Integrative Temporo-Spatial, Mineralogic, Spectroscopic, and Proteomic Analysis of Postnatal Enamel Development in Teeth with Limited Growth

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.

Mapping the Tooth Enamel Proteome and Amelogenin Phosphorylation Onto Mineralizing Porcine Tooth Crowns

“…The spatial distributions of AMELX, AMBN, ENAM, MMP20, and KLK4 (Figures 1D, 4) agree very well with published data on abundance patterns during specific stages of enamel formation (see Moradian-Oldak, 2012; Bartlett, 2013; Lacruz et al, 2017 and references therein). Our findings are consistent with the number and identifications of proteins of recently published proteomics analyses of pooled human and rodent enamel (Chen et al, 1995; Hubbard and Kon, 2002; Jagr et al, 2012, 2014; Eckhardt et al, 2014; Charone et al, 2016; Pandya et al, 2017; De Lima Leite et al, 2018; Mann et al, 2018; Zhang et al, 2018). For instance, we mapped 19 of the 24 proteins recently reported in enamel onto enamel formation stages (Pandya et al, 2017), and localized close homologs for the remaining five proteins (Supplementary Table 1).…”

“…Our findings are consistent with the number and identifications of proteins of recently published proteomics analyses of pooled human and rodent enamel (Chen et al, 1995; Hubbard and Kon, 2002; Jagr et al, 2012, 2014; Eckhardt et al, 2014; Charone et al, 2016; Pandya et al, 2017; De Lima Leite et al, 2018; Mann et al, 2018; Zhang et al, 2018). For instance, we mapped 19 of the 24 proteins recently reported in enamel onto enamel formation stages (Pandya et al, 2017), and localized close homologs for the remaining five proteins (Supplementary Table 1). Our spatial proteomic data for AMBN, ENAM, MMP20, and CA2 conform nearly identically to the abundances observed through western blotting of successive stages of mouse molar enamel mineralization as reported by Pandya et al (2017).…”

“…Changes in AMELX, ENAM, and AMBN abundance and modification occur within a broader proteomic context that helps regulate mineralization. Proteomic studies of enamel, dentin, bone and cementum have identified over 200 unique proteins (Eckhardt et al, 2014; Jagr et al, 2014; Charone et al, 2016; Pandya et al, 2017; De Lima Leite et al, 2018) with functions that include calcium binding, cytoskeletal and cell adhesion, immune function, proteolysis and protease inhibition. Gel electrophoresis and nano-LC-MS/MS analyses identified structural molecules including 20 keratins, collagens, serpins, ubiquitin and serum albumin (Webb et al, 1998; Felszeghy et al, 2000; Acil et al, 2005; Yamakoshi et al, 2006b;Jagr et al, 2012; Salmon et al, 2013; Castiblanco et al, 2015).…”

“…Though recent proteomics analyses of human and murine teeth indicate a high diversity of extracellular proteins during enamel formation, results so far have focused on the analysis of either entire molars, or of secretion and maturation stages of enamel formation in incisors, or have targeted the dentin-enamel junction, but have not addressed spatiotemporal abundance levels in more detail (Castiblanco et al, 2015; Charone et al, 2016; Pandya et al, 2017; De Lima Leite et al, 2018; Jágr et al, 2019). This is in part due to the small size of mouse teeth impeding the separation of enamel from dentin and pulp.…”

Tooth Enamel and Its Dynamic Protein Matrix

“…This approach was leveraged in the use of whole mouse molars. The teeth were harvested at specific and discrete postnatal time points during the first 8 days postnatally, for analyses of their protein composition by western blot and nano LC-MS/MS analysis [ 120 ]. The authors showed how the classic enamel proteins, including ameloblastin, enamelin and Mmp20 decreased one day to the next during tooth development.…”