Scientists have shown how a tiny flaw in a protein results in damaged enamel that is prone to decay

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Scientists have shown how a tiny flaw in a protein results in damaged enamel that is prone to decay, according to a new study published in the Proceedings of the National Academy of Sciences.

People with a condition known as Amelogenesis imperfecta (Al) don’t develop enamel correctly because of a single amino acid defect in the critical enamel protein called amelogenin.

Scientists from Pacific Northwest National Laboratory (PNNL) report that defective amelogenin proteins stick or bind abnormally tightly to the building enamel, failing to clear out when they should, thus hindering the careful growth process through which strong enamel is built.

“The teeth aren’t as strong because the enamel is much thinner and the crystals less ordered,” said Jinhui Tao, the first author of the paper.

“In most people, the enamel is the hardest substance in their body, but that’s not true for patients with AI.”

The genetic defect results in enamel that is discolored, soft, and easily broken.

The defective enamel makes patients more susceptible to tooth decay and gum disease.

To understand what’s happening, Tao and colleagues took a close look at a process known as protein binding – how strongly proteins stick to other substances and to each other, in real time.

The process is crucial for cell signaling and for our health, and binding errors are behind many diseases.

The team combined atomic force microscopy with solid-state nuclear magnetic resonance spectroscopy available through EMSL, the Environmental Molecular Sciences Laboratory, as well as other methods to study mineralization and other processes involving the proteins that form enamel.

Abnormal binding of proteins impedes creation of crystalline enamel structure, which can lead to bad teeth
Tooth enamel X-ray. Credit: Environmental Molecular Sciences Laboratory

They found the defective proteins’ propensity to literally stick too long and too strong to the surface thwarts other molecular players from doing their jobs in creating a solid crystalline structure.

They slow down an enzyme known as MMP20, which removes excess amelogenin from the developing mineral surface. When MMP20 can’t do its job, enamel grows more slowly and is weaker.

The sticky proteins also slow down the formation of hydroxyapatite, the crystalline building block of enamel.

It’s a little bit like bricklaying, only in developing teeth, many molecules work together to do a job similar to that of a bricklayer.

How the mortar sits between bricks is crucial for creating a solid, regular –crystalline – structure.

If mortar is applied inconsistently or sloppily, and if too much mortar remains and hardens into clumps, the bricks don’t fit together tightly, gaps result, and the entire structure is weak and porous and doesn’t grow as thick or as ordered as necessary.

Instead of an impenetrable wall made of mineral crystals, pits and gaps form in the enamel of AI patients, allowing penetration by acids and bacteria that can cause tooth pain and promote decay.

The smooth, solid tooth enamel that most people are born with belies the incredible molecular complexity that makes it possible.

Proteins are constantly interacting with the apatite mineral surface.

This new research shows that how strongly the proteins bind to the mineral structure as well as to each other is a key factor in determining how our teeth develop.

Exploring how proteins use binding energy as currency to accomplish their tasks is relevant to many other areas of science as well.

The current work focuses on naturally occurring genetic disorders, but the understanding that the team gained of how proteins bind to and interplay in a complex environment is something that is relevant to a wide range of material sciences research.

“This works helps us understand why people with these mutations have weak and fragile tooth enamel, but more broadly, it gives us important information about how to control the creation or manipulation of materials for many applications, such as the development of new organic-inorganic hybrid materials for high-performance computing, catalysis research, or energy storage,” Tao said.


Dentin is primarily composed of hydroxyapatite crystals within a rich organic matrix. The organic matrix comprises collagenous structural components, within which a variety of bioactive molecules are sequestered.

During caries progression, dentin is degraded by acids and enzymes derived from various sources, which can release bioactive molecules with potential reparative activity towards the dentin-pulp complex.

While these molecules’ repair activities in other tissues are already known, their biological effects are unclear in relation to degradation events during disease in the dentin-pulp complex.

This study was undertaken to investigate the effects of dentin matrix components (DMCs) that are partially digested by matrix metalloproteinases (MMPs) in vitro and in vivo during wound healing of the dentin-pulp complex. DMCs were initially isolated from healthy dentin and treated with recombinant MMPs.

Subsequently, their effects on the behaviour of primary pulp cells were investigated in vitro and in vivo.

Digested DMCs modulated a range of pulp cell functions in vitro. In addition, DMCs partially digested with MMP-20 stimulated tertiary dentin formation in vivo, which exhibited a more regular tubular structure than that induced by treatment with other MMPs.

