Edited by: Steven Joseph Brookes, Leeds Dental Institute, United Kingdom
Reviewed by: Claudio Cantù, University of Zurich, Switzerland; Zhi Chen, Wuhan University, China; Brad A. Amendt, University of Iowa, United States
*Correspondence: Thomas G. H. Diekwisch
This article was submitted to Craniofacial Biology and Dental Research, a section of the journal Frontiers in Physiology
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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.
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. Allegorically 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 enamel-related enzymes, including MMP20 and KLK4. As amelogenesis progresses, the volume percentage of proteins and water decreases, while the mineral content increases, resulting in a 96% mineral content in the mature enamel layer of adult mammals (Deakins,
For decades, the effect of individual enamel proteins such as amelogenin on enamel crystal growth have been a most intriguing and rewarding subject of study (Lagerström et al.,
While much is known about the major proteins and minerals involved in tooth enamel formation, it has become increasingly obvious that amelogenesis is more complex than a mixture of an aqueous enamel protein solution with a combination of calcium and phosphate ions, subjected to enzymatic protein digestion and gradual removal of water over time. Recent studies have illustrated the importance of ion transport mechanisms for mineral transport (Hubbard,
Development of a synthetic or mimetic model of amelogenesis would greatly benefit from a temporo-spatial integration of the multitude of processes involved in mammalian amelogenesis. Such multi-level and multi-scale data mining commonly requires a systems biology approach. Systems biology of development seeks to integrate bioinformatic data analysis with other molecular, cellular, and tissue-related information to reach a higher-level, multifaceted, and integrative understanding of developmental processes (Bard,
In the present study we have employed first mandibular mouse molar amelogenesis as a model system to systematically map proteomic, spectroscopic, temporo-spatial, and mineralogic events during the first 8 days of postnatal enamel development. The benefit of a model based on teeth with limited growth is the synchronicity of developmental events leading up to maturation of the entire tooth surface by the time of tooth eruption and providing a homogeneous enamel matrix at each stage ideally suited for proteomic and spectroscopic analysis. During the course of this study we have generated sets of spectroscopic, proteomic, and mineralogic data and integrated related events through their common timescale of development. Our analysis provides timing of events, novel proteomic and spectroscopic data, and identification of novel non-hydrophobic groups of proteins and individual proteins that may contribute toward amelogenesis. Future studies will enhance our understanding of the interconnectedness between these processes during the progression of amelogenesis as they contribute to the formation of highly organized tooth enamel.
First mandibular molars of 1, 2, 3, 4, 5, 6, 7, and 8 day postnatal mice (Figures
Panel
Mouse molar enamel birefringence
Enamel thickness was measured on enlarged micrographs generated by a Leica stereo microscope. Enamel thickness was determined by calibration against a metric scale bar imaged at the same magnification. Birefringence of the enamel matrix was assessed by placing the enamel organs between crossed polarizers. The first polarizer was placed between the light source and the tooth organ and the second polarizer was placed between the tooth organ and the camera. The second polarizer was rotated in 45° intervals, and birefringence was identified based on the color changes within the enamel matrix.
For this study, the tooth enamel matrices of molars from days 1 to 8 postnatal mice were analyzed using X-ray powder diffraction in 1-day intervals (excluding samples from 3 day postnatal mice). For each day of analysis, the enamel matrix of four different mouse molars was harvested in distilled water, and samples from all four teeth at each developmental stage were pooled for further analysis. Samples were stored for about 24 h at < 0°C until analyzed at room temperature. Debye-Scherrer data simulations were obtained using a Bruker three-circle (transmission-mode) diffractometer using Mo radiation (0.7107 Å, APEX CCD detector, graphite monochromator, 0.3 mm Monocap capillary collimator, at operating conditions of 45 kV, 25 mA). Analyzed samples were~0.1 mm3 in volume, with each sample mounted on the end of a glass fiber, placed in the X-ray beam and rotated 360° about the glass-fiber axis. Detector positions were at two theta = 0, 20, and 35° using a frame resolution of 1,024 × 1,024 pixels, sample-to-detector distance of 120 mm, and each exposure was for 1,200 s. Data collection using the SMART collection software (Braintree, MA) and initial data processing using the Bruker GADDS software package (Bruker, Billerica, MA). Integration along the Debye rings was performed after data collection with a step (bin) size of 0.02, followed by construction of intensity vs. two-theta plots for each of the three detector positions, followed by a merger of the three plots based on the overlap of adjacent exposures to produce a traditional powder diffraction pattern. Additional pattern processing and phase identification using the Internal Center for Diffraction Data (ICDD, Newtown Square, PA, 2010) powder diffraction file was applied using the JADE software (Materials Data, Inc., Livermore, CA, 2009). Details of the Debye-Scherrer technique have been published in Klug and Alexander (
For FTIR analysis, mice molar enamel matrix samples were transferred to an ATR crystal (PIKE MIRacle single reflection diamond ATR) accessory placed in an FTIR (Bruker Vertex 80) sample compartment. Samples were pressed against the crystal surface with a pressure clamp to form a better contact covering most of the crystal surface. Sample absorbance spectra over the range 4,500–600 cm−1 were collected as an average of 2,048 scans (10 kHz scan speed with a DTGS detector) and processed with 3-term Blackman-Harris apodization and zero filling of 2. Background spectra, collected with same measurement parameters but without sample on the ATR crystal surface, were subtracted as a baseline correction.
