Publications

  • Qichao Ruan, Janet Moradian-Oldak
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    ABSTRACT: Mature tooth enamel is acellular and does not regenerate itself. Developing technologies that rebuild tooth enamel and preserve tooth structure is therefore of great interest. Considering the importance of amelogenin protein in dental enamel formation, its ability to control apatite mineralization in vitro, and its potential to be applied in fabrication of future bio-inspired dental material this review focuses on two major subjects: amelogenin and enamel biomimetics. We review the most recent findings on amelogenin secondary and tertiary structural properties with a focus on its interactions with different targets including other enamel proteins, apatite mineral, and phospholipids. Following a brief overview of enamel hierarchical structure and its mechanical properties we will present the state-of-the-art strategies in the biomimetic reconstruction of human enamel.
    03/2015; DOI:10.1039/C5TB00163C
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    ABSTRACT: The host immune system is known to influence mesenchymal stem cell (MSC)-mediated bone tissue regeneration. However, the therapeutic capacity of hydrogel biomaterial to modulate the interplay between MSCs and T-lymphocytes is unknown. Here it is shown that encapsulating hydrogel affects this interplay when used to encapsulate MSCs for implantation by hindering the penetration of pro-inflammatory cells and/or cytokines, leading to improved viability of the encapsulated MSCs. This combats the effects of the host pro-inflammatory T-lymphocyte-induced nuclear factor kappaB pathway, which can reduce MSC viability through the CASPASE-3 and CASPASE-8 associated proapoptotic cascade, resulting in the apoptosis of MSCs. To corroborate rescue of engrafted MSCs from the insult of the host immune system, the incorporation of the anti-inflammatory drug indomethacin into the encapsulating alginate hydrogel further regulates the local microenvironment and prevents pro-inflammatory cytokine-induced apoptosis. These findings suggest that the encapsulating hydrogel can regulate the MSC-host immune cell interplay and direct the fate of the implanted MSCs, leading to enhanced tissue regeneration.
    Advanced Functional Materials 03/2015; DOI:10.1002/adfm.201500055 · 10.44 Impact Factor
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    ABSTRACT: Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, towards the goal of understanding the mechanisms of amelogenin-cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD) and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (~334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. Though in DLS studies we cannot exclude the possibility of fusion of liposomes as the result of amelogenin addition, NMR and CD studies revealed a disorder-order transition of rP172 in a model membrane environment. Strong FRET from Trp in rP172 to DNS-bound-phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities.
    Biopolymers 02/2015; 103(2). DOI:10.1002/bip.22573 · 2.29 Impact Factor
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    ABSTRACT: Abstract We recently reported an amelogenin-chitosan (CS-AMEL) hydrogel as a promising biomimetic material for future in situ human enamel regrowth. To further optimize the necessary conditions for clinical applicability of CS-AMEL hydrogel, herein we studied the effects of viscosity and supersaturation degree on the size and orientation of synthetic crystals by means of scanning electron microscopy (SEM) and X-ray diffraction (XRD). Raising the hydrogel viscosity by increasing chitosan concentration from 1% to 2% (w/v) improved the orientation of the crystals, while a higher supersaturation (σ(HAp) >10.06, [Ca(2+)] >5 mM) resulted in the formation of random crystals with larger sizes and irregular structures. We conclude that optimal conditions to produce organized enamel-like crystals in a CS-AMEL hydrogel are: 2% (w/v) chitosan, 2.5 mM calcium, and 1.5 mM phosphate (degree of supersaturation = 8.23) and 200 µg/ml of amelogenin.
    Connective Tissue Research 08/2014; 55(S1):150-154. DOI:10.3109/03008207.2014.923856 · 1.98 Impact Factor
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    ABSTRACT: Epithelially-derived ameloblasts secrete extracellular matrix proteins including amelogenin, enamelin, and ameloblastin. Complex intermolecular interactions among these proteins are believed to be important in controlling enamel formation. Here we provide in vitro and in vivo evidence of co-assembly and co-localization of ameloblastin with amelogenin using both biophysical and immunohistochemical methods. We performed co-localization studies using immunofluorescence confocal microscopy with paraffin-embedded tissue sections from mandibular molars of mice at 1, 5, and 8 days of age. Commercially-available ameloblastin antibody (M300) against mouse ameloblastin residues 107-407 and an antibody against full-length recombinant mouse (rM179) amelogenin were used. Ameloblastin-M300 clearly reacted along the secretory face of ameloblasts from days 1-8. Quantitative co-localization was analyzed (QCA) in several configurations by choosing appropriate regions of interest (ROIs). Analysis of ROIs along the secretory face of ameloblasts revealed that at day 1, very high percentages of both the ameloblastin and amelogenin co-localized. At day 8 along the ameloblast cells the percentage of co-localization remained high for the ameloblastin whereas co-localization percentage was reduced for amelogenin. Analysis of the entire thickness on day 8 revealed no significant co-localization of amelogenin and ameloblastin. With the progress of amelogenesis and ameloblastin degradation, there was a segregation of ameloblastin and co-localization with the C-terminal region decreased. CD spectra indicated that structural changes in ameloblastin occurred upon addition of amelogenin. Our data suggest that amelogenin-ameloblastin complexes may be the functional entities at the early stage of enamel mineralization.
