Abstract
Tooth amelogenesis is a complex process beginning with enamel organ cell differentiation and enamel matrix secretion, transitioning through changes in ameloblast polarity, cytoskeletal, and matrix organization, that affects crucial biomineralization events such as mineral nucleation, enamel crystal growth, and enamel prism organization. Here we have harvested the enamel organ including the pliable enamel matrix of postnatal first mandibular mouse molars during the first 8 days of tooth enamel development to conduct a step-wise cross-sectional analysis of the changes in the mineral and protein phase. Mineral phase diffraction pattern analysis using single-crystal, powder sample X-ray diffraction analysis indicated conversion of calcium phosphate precursors to partially fluoride substituted hydroxyapatite from postnatal day 4 (4 dpn) onwards. Attenuated total reflectance spectra (ATR) revealed a substantial elevation in phosphate and carbonate incorporation as well as structural reconfiguration between postnatal days 6 and 8. Nanoscale liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) demonstrated highest protein counts for ECM/cell surface proteins, stress/heat shock proteins, and alkaline phosphatase on postnatal day 2, high counts for ameloblast cytoskeletal proteins such as tubulin β5, tropomyosin, β-actin, and vimentin on postnatal day 4, and elevated levels of cofilin-1, calmodulin, and peptidyl-prolyl cis-trans isomerase on day 6. Western blot analysis of hydrophobic enamel proteins illustrated continuously increasing amelogenin levels from 1 dpn until 8 dpn, while enamelin peaked on days 1 and 2 dpn, and ameloblastin on days 1–5 dpn. In summary, these data document the substantial changes in the enamel matrix protein and mineral phase that take place during postnatal mouse molar amelogenesis from a systems biological perspective, including (i) relatively high levels of matrix protein expression during the early secretory stage on postnatal day 2, (ii) conversion of calcium phosphates to apatite, peak protein folding and stress protein counts, and increased cytoskeletal protein levels such as actin and tubulin on day 4, as well as (iii) secondary structure changes, isomerase activity, highest amelogenin levels, and peak phosphate/carbonate incorporation between postnatal days 6 and 8. Together, this study provides a baseline for a comprehensive understanding of the mineralogic and proteomic events that contribute to the complexity of mammalian tooth enamel development.
Highlights
Enamel development is an integral process of symphonic dimensions that is characterized by a continuous interplay between cells, matrices, minerals, proteins, and signals over the entire period of amelogenesis
Speaking, the key players in this symphony have been known for decades, including a mineral section that undergoes a transition from amorphous calcium phosphate and a protein section made up by classic enamel proteins such as amelogenins, ameloblastin, and enamelin, as they are further processed by enamelrelated enzymes, including MMP20 and KLK4
The key benefit of the postnatal mouse molar model was the suitability of the distal slopes of first molar cusps to harvest sufficient quantities of fresh enamel matrix for proteomic and mineral composition analysis in daily increments
Summary
Enamel development is an integral process of symphonic dimensions that is characterized by a continuous interplay between cells, matrices, minerals, proteins, and signals over the entire period of amelogenesis. Speaking, the key players in this symphony have been known for decades, including a mineral section that undergoes a transition from amorphous calcium phosphate and a protein section made up by classic enamel proteins such as amelogenins, ameloblastin, and enamelin, as they are further processed by enamelrelated enzymes, including MMP20 and KLK4. In addition to deciphering individual aspects of amelogenin function, much progress has been made elucidating the role of the less prominent enamel-related proteins ameloblastin and enamelin on enamel crystal growth and habit (Masuya et al, 2005; Lu et al, 2011; Hu et al, 2014). It has been demonstrated that enamel proteins undergo posttranslational processing by the enamel proteinases MMP20 and KLK4 (Bartlett and Simmer, 1999; Simmer and Hu, 2002; Bartlett, 2013)
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