Low molecular mass amelogenin-related polypeptides extracted from mineralized dentin have the ability to affect the differentiation pathway of embryonic muscle fibroblasts in culture and lead to the formation of mineralized matrix in in vivo implants. The objective of the present study was to determine whether the bioactive peptides could have been amelogenin protein degradation products or specific amelogenin gene splice products. Thus, the splice products were prepared, and their activities were determined in vitro and in vivo. A rat incisor tooth odontoblast pulp cDNA library was screened using probes based on the peptide amino acid sequencing data. Two specific cDNAs comprised from amelogenin gene exons 2,3,4,5,6d,7 and 2,3,5,6d,7 were identified. The corresponding recombinant proteins,

Background/Aims: Tooth root cementum is sensitive to modulation of inorganic pyrophosphate (PP_i), an inhibitor of hydroxyapatite precipitation. Factors increasing PP_i include progressive ankylosis protein (ANK) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) while tissue nonspecific alkaline phosphatase hydrolyzes PP_i. Studies here aimed to define the role of ANK in root and cementum by analyzing tooth development in Ank knock-out (KO) mice versus wild type. Materials and Methods: Periodontal development in KO versus control mice was analyzed by histology, histomorphometry, immunohistochemistry, in situ hybridization, electron microscopy, and nanoindentation. Cementoblast cultures were used in vitro to provide mechanistic underpinnings for PP_i modulation of cell function. Results: Over the course of root development, Ank KO cervical cementum became 8- to 12-fold thicker than control cervical cementum. Periodontal ligament width was maintained and other dentoalveolar tissues, including apical cementum, were unaltered. Cervical cementum uncharacteristically included numerous cells, from rapid cementogenesis. Ank KO increased osteopontin and dentin matrix protein 1 gene and protein expression, and markedly increased NPP1 protein expression in cementoblasts but not in other cell types. Conditional ablation of Ank in joints and periodontia confirmed a local role for ANK in cementogenesis. In vitro studies employing cementoblasts indicated that Ank and Enpp1 mRNA levels increased in step with mineral nodule formation, supporting a role for these factors in regulation of cementum matrix mineralization. Conclusion: ANK, by modulating local PP_i, controls cervical cementum apposition and extracellular matrix. Loss of ANK created a local environment conducive to rapid cementogenesis; therefore, approaches modulating PP_i in periodontal tissues have potential to promote cementum regeneration.

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Embryonic mouse tooth germs were cultured in vitro in the presence of two related amelogenin isoforms to determine their effects on tooth development. Our results show that these individual proteins have specific but quite different effects on epithelial-derived ameloblasts versus mesenchymal-derived odontoblasts.Introduction: Amelogenins, the main protein components of enamel matrix, have been shown to have signaling activity. Amelogenin isoforms differing only by the presence or exclusion of exon 4, designated [A+4] (composed of exons 2, 3, 4, 5, 6d, and 7) and [A-4] (composed of exons 2, 3, 5, 6d, and 7), showed similar, but different, effects both in vitro and in vivo on postnatal teeth. Materials and Methods: Lower first molar tooth germs of E15/16 CD1 mice were microdissected and cultured in vitro in a semisolid media containing either 20% FBS, 2% FBS, or 2% FBS with either 1.5 nM [A+4], [A-4], or both for 6 days. Tooth germs were analyzed by H&E staining and immunohistochemistry for collagen I, dentin matrix protein 2, and DAPI nuclear staining. Results: Teeth cultured in media containing 20% FBS showed normal development with polarized ameloblasts, and odontoblasts producing dentin matrix, and DMP2 expression in odontoblasts and pre-ameloblasts. Culture in 2% FBS media resulted in no ameloblast polarization and modest odontoblast differentiation with scant dentin matrix. Tooth germs cultured with [A+4] in 2% FBS media had well-polarized odontoblasts with robust dentin production and concomitant ameloblast polarization. DMP2 expression was equal to or greater than seen in the 20% FBS culture condition. In cultures with [A-4] in 2% FBS media, odontoblast polarization and dentin production was reduced compared with [A+4]. However, the pre-ameloblast layer was disorganized, with no ameloblast polarization occurring along the dentin surface. DMP2 expression was reduced in the odontoblasts compared with the 20% FBS and [A+4] conditions and was almost completely abrogated in the pre-ameloblasts. Conclusion: These data show different signaling activities of these closely related amelogenin isoforms on tooth development. Here we make the novel observation that [A-4] has an inhibitory effect on ameloblast development, whereas [A+4] strongly stimulates odontoblast development. We show for the first time that specific amelogenin isoforms have effects on embryonic tooth development in vitro and also hypothesize that DMP2 may play a role in the terminal differentiation of both ameloblasts and odontoblasts.