Central Role of Pyrophosphate in Acellular Cementum Formation

Background: Inorganic pyrophosphate (PPi) is a physiologic inhibitor of hydroxyapatite mineral precipitation involved in regulating mineralized tissue development and pathologic calcification. Local levels of PPi are controlled by antagonistic functions of factors that decrease PPi and promote mineralization (tissue-nonspecific alkaline phosphatase, Alpl/TNAP), and those that increase local PPi and restrict mineralization (progressive ankylosis protein, ANK; ectonucleotide pyrophosphatase phosphodiesterase-1, NPP1). The cementum enveloping the tooth root is essential for tooth function by providing attachment to the surrounding bone via the nonmineralized periodontal ligament. At present, the developmental regulation of cementum remains poorly understood, hampering efforts for regeneration. To elucidate the role of PPi in cementum formation, we analyzed root development in knock-out ((-/-)) mice featuring PPi dysregulation. Results: Excess PPi in the Alpl(-/-) mouse inhibited cementum formation, causing root detachment consistent with premature tooth loss in the human condition hypophosphatasia, though cementoblast phenotype was unperturbed. Deficient PPi in both Ank and Enpp1(-/-) mice significantly increased cementum apposition and overall thickness more than 12-fold vs. controls, while dentin and cellular cementum were unaltered. Though PPi regulators are widely expressed, cementoblasts selectively expressed greater ANK and NPP1 along the root surface, and dramatically increased ANK or NPP1 in models of reduced PPi output, in compensatory fashion. In vitro mechanistic studies confirmed that under low PPi mineralizing conditions, cementoblasts increased Ank (5-fold) and Enpp1 (20-fold), while increasing PPi inhibited mineralization and associated increases in Ank and Enpp1 mRNA. Conclusions: Results from these studies demonstrate a novel developmental regulation of acellular cementum, wherein cementoblasts tune cementogenesis by modulating local levels of PPi, directing and regulating mineral apposition. These findings underscore developmental differences in acellular versus cellular cementum, and suggest new approaches for cementum regeneration

Pyrophosphate does not affect cementoblast proliferation or collagen synthesis, <i>in vitro</i>.

<p>(A) Cell proliferation was assayed by MTS assay where absorbance at 570 nm is proportional to the number of living cells in culture. No difference in OCCM.30 cementoblast cell number was found between non-mineralizing (AA) and mineralizing (AA + BGP) treatments at concurrent time points, including with doses of 10 or 100 µM PP_i. (B) Picrosirius red dye was used to stain collagen deposited by cementoblasts at days 3, 5, and 7. (C) Quantification of the collagen-binding assay did not identify any treatment differences for collagen deposition at any of the time points. For both (A) and (C), graphs show mean +/− SD for n = 3 samples, and no intergroup significant differences (at the same time point) were identified by one-way ANOVA and post-hoc Tukey analysis, for α = 0.05.</p

Increased cementum apposition in <i>Ank</i> and <i>Enpp1</i>^−/− teeth.

<p>(A) During early root formation between 14 and 26 dpn, both <i>Ank</i> and <i>Enpp1</i>^−/− molars featured at least 10-fold greater cementogenesis compared to controls. From 26 to 60 dpn, <i>Ank</i> and <i>Enpp1</i>^−/− cementum continued to increase at a rate of 0.2–0.7 µm/day, while <i>Ank</i> and <i>Enpp1</i>^+/+ controls featured tightly controlled apposition at the pace of 0.01–0.05 µm/day. (B) Histomorphometry confirmed <i>Ank</i> or <i>Enpp1</i>^−/− cervical cementum was significantly increased compared to controls, while PDL width was maintained and alveolar bone thickness tended towards reduction. Values with the same letter were not significantly different, while different letters indicate a statistically significant intergroup (genotype) difference (p<0.05) as tested by ANOVA followed by the Tukey test for direct pair-wise comparisons.</p

Reduced pyrophosphate alters gene expression in cervical cementoblasts.

<p><i>Opn</i> mRNA is markedly increased in root-lining cementoblasts in both (B) <i>Ank</i> and (C) <i>Enpp1</i>^−/−, compared to (A) <i>Ank</i> and <i>Enpp1</i>^+/+ controls. Increased numbers of cells associated with the thick cervical cementum express <i>Dmp1</i> in (E) <i>Ank</i> and (F) <i>Enpp1</i>^−/− molars, compared to (D) ^+/+ controls. <i>Bsp</i> gene expression was not different in cementoblasts in (H) <i>Ank</i> and (I) <i>Enpp1</i>^−/− vs. (G) ^+/+ controls. Black arrowheads indicate regions of positively stained cells. All panels are samples from mice at 14 dpn. Abbreviations: d = dentin, c = (cervical) cementum; p = periodontal ligament; b = bone. Scale bar = 100 µm.</p

Pyrophosphate regulates cementoblast mineralization-coupled gene expression.

