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Amelotin Promotes Mineralization and Adhesion in Collagen-Based Systems

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Abstract

Introduction

Periodontitis is characterized by the destruction of tooth-supporting tissues including the alveolar bone. Barrier membranes are used in dentistry for tissue regenerative therapy. Nevertheless, conventional membranes have issues related to membrane stability and direct induction of bone mineralization. Amelotin (AMTN), an enamel matrix protein, regulates hydroxyapatite crystal nucleation and growth. To apply an AMTN membrane in clinical practice, we investigated the mineralizing and adhesive effects of recombinant human (rh) AMTN in vitro using a collagen-based system.

Methods

Collagen hydrogel incorporated with rhAMTN (AMTN gel) and rhAMTN-coated dentin slices were prepared. AMTN gel was then applied on a commercial membrane (AMTN membrane). Samples were incubated for up to 24 h in mineralization buffer, and the structures were observed. The peak adhesive tensile strength between the dentin and AMTN membrane was measured. Using an enzyme-linked immunosorbent assay, the release kinetics of rhAMTN from the membrane were investigated.

Results

The AMTN gel resulted in the formation of hydroxyapatite deposits both onto and within the collagen matrix. Furthermore, coating the dentin surface with rhAMTN promoted the precipitation of mineral deposits on the surface. Interestingly, site-specific mineralization was observed in the AMTN membrane. Only 1% of rhAMTN was released from the membrane. Hence, the AMTN membrane adhered to the dentin surface with more than twofold greater tensile strength than that detected for a rhAMTN-free barrier membrane.

Conclusions

RhAMTN can accelerate mineralization and adhesion in collagen-based systems. Furthermore, the AMTN membrane could inform the optimal design of calcified tissue regenerative materials.

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Abbreviations

AMTN:

Amelotin

BMP:

Bone morphogenetic protein

DMP1:

Dentin matrix protein 1

ELISA:

Enzyme-linked immunosorbent assay

GTR:

Guided tissue regeneration

HA:

Hydroxyapatite

JE:

Junctional epithelium

PBS:

Phosphate-buffered saline

PBST:

Phosphate-buffered saline containing 0.1% Tween® 20

Rh:

Recombinant human

SAED:

Selected area electron diffraction

SBF:

Modified simulated body fluid

SEM:

Scanning electron microscopy

SIBLING:

Small integrin-binding ligand N-linked glycoprotein

TEM:

Transmission electron microscopy

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Acknowledgments

This project was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC, Operating grant #490975) to BG, the Canadian Institutes of Health Research (CIHR, Operating Grant MOP-492418) to BG and JSPS KAKENHI Grant (Grant-in-Aid for Early-Career Scientists 18K17040) to YI. We would like to thank Douglas Holmyard (Advanced Bioimaging Centre, Mount Sinai Hospital, Toronto, Ontario, Canada) for his expert assistance with the TEM imaging. The authors also would like to thank Enago (www.enago.jp) for the English language review.

Author Contributions

YI, JH and BG designed the experiments. YI conducted the experiments. YI analyzed the data. YI, JH, EI and BG interpreted the data. YI, JH, EI and BG drafted the final version of the manuscript. YI takes responsibility for the integrity of the data analysis.

Conflict of interest

YI and BG declare a Canadian Patent (#CA2,968,134 C) and a US Patent (#10,596,301), which are relevant to this study. JH and EI state that they have no conflicts of interest.

Ethical Approval

No human studies nor animal studies were carried out by the authors for this article.

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Authors and Affiliations

  1. Faculty of Dentistry and Institute of Biomedical Engineering, University of Toronto, Toronto, Canada

    Yuichi Ikeda, James Holcroft, Eri Ikeda & Bernhard Ganss

  2. Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo, 113-8549, Japan

    Yuichi Ikeda & Eri Ikeda

  3. Molecular Immunology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan

    Eri Ikeda

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  1. Yuichi Ikeda
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  2. James Holcroft
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  4. Bernhard Ganss
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Correspondence to Yuichi Ikeda.

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Associate Editor Mohammad R. K. Mofrad oversaw the review of this article.

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Ikeda, Y., Holcroft, J., Ikeda, E. et al. Amelotin Promotes Mineralization and Adhesion in Collagen-Based Systems. Cel. Mol. Bioeng. 15, 245–254 (2022). https://doi.org/10.1007/s12195-022-00722-2

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