    Frontiers in Physiology 07/2014; 5:274. DOI:10.3389/fphys.2014.00274
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    ABSTRACT: Objectives We recently demonstrated a significant correlation between enamel delamination and tooth-level radiation dose in oral cancer patients. Since radiation can induce the synthesis and activation of matrix metalloproteinases, we hypothesized that irradiated teeth may contain active matrix metalloproteinases. Materials and Methods: Extracted teeth from oral cancer patients treated with radiotherapy and from healthy subjects were compared. Extracted mature third molars from healthy subjects were irradiated in vitro and/or incubated for 0 to 6 months at 37 °C. All teeth were then pulverized, extracted, and extracts subjected to proteomic and enzymatic analyses. Results: Screening of irradiated crown extracts using mass spectrometry identified MMP-20 (enamelysin) which is expressed developmentally in dentin and enamel but believed to be removed prior to tooth eruption. MMP-20 was composed of catalytically active forms at Mr = 43, 41, 24 and 22 kDa and was immunolocalized predominantly to the morphological dentin enamel junction. The proportion of different sized MMP-20 forms changed with incubation and irradiation. While the pattern was not altered directly by irradiation of healthy teeth with 70 G, subsequent incubation at 37 °C for 3-6 months with or without prior irradiation caused the proportion of Mr = 24-22 kDa MMP-20 bands to increase dramatically. Extracts of teeth from oral cancer patients who received >70 Gy radiation also contained relatively more 24 and 22 kDa MMP-20 than those of healthy age-related teeth. Conclusion: MMP-20 is a radiation-resistant component of mature tooth crowns enriched in the dentin-enamel. We speculate that MMP-20 catalyzed degradation of organic matrix at this site could lead to enamel delamination associated with oral cancer radiotherapy.
    Journal of dentistry 05/2014; DOI:10.1016/j.jdent.2014.02.013 · 3.20 Impact Factor
  • P. MAZUMDER, V. GALLON, L. CHEN, S. PRAJAPATI, J. MORADIAN-OLDAK
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    ABSTRACT: We recently reported interactions between amelogenin and enamelin using both in vitro and in vivo strategies [J Struct Biol. 183, 239-49]. Objectives: To quantitatively analyze co-localization of ameloblastin and amelogenin providing evidence for their interactions during enamel formation using immunofluorescence confocal microscopy. Methods: Fluorescent-IHC staining has been performed on paraffin-embedded tissue sections from 1 to 8 days mouse mandibular molars. Commercially available antipeptide antibodies against N-terminal (N18) and C-terminal (M300) of ameloblastin, and an antibody against the full-length recombinant mouse (rM179) amelogenin were used. Co-localization patterns were analyzed using two different regions of interests (ROIs): a large (40 µm height) and a small (7 µm diameter). Results: Ameloblastin-M300 clearly reacted along the secretary face of ameloblasts from day 2-8, while significant reaction against ameloblastin-N18 was observed throughout the entire thickness of enamel from day 5-8. Co-localization analysis by small ROIs along the secretary face of ameloblasts reveled that at day 2, equal amounts (~80%) of both the ameloblastin C-terminal and amelogenin co-localized. Co-localization decreased at day 8 when only ~66% of amelogenin and ~57% of ameloblastin-C-terminal co-localized. Analysis of the entire thickness from day 1 to 5 using the N-terminal antibody revealed lower and different values of co-localization for amelogenin and ameloblastin (48% and 30% respectively) while on day 8 co-localization was low and the same for both (~40%). Conclusions: Amelogenin and ameloblastin co-localize at the secretary face of ameloblasts at the early stage of amelogenesis, suggesting that they are secreted simultaneously. With the progress of amelogenesis and ameloblastin degradation co-localization pattern changes as following: 1) there is a segregation of ameloblastin C- and N-terminal, 2) co-localization with C-terminal decreases while co-localization with the N-terminal is low and does not change. Our data suggest that amelogenin-ameloblastin complexes may be the functional entities at the early stage of enamel mineralization.
    AADR Annual Meeting & Exhibition 2014; 03/2014
  • Qichao Ruan, Janet Moradian-Oldak
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    ABSTRACT: Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.
    Journal of Visualized Experiments 01/2014; DOI:10.3791/51606
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    ABSTRACT: Amelogenin, the major extracellular matrix protein of developing tooth enamel is intrinsically disordered. Through its interaction with other proteins and mineral, amelogenin assists enamel biomineralization by controlling the formation of highly organized enamel crystal arrays. We used circular dichroism (CD), dynamic light scattering (DLS), fluorescence and NMR spectroscopy to investigate the folding propensity of recombinant porcine amelogenin rP172 following its interaction with SDS, at levels above critical micelle concentration. The rP172-SDS complex formation was confirmed by DLS, while an increase in the structure moiety of rP172 was noted through CD and fluorescence experiments. Fluorescence quenching analyses performed on several rP172 mutants where all but one Trp was replaced by Tyr at different sequence regions confirmed that the interaction of amelogenin with SDS micelles occurs via the N-terminal region close to Trp25 where helical segments can be detected by NMR. NMR spectroscopy and structural refinement calculations using CS-Rosetta modelling confirm that the highly conserved N-terminal domain is prone to form helical structure when bound to SDS micelles. Our findings reported here reveal interactions leading to significant changes in the secondary structure of rP172 upon treatment with SDS. These interactions may reflect the physiological relevance of the flexible nature of amelogenin and its sequence specific helical propensity that might enable it to structurally adapt with charged and potential targets such as cell surface, mineral, and other proteins during enamel biomineralization.
