Dental enamel forms through a protein controlled mineralization and degradation process with a nanoscale precision that new technologies and human engineering may be able to mimic. The unique enamel microstructure is a result of protein-guided growth of highly anisotropic apatite crystals in a three-dimensional organic framework generated by the self-assembly of amelogenin proteins that hydrolyze in coordination with an advancing mineralization to transform into a tissue almost entirely comprised of mineral. The overall objective of the proposed research is to generate nanofibrous apatite similar to crystals in enamel through the design of a recombinant protein matrix framework that guides crystal growth and is susceptible to enzymatic digestion. Cloning of proteins and proteases of the extracellular matrix of enamel provides the opportunity to generate an artificial environment that can mimic the biochemistry of the forming enamel. New titration electronics allow the accurate addition of nanoliters of mineralizing solutions to maintain an ionic microenvironment at constant levels over long periods of time similar to the in-vivo process. Thus, we propose that two major activities of ameloblast cells, e.g. the expression and provision of matrix proteins and proteases and the precise control over ionic concentration can be achieved through human engineering, providing us with the ability to mimic enamel formation. The hypothesis is that fibrous apatite nanocrystals can be generated by the coordination of the self-assembly of a recombinant protein matrix and its enzymatic degradation with the growth of hydroxyapatite crystals from a saturated solution of constant composition. This hypothesis will be tested by the following specific aims: (1) To determine the physicalchemical and biochemical parameters that enable self-assembly of amelogenin proteins into a supramolecular framework;(2) To induce apatite crystallization on nucleating surfaces in synchronization with amelogenin supramolecular self-assembly and (3) To mimic enamel maturation by gradual degradation of the protein scaffold by MMP-20 and/or serine proteases while mineralization proceeds. This project will result in an improved understanding of biomineralization in dental enamel including the functions of some of the enamel matrix proteins and proteases at the molecular level. The methodology and knowledge gained will provide the basis for similar biomimetic approaches to understand mineralization in dentin, bone, shells and other calcified tissues. Importantly, comprehending nanoscale control over this complex process will provide a basis for development of novel methods for mineralized tissue repair.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE017529-04
Application #
7803703
Study Section
Musculoskeletal Tissue Engineering Study Section (MTE)
Program Officer
Drummond, James
Project Start
2007-07-06
Project End
2012-04-30
Budget Start
2010-05-01
Budget End
2012-04-30
Support Year
4
Fiscal Year
2010
Total Cost
$378,180
Indirect Cost
Name
University of California San Francisco
Department
Dentistry
Type
Schools of Dentistry
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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Martinez-Avila, Olga M; Wu, Shenping; Cheng, Yifan et al. (2011) Self-assembly of amelogenin proteins at the water-oil interface. Eur J Oral Sci 119 Suppl 1:75-82
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Uskokovi?, Vuk; Li, Wu; Habelitz, Stefan (2011) Biomimetic Precipitation of Uniaxially Grown Calcium Phosphate Crystals from Full-Length Human Amelogenin Sols. J Bionic Eng 8:114-121
Uskokovic, Vuk; Odsinada, Roselyn; Djordjevic, Sonia et al. (2011) Dynamic light scattering and zeta potential of colloidal mixtures of amelogenin and hydroxyapatite in calcium and phosphate rich ionic milieus. Arch Oral Biol 56:521-32
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Zhu, Li; Uskokovic, Vuk; Le, Thuan et al. (2011) Altered self-assembly and apatite binding of amelogenin induced by N-terminal proline mutation. Arch Oral Biol 56:331-6
Bechtle, Sabine; Habelitz, Stefan; Klocke, Arndt et al. (2010) The fracture behaviour of dental enamel. Biomaterials 31:375-84
Uskokovic, V; Castiglione, Z; Cubas, P et al. (2010) Zeta-potential and particle size analysis of human amelogenins. J Dent Res 89:149-53

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