Our results indicate that MMP-20 may be especially effective in stimulating wound healing of the dentin-pulp complex.

Discussion

Traditional direct pulp capping materials, such as Ca (OH)2 or MTA, have a 60%–80% success rate, as reported in clinical studies4,23,24.

However, these materials were developed on a more empirical basis, rather than through specific understanding of the wound-healing mechanisms of the dentin-pulp complex.

Recent studies have aimed to introduce bio-mimicked tissue healing with growth factors as a direct pulp-capping material2527.

However, limited effects have been observed regarding the healing of wounded pulp tissues as the wound healing mechanisms of the dentin-pulp complex remain not entirely clear.

MMPs are calcium- and zinc-dependent, host-derived enzymes28 that can hydrolyse ECM components29. ECM integrity is affected by MMPs and TIMPs, which play critical roles during matrix remodelling911.

Several MMPs have been reported to be sequestered in mineralized dentin30, and recent studies revealed that host-derived MMPs contribute to the breakdown of collagenous matrices during dental caries30.

Conversely, MMPs have been reported to be involved in wound healing processes, such as cell growth, cell migration, tissue remodelling and angiogenesis31,32. We also previously reported upregulated MMP gene expression after pulp injury8 and MMPs have been detected in the dentin matrix in an inactive state33,34.

The distribution of MMP molecules has been reported at deeper sites within the dentin, where they exhibit gradients of activity3537.

The presence of these molecules within the dentin-pulp complex enable them to activate DMCs released by bacterial acids or restorative materials.

These activated molecules may subsequently act in biological defence and repair processes within the tooth. Therefore, in the present study, we focused on MMPs that might facilitate matrix remodelling of dental pulp tissues or digested DMCs which promote wound healing21,37.

Dentin is a major component of the tooth and covers the dental pulp. It is considered to be part of the ECM of the combined dentin-pulp complex, rather than being a distinct tissue, as it originates from the same mesenchymal tissue38,39.

However, the complete range of functions of dentin is still under investigation. During caries progression, dentin is demineralized by acids secreted by caries-associated bacteria, and DMCs may be digested enzymatically during this process40.

Therefore, we investigated the effects of digested DMCs on wound healing of the dentin-pulp complex both in vitro and in vivo.

Protein analysis by SDS-PAGE showed DMCs were digested with MMPs-1, -2, -3, -8, -9, -13, and -20. The resultant protein profiles differed among MMPs (Fig. 1), which was as expected because of their differing substrate specificities.

Generally, MMP-1 has been considered as a collagenase and MMP-2 is reported as a gelatinase. However, MMP-1 is also able to degrade proteoglycans41 and MMP-2 reportedly can degrade and activate the DMP-1 protein, which is a key dentin matrix signalling protein42.

In addition, non-canonical substrates have been identified as targets of these MMPs41.

Similarly, MMP20 has been recognized as enamelysin and cleaves enamel-related proteins; however, further substrates could exist.

Recently, MMP20 was reported to associate with the activation of DSPP, which is a member of the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family43.

Several previous reports have demonstrated that various proteins from the SIBLING family, along with several growth factors, are present in DMCs4447.

Therefore, DMCs may contain SIBLING substrates for MMPs.

Subsequently, we performed SDS-PAGE analysis and confirmed that DMCs extracted with EDTA could react and respond to the MMP molecules.

These results suggested that MMPs may hydrolyse or facilitate dentin decomposition, consistent with the findings from these previous studies48.

Next, we examined the effects of DMCs digested with MMPs on the functions of primary pulp cells in vitro. DMCs digested with some MMPs promoted angiogenesis, migration, proliferation, migration, osteogenic differentiation and hard tissue formation.

However, MMP molecules that promote wound healing in dental pulp cells may operate differently in vivo and the Cellular responses may be due to the target cells expressing different substrate binding sites for the MMP-digested DMCs.

These in vivo outcomes may indicate variations in functionality of the digested products from the same substrates according to the enzymes used to digest them; and this variation also agrees with previous reports49.

Indeed, whilst we observed that DMCs digested with MMPs up-regulated some pulp cell responses in vitro (as described above) which associate with the wound healing response in the pulp tissue, it was however important to directly investigate whether MMP-digested DMCs invoked tissue healing responses in vivo, by using a direct pulp capping approach.

Such an approach enables the evaluation of a more complete and simulated form of dental tissue repair and may also identify translational opportunities.

Thus, we investigated the effects of digested DMCs on wound healing in pulp tissue by using direct pulp capping in vivo.

