# Enamel matrix 3D organization and maturation stage ion flow

> **NIH NIH R01** · ADA FORSYTH INSTITUTE, INC. · 2020 · $497,500

## Abstract

The goal of this project is to advance our understanding of enamel formation by overcoming an apparent
impasse in the field. Although the need for multi-protein studies is clearly recognized, in vitro studies have heavily
focused on amelogenin as the key enamel matrix protein. However, the two other matrix proteins ameloblastin
and enamelin occur in much lower abundance in the matrix and have extensive posttranslational modifications.
Their low abundance precludes purification from animal teeth and attempts to express these proteins in
recombinant form have failed because they are not properly post-translationally modified. This has resulted in a
limited view of enamel formation because in vitro studies have difficulty addressing the role ameloblastin and
enamelin play in enamel formation. Mouse models have shown that both ameloblastin and enamelin are critical
for enamel formation and that no enamel forms if either protein is absent. Therefore, a critical need exists to
better understand ameloblastin and enamelin function in enamel development. We address this problem by
proposing three specific aims and working hypotheses. 1) Enamelin and/or ameloblastin facilitate the conversion
of amorphous calcium phosphate (ACP) to hydroxyapatite (HAP) and their cleavage products become part of a
support structure for growing crystallites. 2) In the absence of amelogenin, the enamelin and/or ameloblastin
proteins still regulate the mineral phase. 3) The inability to remove matrix proteins from Klk4 knockout enamel
prevents proper ion movement that then compromises enamel maturation. We will test these hypotheses by
characterizing the matrix architecture in situ relative to mineral phase at the mineralization front in wild type and
amelogenin ablated mice. Likewise, we will analyze pH, expression levels of ion exchangers and their protein
levels, and compare the enamel matrix composition between Klk4 KO mouse versus wild type using mass
spectrometry LC-MS/MS. Taking advantage of recent advances in microscopy techniques, we can now perform
in situ fluorescence microscopy of specifically labeled enamel matrix proteins correlated with scanning electron
microscopy in undermineralized samples. This allows visualization of both protein and crystallites and to extend
the scale of analytical resolution from micrometers to nanometers for the study of protein and mineral phase.
Importantly, we will be able to address questions of protein distribution around crystallites within prisms and
identify mineral phase by further extending the analytic resolution to an atomic scale through in situ atom probe
analyses. To examine and visualize the three dimensional organization of matrix and developing crystallites in
situ with unprecedented depth of field, we will perform high resolution helium ion microscopy. The realization of
this project will substantially advance the field of enamel research by integrating the distribution of all three
structural matrix proteins relative ...

## Key facts

- **NIH application ID:** 9965886
- **Project number:** 5R01DE025865-05
- **Recipient organization:** ADA FORSYTH INSTITUTE, INC.
- **Principal Investigator:** Felicitas B Bidlack
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $497,500
- **Award type:** 5
- **Project period:** 2016-07-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9965886

## Citation

> US National Institutes of Health, RePORTER application 9965886, Enamel matrix 3D organization and maturation stage ion flow (5R01DE025865-05). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/9965886. Licensed CC0.

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