<p>Mineralizing cultures (AA + BGP) increased expression of <i>Ank</i>, <i>Enpp1</i>, <i>Opn</i>, and <i>Dmp1</i> at days 3 and 5, concurrent with mineralization. The higher dose of 100 µM PP_i significantly depressed expression of all four genes on day 3 compared to mineralizing cultures; <i>Ank</i>, <i>Enpp1</i>, and <i>Opn</i> suppression was maintained on day 5. The lower dose of 10 µM PP_i showed milder effects on all four genes at day 3. Differences were not maintained by day 7. Unlike expression of <i>Ank</i>, <i>Enpp1</i>, <i>Opn</i>, and <i>Dmp1</i>, where PP_i was able to block mineralization-associated induction, additional markers <i>Alpl</i>, <i>Bsp</i>, and <i>Col1</i> were not regulated in coordinated fashion by mineralization or inclusion of either dose of PP_i. Graphs show mean +/− SD for n = 3 samples. Lowercase letters indicate treatment comparison at each time point, where different letters indicate a statistically significant intergroup difference. Uppercase letters indicate comparisons over time in the same treatment group, where different letters indicate a statistically significant intragroup difference. Values sharing the same uppercase or lowercase letter in were not significantly different. Means were compared by ANOVA (p<0.05) followed by the Tukey test for direct pair-wise comparisons.</p

Pyrophosphate regulates cementoblast mineralization and nucleotide pyrophosphohydrolase (NTPPPH) activity, <i>in vitro</i>.

<p>(A) By von Kossa staining, OCCM.30 cells cultured with 5 mM BGP produced mineral nodules by days 6 and 8, while cells receiving only AA did not mineralize. The low dose of 10 µM PP_i did not affect mineral nodule precipitation, however, the higher dose of 100 µM was a potent inhibitor of mineral nodules. (B) Quantitative calcium assay performed on days 6 and 8 confirmed visual mineral nodule staining by von Kossa. (C) Relative ALP enzyme activity was not affected by inhibition of mineralization by 100 µM PP_i. (D) NTPPPHase activity was increased under mineralizing conditions, but inclusion of 100 µM PP_i brought activity back to basal levels of non-mineralizing cultures. Graphs show mean +/− SD for n = 3 samples. Lowercase letters indicate treatment comparison at each time point, where different letters indicate a statistically significant intergroup difference. Uppercase letters indicate comparisons over time in the same treatment group, where different letters indicate a statistically significant intragroup difference. Values sharing the same uppercase or lowercase letter in were not significantly different. Means were compared by ANOVA (p<0.05) followed by the Tukey test for direct pair-wise comparisons.</p

Pyrophosphate homeostasis in the extracellular space.

<p>Inorganic phosphate (P_i) is a component of mineral hydroxyapatite (HAP), while pyrophosphate (PP_i) is a potent inhibitor of HAP crystal precipitation and growth. The enzyme tissue nonspecific alkaline phosphatase (TNAP) hydrolyzes PP_i to release ionic P_i, creating conditions conducive for mineralization. Local PP_i is increased by the functions of the progressive ankylosis protein (ANK) and ectonucleotide pyrophosphatase phosphodiesterase 1 (NPP1), which act to keep the mineralization process in check.</p

Reduced pyrophosphate alters acellular cementum matrix composition.

<p>IHC was performed on <i>Ank</i> and <i>Enpp1</i>^+/+ (Control) and ^−/− tissues at 26 dpn. OPN defines the acellular cementum layer in (A) wild-type cervical cementum, and is intensely localized to the thick AEFC in (B, C) both ^−/− models. DMP1 did not label acellular cementum in (G) ^+/+ controls, but was increased dramatically in the thickened cervical cementum of (H, I) both ^−/− models. BSP was present in (M) control AEFC, as well as in (N, O) <i>Ank</i> and <i>Enpp1</i>^−/− AEFC in diluted concentrations. Localization of OPN, DMP1, and BSP was not different in cellular cementum of null models vs. controls (D–F, J–L, and P–R). Abbreviations: d = dentin; c = acellular cementum; p = periodontal ligament; b = bone. Scale bar = 100 µm.</p