Ablation of Pyrophosphate Regulators Promotes Periodontal Regeneration

Abstract

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.

Delivery of Alkaline Phosphatase Promotes Periodontal Regeneration in Mice

“…Accumulative data have revealed that the periodontium, particularly cementum, is sensitive to regulators of P i /PP i metabolism (Zweifler et al 2015; Thumbigere-Math et al 2018; Chu et al 2020; Nagasaki et al 2020). We determined whether pharmacologic modulation of P i /PP i through delivery of mineralization-promoting enzyme TNAP could promote cementogenesis using a novel Ibsp −/− mouse model of periodontal breakdown.…”

“…Identification of therapeutic approaches inspired by understanding the developmental biology of periodontal tissues may lead to improved therapeutic outcomes. Studies from our group and others revealed that appropriate balance in levels of inorganic phosphate (P i ), a promoter of hydroxyapatite formation, and pyrophosphate (PP i ), a potent inhibitor of hydroxyapatite crystal growth, is crucial for proper mineralization and that acellular cementum is particularly sensitive to this mechanism of regulation (Foster et al 2012; Ao et al 2017; Thumbigere-Math et al 2018; Chu et al 2020; Nagasaki et al 2020). Three P i /PP i regulators work in concert to direct mineralization of periodontal tissues.…”

“…We reported that genetic reduction of PP i by crossing Ank −/− mice with Alpl −/− mice corrected cementum developmental defects (Chu et al 2020). Furthermore, loss of ANK or ENPP1 promotes cementum regeneration in a periodontal fenestration model (Rodrigues et al 2011; Nagasaki et al 2020), implicating modifiers of P i /PP i metabolism as strategies to promote periodontal regeneration.…”

Loss of function mutation in progressive ankylosis gene causes aberrant mineralization and acquisition of osteoblast-like-phenotype by the cells of the intervertebral disc

“…The role of ANK has been studied in bone, teeth, and cartilage, which has provided insights into its biological functions, including stimulator of marrow stromal cell and osteoblast differentiation into osteocytes 12,13,28 , a suppressor of acellular cementum mineralization [29][30][31][32] , a protector of chondrocyte phenotype 33 , and indirect stimulator of mineralization of cartilaginous tissue 34 . These studies have highlighted that ANK has broader context dependent biological functions than simply as an inhibitor of calci ed mineralization.…”

Between a rock and a hard place: Regulation of mineralization in the periodontium

“…As a first proof‐of‐concept, a fenestration defect model showed that Ank −/− mice regenerated increased amounts of acellular cementum compared to WT (Rodrigues, Nagatomo, Foster, Nociti, & Somerman, 2011 ). Building on that study and using the same fenestration model, genetic ablation of Ank , Enpp1 , or both factors increased cementum regeneration compared to controls; however, no differences were found in regenerated alveolar bone between genotypes (Nagasaki et al, 2021 ). Differences in cementum and alveolar bone in response to reduced PP i underscore the need to consider tissue‐specific responses in strategies aiming to regenerate the entire periodontal complex.…”