth including the ameloblasts. The formation of enamel, termed amelogenesis, occurs through the cytodifferentiation of ameloblasts, ultimately leading to apoptosis and necrosis of these cells with eruption. Therefore, studies regarding enamel matrix formation and bioengineering have been limited. In this study, we establish and characterize two mouse immortalized ameloblast-like cell lines using human papillomavirus 16 (HPV16) E6/E7 oncogenes for the first time. Setting and Sample Population: Two mouse EOE dental cell lines (EOE-2M and EOE-3M). Material and Methods: Isolated EOE primary cells were used to establish clonal cell lines and immortalized using the HPV16 E6/E7 gene platform. Two established cell lines were characterized by growth rate (Cell Proliferation Assay, MTS), gene (quantitative RT-PCR) and protein (immunocytochemistry) expression profiles, and mineralization potential (in situ alkaline phosphatase (ALP) histochemistry and Xylene Orange staining) in media supplemented with ascorbic acid and β-glycerophosphate. Gene and protein expression analyses included specific enamel matrix and ameloblast cell markers: Amel, Ambn, Enam, Amtn, ODAM, MMP20, Krt14 and DLX3. Results: Both cell lines were maintained in excess of 30 passages, with EOE-3M cells proliferating at a slightly higher rate. The cell lines expressed all tested enamel matrix markers and produced a mineralized ECM demonstrating an ameloblast-like profile. Conclusions: Two mouse ameloblasts-like immortalized cell lines have been characterized that will be useful tool for studies regarding enamel bioengineering.