Distal slope enamel matrices of 50 first mouse mandibular molars were prepared from 2, 4, and 6 dpn mice, and four sets of samples per time point were chosen. Enamel matrices from mice earlier than 2 dpn were not harvested due to a lack of overall quantity and enamel matrices from mice later than 6 dpn were omitted due to the advanced mineralization of those samples. Following cold acetone/trichloric acid precipitation, samples were redissolved in fifty microliters of 8 M urea. Reduction and alkylation of cysteines was accomplished by adding 1/10 volume of 45 mM DTT to the sample, followed by 45 min incubation at 37°C. After samples were cooled to room temperature, 1/10 volume of 100 mM iodoacetamide were added to the solution and samples were placed in dark at room temperature for 30 min. To equilibrate the sample for trypsin digestion, sufficient water was added to dilute the original 8 M urea/0.4 M ammonium bicarbonate solution 4-fold. A total of 1 μg trypsin was added and the sample was incubated at 37°C for 18–24 h. The trypsin digest was stopped by freezing until nanoLC-MS/MS analysis.
Nano-scale liquid chromatography was performed using a Dionex Ultimate 3000 system. Mobile phase A was water/acetonitrile (95:5) with 0.1% formic acid. Mobile phase B was water: acetonitrile (5:95) with 0.1% formic acid. Digested sample was loaded offline onto a Thermo Scientific C18 PepMap100 peptide trap (300 μm ID × 5 mm, 5 μm, 100 A) with 100% mobile phase A flowing at 50 μL per minute. After allowing the peptides to concentrate and desalt for 10 min., the trap was switched inline with an Agilent Zorbax 300SB C18 nanoLC column (3.5 μm, 150 mm × 75 μm ID). The peptides were then resolved using a linear gradient from 5% B to 35% B over 60 min. The flow rate through the column was 250 nL per minute.
The instrument used for mass spectrometry was an LTQ Orbitrap Velos Pro (Thermo Fisher) equipped with a Thermo LTQ nanospray source, which was operated at an ion spray voltage of 1.8 kV and a heated capillary temperature of 275°C. Full scan mass data were obtained between 400–1,800 Da and the Orbitrap resolution was 30,000. The Orbitrap was operated in data dependent acquisition mode with dynamic exclusion (120 s). Twenty most intense ions above the minimum signal threshold with charge states greater than or equal to 2 were selected for low-energy CID in the ion trap. Other operating parameters included a minimum signal threshold of 25,000 and an activation time of 30.0 ms.
Raw data files were processed using the Mass Matrix Conversion tool to generate Mascot generic files (MGFs) for the protein database search. Mascot 2.2 was used as a search engine and NCBI Mascot search results were imported into Scaffold.
Cheesy enamel matrix from 1 to 8 days postnatal mouse molars was scraped off and the proteins were homogenized and extracted using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Equal amounts of the extracted proteins were loaded and separated on a 10% SDS-PAGE gel. From the gel, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane in a semi-dry blotting apparatus at 18 V for 40 min. The membrane was blocked for 1 h with 5% milk powder after which it was incubated with anti-AMEL (1:200, custom made full-length), anti-AMBN (ab72776 1:200, Abcam), anti-ENAM (sc-33107, 1:100, Santa Cruz), anti-MMP20 (ab84737, 1:50, Abcam), and anti-carbonic anhydrase 2 (ab191343, 1:100, Abcam) primary antibodies for 1 h. Following primary antibody incubation, the membrane was washed three times with washing buffer (TBS-T) for 15 min each and then incubated with HRP conjugated secondary anti-chicken, anti-mouse, or anti-rabbit antibodies. To detect HRP, a chemiluminescent substrate (Thermo Scientific) was used. Positive bands were quantitatively assessed using densitometry analysis using the Image J software.
