Ablation of Pyrophosphate Regulators Promotes Periodontal Regeneration

Abstract

Biomineralization is regulated by inorganic pyrophosphate (PP_i), a potent physiological inhibitor of hydroxyapatite crystal growth. Progressive ankylosis protein (ANK) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) act to increase local extracellular levels of PP_i, inhibiting mineralization. The periodontal complex includes 2 mineralized tissues, cementum and alveolar bone (AB), both essential for tooth attachment. Previous studies demonstrated that loss of function of ANK or ENPP1 (reducing PP_i) resulted in increased cementum formation, suggesting PP_i metabolism may be a target for periodontal regenerative therapies. To compare the effects of genetic ablation of <i>Ank, Enpp1</i>, and both factors concurrently on cementum and AB regeneration, mandibular fenestration defects were created in <i>Ank</i> knockout (<i>Ank</i> KO), <i>Enpp1</i> mutant (<i>Enpp1^asj/asj</i>), and double KO (dKO) mice. Genetic ablation of <i>Ank, Enpp1</i>, or both factors increased cementum regeneration compared to controls at postoperative days (PODs) 15 and 30 (<i>Ank</i> KO: 8-fold, 3-fold; <i>Enpp1^asj/asj</i>: 7-fold, 3-fold; dKO: 11-fold, 4-fold, respectively) associated with increased fluorochrome labeling and expression of mineralized tissue markers, dentin matrix protein 1 (<i>Dmp1</i>/DMP1), osteopontin (<i>Spp1</i>/OPN), and bone sialoprotein (<i>Ibsp</i>/BSP). Furthermore, dKO mice featured increased cementum thickness compared to single KOs at POD15 and <i>Ank</i> KO at POD30. No differences were noted in AB volume between genotypes, but osteoblast/osteocyte markers were increased in all KOs, partially mineralized osteoid volume was increased in dKO versus controls at POD15 (3-fold), and mineral density was decreased in <i>Enpp1^asj/asj</i> and dKOs at POD30 (6% and 9%, respectively). Increased numbers of osteoclasts were present in regenerated AB of all KOs versus controls. These preclinical studies suggest PP_i modulation as a potential and novel approach for cementum regeneration, particularly targeting ENPP1 and/or ANK. Differences in cementum and AB regeneration in response to reduced PP_i conditions highlight the need to consider tissue-specific responses in strategies targeting regeneration of the entire periodontal complex.