Supplementary Material for: The Progressive Ankylosis Protein Regulates Cementum Apposition and Extracellular Matrix Composition

<i>Background/Aims:</i> Tooth root cementum is sensitive to modulation of inorganic pyrophosphate (PP<sub>i</sub>), an inhibitor of hydroxyapatite precipitation. Factors increasing PP<sub>i</sub> include progressive ankylosis protein (ANK) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) while tissue nonspecific alkaline phosphatase hydrolyzes PP<sub>i</sub>. Studies here aimed to define the role of ANK in root and cementum by analyzing tooth development in <i>Ank</i> knock-out (KO) mice versus wild type. <i>Materials and Methods:</i> Periodontal development in KO versus control mice was analyzed by histology, histomorphometry, immunohistochemistry, in situ hybridization, electron microscopy, and nanoindentation. Cementoblast cultures were used in vitro to provide mechanistic underpinnings for PP<sub>i</sub> modulation of cell function. <i>Results:</i> Over the course of root development, <i>Ank</i> KO cervical cementum became 8- to 12-fold thicker than control cervical cementum. Periodontal ligament width was maintained and other dentoalveolar tissues, including apical cementum, were unaltered. Cervical cementum uncharacteristically included numerous cells, from rapid cementogenesis. <i>Ank </i>KO increased osteopontin and dentin matrix protein 1 gene and protein expression, and markedly increased NPP1 protein expression in cementoblasts but not in other cell types. Conditional ablation of <i>Ank</i> in joints and periodontia confirmed a local role for ANK in cementogenesis. In vitro studies employing cementoblasts indicated that <i>Ank</i> and <i>Enpp1</i> mRNA levels increased in step with mineral nodule formation, supporting a role for these factors in regulation of cementum matrix mineralization. <i>Conclusion: </i>ANK, by modulating local PP<sub>i</sub>, controls cervical cementum apposition and extracellular matrix. Loss of ANK created a local environment conducive to rapid cementogenesis; therefore, approaches modulating PP<sub>i</sub> in periodontal tissues have potential to promote cementum regeneration.