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. 2012 Jun;4(2):69-77.
doi: 10.1038/ijos.2012.40. Epub 2012 Jun 29.

Cementomimetics-constructing a Cementum-Like Biomineralized Microlayer via Amelogenin-Derived Peptides

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Free PMC article

Cementomimetics-constructing a Cementum-Like Biomineralized Microlayer via Amelogenin-Derived Peptides

Mustafa Gungormus et al. Int J Oral Sci. .
Free PMC article

Abstract

Cementum is the outer-, mineralized-tissue covering the tooth root and an essential part of the system of periodontal tissue that anchors the tooth to the bone. Periodontal disease results from the destructive behavior of the host elicited by an infectious biofilm adhering to the tooth root and left untreated, may lead to tooth loss. We describe a novel protocol for identifying peptide sequences from native proteins with the potential to repair damaged dental tissues by controlling hydroxyapatite biomineralization. Using amelogenin as a case study and a bioinformatics scoring matrix, we identified regions within amelogenin that are shared with a set of hydroxyapatite-binding peptides (HABPs) previously selected by phage display. One 22-amino acid long peptide regions referred to as amelogenin-derived peptide 5 (ADP5) was shown to facilitate cell-free formation of a cementum-like hydroxyapatite mineral layer on demineralized human root dentin that, in turn, supported attachment of periodontal ligament cells in vitro. Our findings have several implications in peptide-assisted mineral formation that mimic biomineralization. By further elaborating the mechanism for protein control over the biomineral formed, we afford new insights into the evolution of protein-mineral interactions. By exploiting small peptide domains of native proteins, our understanding of structure-function relationships of biomineralizing proteins can be extended and these peptides can be utilized to engineer mineral formation. Finally, the cementomimetic layer formed by ADP5 has the potential clinical application to repair diseased root surfaces so as to promote the regeneration of periodontal tissues and thereby reduce the morbidity associated with tooth loss.

Figures

Figure 1
Figure 1
Identification of ADPs. (a) Flowchart showing the design steps for identifying the ADPs. (b) High- and low-similarity amino-acid domains among the rM180 and two experimentally selected HABP sets. Each bar represents one amino acid and the amino-acid domains above the baseline represent the high similarity, while those below represent low similarity regions. The overlapped plot shows the potential calcium ion-binding domains (red arrows). Note that the majority of the highest potential domains coincide with the high-similarity regions (arrow heads). (c) Computationally determined molecular structure for rM180 amelogenin showing position of (folded) ADP7 (red) within rM180 (see Supplementary Information for further details). (d) Positions of the ion-binding domains (blue circles) on rM180. (e) The locations of the ADPs along rM180 (blue) with red colored segments represent the high-similarity regions and green colored segments represent the low-similarity regions. ADP, amelogenin-derived peptide; HABP, hydroxyapatite-binding peptide; rM180, recombinant mouse 180 amino acid long amelogenin.
Figure 2
Figure 2
Procedure for in vitro, cell-free synthesis of cementomimetic layer by ADP5 on human root surfaces. Extracted human teeth are cleaned of any contaminating material and cylindrical pieces are cut right below the cement–enamel junction. An aqueous solution of ADP5, the mineralization directing peptide, is applied on the demineralized root surface. The specimen is immersed into a mineralization solution containing calcium and phosphate ions. Cell adhesion and proliferation is investigated on the re-mineralized root surfaces. ADP, amelogenin-derived peptide.
Figure 3
Figure 3
Binding, mineralization, and structural characterization of mineral products of the ADPs. (a) Binding constants, KD, of the ADPs determined by QCM (see Supplementary Information). (b) Calcium consumption rates of mineralization in the presence of ADP5 and ADP7 and rM180. (c) Calcium phosphate minerals formed in solution in the presence of ADPs. (d) The mineral product of ADP7 resembles those formed in the presence of rM180 in solution and these acicular crystallites are consistent with HAp morphology. (e) XRD patterns of the minerals formed by ADPs. Materials formed by ADP7 and amelogenin display the characteristic peaks belonging to the HAp crystal structure, while all minerals formed by other ADPs display weak diffraction peaks, consistent with amorphous, or only loosely crystalline outcomes shown in (c). ADP, amelogenin-derived peptide; HABP, hydroxyapatite-binding peptide; HAp, hydroxyapatite; rM180, recombinant mouse 180 amino acid long amelogenin; XRD, X-ray diffraction.
Figure 4
Figure 4
Structural and functional characteristics of the cementomimetic layer formed on the root of human tooth by ADP5. (a and b) SEM images of the demineralized (a) and (b) ADP5-formed cementomimetic layer revealing uniform nanocrystals with a Ca/P ratio of 1.67 obtained from EDX () spectra (insets). (c) TEM images and the electron diffraction pattern of the newly formed cementomimetic mineral layer in cross-section showing HAp crystallites. (d) SEM image of mechanically separated cementomimetic mineral layer displaying uniform thickness of crystallized HAp. (e) Attachment of hPDL cells on control and cementomimetic mineral layer. (f) Proliferation of the hPDL cells on control, uncoated, root stock compared to ADP-induced cementomimetic mineral layer. (g and h) Fluorescent microscopy image showing F-actin. Cell attachment without formation of organized actin network on control surface (g) is compared to those on ADP-induced cementomimetic mineral layer that reveals a well-organized actin cytoskeleton and lamellapodia (h). EDX, energy dispersive X-ray; HAp, hydroxyapatite; hPDL, human periodontal ligament; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

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