For X-ray diffraction and ATR-FTIR studies, enamel matrix from four different mice of the same position and developmental stage was pooled, and pooled enamel matrix was used for further analysis. Mass-spectroscopy data and proteomics analysis were based on separately collected biological quadruplicates. All other data (thickness measurements and Western blot analyses) were based on triplicates. Data analysis was performed using SPSS software. Statistical significance was assessed using the non-parametric Mann-Whitney
First mandibular molars of 1, 2, 3, 4, 5, 6, 7, and 8 day postnatal mice (Figures
Previous studies have indicated that the mineral phase of mouse molar enamel transitions from calcium carbonate, tri- and octacalcium phosphate precursors to partially fluoride substituted hydroxyapatite (Diekwisch et al.,
Analysis of the postnatal mouse molar enamel mineral layer.
The ATR spectra were measured for teeth obtained at four stages of development (2, 4, 6, and 8 days postnatal, labeled as 2, 4, 6, and 8 dpn) of which 2, 6, and 8 dpn are shown in Figure
Enamel organ proteomics analysis by nanoLC-MS/MS resulted in discrete peptide identification patterns distinguished between enamel organ/enamel matrix protein complex samples from postnatal days 2, 4, and 6. In these samples, individual proteins were identified using the Mascot search software and ranked based on quantitative Orbitrap counts. Mascot data analysis yielded five protein groups with high spectral counts (Figure
Comparison between spectral counts of individual proteins identified during postnatal mouse molar enamel development (days 2, 4, and 6 dpn)(99% probability, 2 peptide minimum). Proteins were grouped into four categories:
Among the biomineralization proteins, alkaline phosphatase 2 peaked on day 2 and significantly decreased by 0.9-fold (
Four cytoskeleton related proteins displayed an increase in expression levels between days 2 and 4 and then decreased on day 6. Tubulin β5, β-actin and vimentin were detected at high levels on day 4 with a significant 10-fold (
All of the proteins related to extracellular matrix/cell surface displayed an identical trend of high levels on day 2, followed by a steep decrease through day 6. Catenin α1, vinculin, integrin β1, laminin B1, cadherin 1, and cadherin 2 all exhibited a significant decrease from day 2 to day 4 by 0.9-fold (
High levels of stress proteins 60 kDa heat shock protein and 78 kDa glucose regulated protein were detected on day 2, and then gradually decreased through day 6 with a 0.8-fold (
As an alternative strategy and because of the hydrophilic bias of our proteomics technology, known enamel organ/enamel matrix protein complex samples were assayed using classic Western blot methodology (Figures
Changes in mouse molar enamel matrix protein levels from postnatal day 1–8.