The result of micro-CT analysis of hard tissue formation indicated that DMCs digested with MMP-20 induced highly condensed tertiary dentin beneath the pulp-capping materials applied (Fig. 7H,I) and histological images from the same specimen showed appropriate tubular dentin morphology (Fig. 8I).

In contrast, DMCs digested with other MMPs induced tertiary dentin with more defects and void structures (Fig. 7A,E,F; Tables 1 and ​and2).2). These results suggest that MMP20 may play an essential role in the pulpal healing process, compared with other MMPs in vivo.

Given that wound healing in vivo is affected by a variety of complex and interacting factors, the results of in vitro experiments might not always mirror the in vivo processes.

Furthermore, the source of MMPs in vivo is unclear and could be derived from variety of sources and locations, such as resident cells within the pulp and/or the inflammatory infiltrate.

A further potential explanation for the differences observed in vivo and in vitro may be due to the presence of other environmental factors, as it has been shown that ECM digested with several enzymes stimulated different actions, according to the environment studied50.

Most reports concerning MMP-20 have found that this enzyme appears to be predominantly expressed within the tooth51,52.

Indeed, during tooth development, MMP-20 may contribute to enamel formation by digesting enamel-related proteins53.

Additionally, MMP-20 is present in the dentin matrix and is expressed by odontoblasts in mature teeth and activated by dental caries progression17.

Others have reported that MMP-20 is present in carious dentin compared with sound dentin33.

More specifically these reports relate to the localization of MMP20 in the dentin-pulp complex; however, there appear to be no reports of its biological role in this location.

Data from the present study indicate that MMP-20 might be involved in a specific pulpal repair system enabling tertiary dentin formation.

For in vitro and in vivo experiments, MMP molecules alone were also used to investigate their direct effects; however, no direct effects on pulp cell function or pulpal tissue repair were observed.

This outcome is likely due to the MMP molecule exerting no direct effect on these responses, or the inability to generate sufficient substrates in this environment due to the target molecules being embedded within the mineralized ECM.

In addition, the most effective ratio of MMPs and DMCs is unknown.

The concentration of MMPs in this study may not reflect the true biological environment as is present in dental caries.

However, this study does indicate a potential role for MMP molecules in modifying or improving the local healing response of the dentin-pulp complex, via its action on DMCs. This model may therefore provide novel insight enabling the future elucidation of the mechanism involved in pulpal healing.

The different MMPs may also function at different doses; therefore, future studies are needed to determine optimal doses required. As MMPs have been reported to directly upregulate dental pulp through protease-activated receptors54,55, further investigation is also necessary to address this.

In addition, other enzymes (e.g., serine or cysteine proteinases) are present in the dentin matrix56 and these proteinases may further degrade DMCs57.

Thus, the effects of other endogenous enzymes during repair of the dentin-pulp complex should be examined.

Based on our findings, host-derived MMP may be involved in the wound healing process of the dentin-pulp complex (Fig. 9).

During caries progression, initially enamel and then dentin is decalcified by bacterial acids and the organic dentin matrix is exposed (Fig. 9A).

Subsequently, the dentin matrix is digested with MMPs, which may be present endogenously (Fig. 9B), and dental pulp cells are activated by these MMP-digested DMCs (Fig. 9C).

DMCs digested with MMP-1, -9, -13, and -20 facilitated wound healing of the dentin-pulp complex in our system (Fig. 9D).

To our knowledge, this study is the first to examine the wound healing potential of the dentin-pulp complex by using DMCs digested with MMP-1, -2, -3, -8, -9, -13, and -20; the data identify a significant role for MMP-20 in mature teeth. Further studies are necessary to analyse the components generated by digested DMCs and identify which molecules are critical for wound healing of this tissue.

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Figure 9
Proposed model for wound healing in pulp tissue following dental caries. Dentin decalcification initially releases matrix metalloproteinases (MMPs) from the dentin matrix during caries progression (A). The dentin matrix is then digested with MMPs (B). Dental pulp cells are subsequently activated by digested dentin matrix components (DMCs) (C). DMCs digested by MMPs, particularly MMP-20, stimulate wound healing in the dentin-pulp complex (D).

More information: Jinhui Tao et al. The energetic basis for hydroxyapatite mineralization by amelogenin variants provides insights into the origin of amelogenesis imperfecta, Proceedings of the National Academy of Sciences (2019). DOI: 10.1073/pnas.1815654116

Journal information: Proceedings of the National Academy of Sciences
Provided by Environmental Molecular Sciences Laboratory

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