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Central role of pyrophosphate in acellular cementum formation.

Foster BL, Nagatomo KJ, Nociti FH, Fong H, Dunn D, Tran AB, Wang W, Narisawa S, Millán JL, Somerman MJ - PLoS ONE (2012)

Bottom Line: Though PP(i) 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 PP(i) output, in compensatory fashion.In vitro mechanistic studies confirmed that under low PP(i) mineralizing conditions, cementoblasts increased Ank (5-fold) and Enpp1 (20-fold), while increasing PP(i) inhibited mineralization and associated increases in Ank and Enpp1 mRNA.These findings underscore developmental differences in acellular versus cellular cementum, and suggest new approaches for cementum regeneration.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Oral Connective Tissue Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, United States of America. brian.foster@nih.gov

ABSTRACT

Background: Inorganic pyrophosphate (PP(i)) is a physiologic inhibitor of hydroxyapatite mineral precipitation involved in regulating mineralized tissue development and pathologic calcification. Local levels of PP(i) are controlled by antagonistic functions of factors that decrease PP(i) and promote mineralization (tissue-nonspecific alkaline phosphatase, Alpl/TNAP), and those that increase local PP(i) 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 PP(i) in cementum formation, we analyzed root development in knock-out ((-/-)) mice featuring PP(i) dysregulation.

Results: Excess PP(i) 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 PP(i) 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 PP(i) 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 PP(i) output, in compensatory fashion. In vitro mechanistic studies confirmed that under low PP(i) mineralizing conditions, cementoblasts increased Ank (5-fold) and Enpp1 (20-fold), while increasing PP(i) 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 PP(i), directing and regulating mineral apposition. These findings underscore developmental differences in acellular versus cellular cementum, and suggest new approaches for cementum regeneration.

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Pyrophosphate does not affect cementoblast proliferation or collagen synthesis, in vitro.(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 PPi. (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.
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pone-0038393-g011: Pyrophosphate does not affect cementoblast proliferation or collagen synthesis, in vitro.(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 PPi. (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.

Mentions: Cells were introduced to exogenous PPi to create culture conditions of low (10 µM) and high (100 µM) PPi. The lower dose of 10 µM PPi did not affect mineralization, while the higher dose of 100 µM was confirmed as an inhibitor of mineral nodule formation under these conditions. While PPi is an inhibitor of HAP crystal precipitation, it has also been reported to have cell signaling effects in osteoblasts [4], [14], [16]. Neither dose of PPi affected OCCM.30 cell proliferation, viability, or collagen synthesis compared to controls (Figure 11), therefore these processes were not indirectly affecting mineralization. Cementoblast ALP enzyme activity was uniform across treatments and times, and added 100 µM PPi did not appreciably affect ALP (Figure 10C), indicating the effect of PPi on mineralization was not by inhibition of TNAP. An enzymatic assay for 5′-nucleotide phosphodiesterase I and nucleotide pyrophosphohydrolase (NTPPPH) activity demonstrated significantly increased NPP1 function with mineralization at days 4, 6, and 8, while 100 µM PPi brought activity back to basal levels of non-mineralizing cultures (Figure 10D).


Central role of pyrophosphate in acellular cementum formation.

Foster BL, Nagatomo KJ, Nociti FH, Fong H, Dunn D, Tran AB, Wang W, Narisawa S, Millán JL, Somerman MJ - PLoS ONE (2012)

Pyrophosphate does not affect cementoblast proliferation or collagen synthesis, in vitro.(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 PPi. (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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366957&req=5

pone-0038393-g011: Pyrophosphate does not affect cementoblast proliferation or collagen synthesis, in vitro.(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 PPi. (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.
Mentions: Cells were introduced to exogenous PPi to create culture conditions of low (10 µM) and high (100 µM) PPi. The lower dose of 10 µM PPi did not affect mineralization, while the higher dose of 100 µM was confirmed as an inhibitor of mineral nodule formation under these conditions. While PPi is an inhibitor of HAP crystal precipitation, it has also been reported to have cell signaling effects in osteoblasts [4], [14], [16]. Neither dose of PPi affected OCCM.30 cell proliferation, viability, or collagen synthesis compared to controls (Figure 11), therefore these processes were not indirectly affecting mineralization. Cementoblast ALP enzyme activity was uniform across treatments and times, and added 100 µM PPi did not appreciably affect ALP (Figure 10C), indicating the effect of PPi on mineralization was not by inhibition of TNAP. An enzymatic assay for 5′-nucleotide phosphodiesterase I and nucleotide pyrophosphohydrolase (NTPPPH) activity demonstrated significantly increased NPP1 function with mineralization at days 4, 6, and 8, while 100 µM PPi brought activity back to basal levels of non-mineralizing cultures (Figure 10D).

Bottom Line: Though PP(i) 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 PP(i) output, in compensatory fashion.In vitro mechanistic studies confirmed that under low PP(i) mineralizing conditions, cementoblasts increased Ank (5-fold) and Enpp1 (20-fold), while increasing PP(i) inhibited mineralization and associated increases in Ank and Enpp1 mRNA.These findings underscore developmental differences in acellular versus cellular cementum, and suggest new approaches for cementum regeneration.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Oral Connective Tissue Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, United States of America. brian.foster@nih.gov

ABSTRACT

Background: Inorganic pyrophosphate (PP(i)) is a physiologic inhibitor of hydroxyapatite mineral precipitation involved in regulating mineralized tissue development and pathologic calcification. Local levels of PP(i) are controlled by antagonistic functions of factors that decrease PP(i) and promote mineralization (tissue-nonspecific alkaline phosphatase, Alpl/TNAP), and those that increase local PP(i) 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 PP(i) in cementum formation, we analyzed root development in knock-out ((-/-)) mice featuring PP(i) dysregulation.

Results: Excess PP(i) 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 PP(i) 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 PP(i) 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 PP(i) output, in compensatory fashion. In vitro mechanistic studies confirmed that under low PP(i) mineralizing conditions, cementoblasts increased Ank (5-fold) and Enpp1 (20-fold), while increasing PP(i) 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 PP(i), directing and regulating mineral apposition. These findings underscore developmental differences in acellular versus cellular cementum, and suggest new approaches for cementum regeneration.

Show MeSH
Related in: MedlinePlus