Biomineralization is regulated by inorganic pyrophosphate (PPi), 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 PPi, 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 PPi) resulted in increased cementum formation, suggesting PPi metabolism may be a target for periodontal regenerative therapies. To compare the effects of genetic ablation of Ank, Enpp1, and both factors concurrently on cementum and AB regeneration, mandibular fenestration defects were created in Ank knockout ( Ank KO), Enpp1 mutant ( Enpp1asj/asj), and double KO (dKO) mice. Genetic ablation of Ank, Enpp1, or both factors increased cementum regeneration compared to controls at postoperative days (PODs) 15 and 30 ( Ank KO: 8-fold, 3-fold; Enpp1asj/asj: 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 ( Dmp1/DMP1), osteopontin ( Spp1/OPN), and bone sialoprotein ( Ibsp/BSP). Furthermore, dKO mice featured increased cementum thickness compared to single KOs at POD15 and Ank 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 Enpp1asj/asj 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 PPi 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 PPi conditions highlight the need to consider tissue-specific responses in strategies targeting regeneration of the entire periodontal complex.

Factors regulating the ratio of pyrophosphate (PPi) to phosphate (Pi) modulate biomineralization. Tissue-nonspecific alkaline phosphatase (TNAP) is a key promineralization enzyme that hydrolyzes the potent mineralization inhibitor PPi. The goal of this study was to determine whether TNAP could promote periodontal regeneration in bone sialoprotein knockout mice ( Ibsp−/− mice), which are known to have a periodontal disease phenotype. Delivery of TNAP was accomplished either systemically (through a lentiviral construct expressing a mineral-targeted TNAP-D10 protein) or locally (through addition of recombinant human TNAP to a fenestration defect model). Systemic TNAP-D10 delivered by intramuscular injection at 5 d postnatal (dpn) increased circulating alkaline phosphatase (ALP) levels in Ibsp−/− mice by 5-fold at 30 dpn, with levels returning to normal by 60 dpn when tissues were evaluated by micro–computed tomography and histology. Local delivery of recombinant human TNAP to fenestration defects in 5-wk-old wild type (WT) and Ibsp−/− mice did not alter long-term circulating ALP levels, and tissues were evaluated by micro–computed tomography and histology at postoperative day 45. Systemic and local delivery of TNAP significantly increased alveolar bone volume (20% and 37%, respectively) and cementum thickness (3- and 42-fold) in Ibsp−/− mice, with evidence for periodontal ligament attachment and bone/cementum marker localization. Local delivery significantly increased regenerated cementum and bone in WT mice. Addition of 100-μg/mL bovine intestinal ALP to culture media to increase ALP in vitro increased media Pi concentration, mineralization, and Spp1 and Dmp1 marker gene expression in WT and Ibsp−/− OCCM.30 cementoblasts. Use of phosphonoformic acid, a nonspecific inhibitor of sodium Pi cotransport, indicated that effects of bovine intestinal ALP on mineralization and marker gene expression were in part through Pi transport. These findings show for the first time through multiple in vivo and in vitro approaches that pharmacologic modulation of Pi/PPi metabolism can overcome periodontal breakdown and accomplish regeneration.

IntroductionBone sialoprotein (BSP) is a key regulator of mineralized tissue formation. Previously, we generated BSP-KAE knock-in mice (KAEKI mice) by substituting a non-function KAE (lysine-alanine-glutamic acid) for the integrin-binding RGD (arginine-glycine-aspartic acid) sequence and reported a vital role of the BSP-RGD motif in modulating the periodontal ligament (PDL). Specifically, histologically a disorganization of the PDL was noted, resulting in a weakened function of the PDL as measured by dynamic mechanical analysis. Intriguingly, also noted was a weight gain as KAEKI mice aged. While several proteins associated with mineralized tissues are reported to affect energy metabolism, the metabolic role of the BSP-RGD region has yet to be elucidated. Here we focus on defining the role of the BSP-RGD region in metabolic activity.MethodsBody weight, body composition, and caloric intake were measured in wild type (WT) and KAEKI mice. Energy expenditure was estimated using energy balance technique. Epididymal fat, interscapular fat, and liver were harvested for histological analysis. Systemic metabolic phenotype was assessed by sera analyses, insulin tolerance and glucose tolerance tests.ResultsThe results showed that KAEKI mice developed mild obesity starting from 13 weeks postnatal (wpn). The increase in body weight correlated with an increase in lean mass and visceral adiposity. Histological examination revealed adipocyte hypertrophy in white epididymal fat and interscapular brown fat in KAEKI vs. WT mice at 17 wpn. Metabolic profiling indicated that KAEKI mice had dyslipidemia and hyperleptinemia but no significant changes in glucose metabolism. Energy balance analyses revealed that hyperphagia preceded weight gain in KAEKI mice.ConclusionThese data suggest that the RGD region of BSP affects energy metabolism by regulating food intake, with further studies warranted to uncover the underlying mechanisms.