Negative regulation by pyrophosphate dictates cementogenesis

Objectives: Pyrophosphate (PPi) functions as a regulator of physiological mineralization and pathologic calcification by inhibiting hydroxyapatite crystal precipitation. During tooth root development, diminution of PPi causes increased cementum. In order to study mechanisms by which PPi governs cementogenesis, we employed in vivo models featuring reduced PPi levels in progressive ankylosis (Ank) or ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) knock-out (KO) mice, as well as in vitro cell culture experiments. Methods: Periodontal development in KO vs. control mice was analyzed by histology, histomorphometry, polarized light microscopy, and immunohistochemistry. Cementoblast cultures were used in parallel to provide mechanistic underpinnings for PPi modulation of cell function. Results: Over the course of root development, Ank and Enpp1 KO cervical cementum (acellular extrinsic fiber cementum, AEFC) featured significantly increased appositional rates, measured 8-60 days postnatal. In the mature molar, KO cementum was increased 12-fold vs. controls, and numerous cells were incorporated during the rapid apposition. In contrast, apically located cellular cementum (cellular intrinsic fiber cementum, CIFC) was unaffected by low PPi. Histomorphometry confirmed that dentin was unchanged and periodontal ligament space was maintained in both knock-outs. Polarized light revealed a high density of Sharpey's fiber insertions in KO cervical cementum, confirming its extrinsic fiber origins. KO cervical cementum exhibited increased mineral-associated proteins osteopontin (OPN) and dentin matrix protein-1 (DMP1) in conjunction with increased thickness. In vitro studies showed that low PPi concentrations promoted increased mineralization, coincident with increased Ank, Enpp1, Opn, and Dmp1 mRNAs, linking PPi metabolism with both mineral apposition and cellular expression of mineralization-related proteins. Conclusions: ANK and NPP1 govern concentrations of microenvironmental PPi, a central negative regulator of AEFC apposition and extracellular matrix composition. Results from these studies illuminate mechanisms which shape AEFC vs. CIFC formation, and underscore the concept that PPi modulation has potential to promote cementum regeneration. NIH R01DE15109(MJS), R01AR47908(JLM)