Here we have used the developing mouse mandibular molar as a model system to track changes in protein and mineral composition during postnatal amelogenesis and to correlate findings from individual protein and mineral analyses to synthesize an integrated systems perspective of enamel formation in the mouse molar. 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. Moreover, the mouse mandibular molar has a long history as a model for morphogenesis, cytodifferentiation, and tissue specific biomineralization (Gaunt,
We began our study by documenting the daily incremental increase in the thickness of enamel covering the distal slopes of the molar cusps between 1 dpn until 8 dpn. During this time, the semi-transparent enamel layer increased in thickness from 1 μm covering to 75 μm. Throughout those 8 days, the matrix was pliable and ideally suited for further biochemical analysis as it allowed for harvesting of the matrix and the attached enamel organ in bulk and in daily increments. The pliable nature of the developing enamel matrix has impressed naturalists and early biochemists since John Hunter's time (Hunter,
X-ray diffraction analysis unambiguously identified enamel matrix diffraction patterns from day 4 to day 8 postnatal as partially fluoride substituted hydroxyapatite, and apatite electron diffraction patterns of 6 and 7 dpn mouse molar enamel were confirmed in the present study and in earlier electron diffraction studies (Diekwisch et al.,
Our ATR data were interpreted according to previously published band assignments (Vignoles-Montrejaud,
Our ATR data revealed dramatic changes in the phosphate region (800–1,100 cm−1) in the postnatal mouse molar develop enamel matrix. Specifically, our data demonstrated a transition from a contoured plateau (1,000–1,100 cm−1) indicative of amorphous calcium phosphate at 2 dpn to a single sharp and highly elevated peak (1,015 cm−1) with shoulders at 1,105 and 958 cm−1 at 8 dpn representative of high crystalline hydroxyapatite with crystals featuring long c-axis dimensions (Gadaleta et al.,
Moreover, there was strong evidence for carbonate substitution in the enamel matrix between postnatal days 6 and 8, as the peaks at 880 cm−1 (A-type carbonate) and 1,405 and 1,445 cm−1 (B-type carbonate) indicated. Carbonate is known to replace phosphate in biological apatites (Zapanta-Legeros,
Our proteomic analysis provided only low counts for classic enamel proteins, which are known to be of hydrophobic nature (Eastoe,
Disregarding the moderate counts for classic enamel proteins such as amelogenin, ameloblastin, and enamelin, our proteomics analysis detected a number of other proteins relevant for enamel mineralization during our 2–6 days postnatal mouse molar enamel matrix sampling window. Among these was the protein with the highest number of counts in our analysis, alkaline phosphatase, which peaked at day 2 during the early secretory stage. At the onset of amelogenesis, alkaline phosphatase may be involved in transporting phosphate from blood vessels near the stratum intermedium into the enamel organ by increasing local phosphate concentrations in the stratum intermedium via hydrolysis of phosphorylated substrates. Phosphatase mediated hydrolysis of pyrophosphate may also be involved in the generation of other phosphorylated macromolecules (Woltgens et al.,
Four of the six high-scoring cytoskeletal proteins in the enamel organ, including actin, tubulin, tropomyosin, and vimentin isoforms, peaked at postnatal day 4 during the late secretory stage. Increased presence of cytoskeletal proteins during the late secretory stage is likely indicative of their role in cell polarization and vesicular secretion (Manneville et al.,
Our proteomics data indicated that six high-scoring extracellular matrix/cell surface molecules identified in the present study all peaked on day 2 postnatal at the onset of the secretory stage. This group included the classic extracellular matrix molecule laminin, the integrin β1 cell surface receptor, the β-integrin binding extracellular matrix adhesion molecule vinculin, and three cell adhesion molecules of the catenin/cadherin complex. The presence of laminin as part of the ameloblast basal lamina at the dentin-enamel junction has been demonstrated to play a role in terminal odontoblast differentiation (Lesot et al.,
Three heat shock/stress proteins also were among the high-scoring proteins that peaked at the onset of the secretory stage. These proteins included the 60 kDa heat shock protein (Hspd1), the 78 kDa glucose regulated protein (Grp-78), and the chaperone stress 70 protein (Hsp70). All three of these proteins are involved in macromolecular assembly, the prevention of misfolding, as well as the prevention of aggregation. One of the major challenges of amelogenesis is the transport of amelogenin, a protein prone to self-assembly (Zhang et al.,
Three isomerases joined the list of high-scoring enamel proteins, including the protein disulfide isomerases A3 and A6, and the peptidyl-prolyl cis-trans isomerase. Disulfide isomerases (PDIs) catalyze protein folding by facilitating disulfide bond formation and arrangement (Kersteen and Raines,
In conclusion, this integrative proteomic/cell biological analysis of postnatal mouse molar enamel development identifies many of the substantial changes in the enamel matrix protein and mineral phase that take place during postnatal mouse molar amelogenesis, including (i) relatively high levels of matrix protein secretion 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 (Figure
All animal experiments were approved by the IACUC committees at the University of Illinois, Chicago and Texas A&M College of Dentistry.
MP, WZ, and TD wrote the manuscript. SG, TK, XL and TD designed the experiments. MP, HL, SD, LL,WZ, SP, and RD performed the experiments. MA designed and performed the statistical analysis.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Ms. C. Beck and Ms. K. Kuc for help with the X-ray analysis. Proteomics and informatics services were performed at the CBC-UIC Research Resources Center Mass Spectrometry, Metabolomics, and Proteomics Facility established in part by a grant from Searle Funds at the Chicago Community trust to the Chicago Biomedical Consortium.