    Biopolymers 10/2013; DOI:10.1002/bip.22415 · 2.29 Impact Factor
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    ABSTRACT: Biomimetic reconstruction of tooth enamel is a significant topic of study in material science and dentistry as a novel approach for prevention, restoration, and treatment of defective enamel. We developed a new amelogenin-containing chitosan hydrogel for enamel reconstruction that works through amelogenin supramolecular assembly, stabilizing Ca-P clusters and guiding their arrangement into linear chains. These amelogenin Ca-P composite chains further fuse with enamel crystals and eventually evolve into enamel-like co-aligned crystals, anchoring to the natural enamel substrate through a cluster growth process. A dense interface between the newly-grown layer and natural enamel was formed and the enamel-like layer had improved hardness and elastic modulus compared to etched enamel. We anticipate that chitosan hydrogel will provide effective protection against secondary caries because of its pH-responsive and antimicrobial properties. Our studies introduce amelogenin-containing chitosan hydrogel as a promising biomaterial for enamel repair and demonstrate the potential of applying protein-directed assembly to biomimetic reconstruction of complex biomaterials.
    Acta biomaterialia 04/2013; 9(7). DOI:10.1016/j.actbio.2013.04.004 · 5.68 Impact Factor
  • Victoria Gallon, Lisha Chen, Xiudong Yang, Janet Moradian-Oldak
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    ABSTRACT: Enamelin and amelogenin are vital proteins in enamel formation. The cooperative function of these two proteins controls crystal nucleation and morphology in vitro. We quantitatively analyzed the co-localization between enamelin and amelogenin by confocal microscopy and using two antibodies, one raised against a sequence in the porcine 32 kDa enamelin region and the other raised against full-length recombinant mouse amelogenin. We further investigated the interaction of the porcine 32 kDa enamelin and recombinant amelogenin using immuno-gold labeling. This study reports the quantitative co-localization results for postnatal days 1, 2, 3, 4, 5, 6, 7 and 8 mandibular mouse molars. We show that amelogenin and enamelin are secreted into the extracellular matrix on the cuspal slopes of the molars at day 1 and that secretion continues to at least day 8. Quantitative co-localization analysis (QCA) was performed in several different configurations using large (45 μm height, 33 μm width) and small (7 μm diameter) regions of interest to elucidate any patterns. Co-localization patterns in day 8 samples revealed that enamelin and amelogenin co-localize near the secretory face of the ameloblasts and appear to be secreted approximately in a 1:1 ratio. The degree of co-localization decreases as the enamel matures, both along the secretory face of ameloblasts and throughout the entire thickness of the enamel. Immuno-reactivity against enamelin is concentrated along the secretory face of ameloblasts, supporting the theory that this protein together with amelogenin is intimately involved in mineral induction at the beginning of enamel formation.
    Journal of Structural Biology 04/2013; DOI:10.1016/j.jsb.2013.03.014 · 3.37 Impact Factor
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    ABSTRACT: Objectives: To identify MMP-20 proteinase and to detect its activity in extracts of mature human teeth. Methods: Individual third molar crowns, without caries or restorations, were pulverized and extracted with a guanidine chloride/ethylenediaminetetraacetic acid cocktail containing protease inhibitors, exchanged into 5% acetic acid and extracts were lyophilized. Western blotting for MMP-20 was done using either antibodies specific for the C-terminus, where the hemopexin domain is present, or for the N-terminus, where the catalytic domain is present. Immunoprecipitation of MMP-20 was performed using the N-terminus antibody. Enzymatic activity in dental extracts was analyzed with zymography using casein, gelatin, or porcine recombinant amelogenin (rP172) gels. Results: Western blotting with the N-terminal antibody routinely detected six bands at 53, 43, 41, 22, 19 and 11 kDa; the first five bands are identical with the sizes of MMP-20 precursor and four activated forms. The C-terminal antibody only recognized the 53 kDa precursor. Casein zymography with crown extracts detected three major bands at 43 kDa, 41 kDa and 22 kDa and two minor bands at 31 and 14 kDa. After immunoprecipitation, only the three major bands at 43, 41 and 22 kDa were observed identifying these forms as catalytically active MMP-20. The relative activity of each band was 33%, 33% and 34%, respectively. Consistent with this assignment, none of the MMP-20 forms were detected in gelatin gels and activity was only detected in the presence of calcium and zinc. Finally, when the dental extracts were analyzed for enzymatic activity using an amelogenin containing gel, three active bands were also observed at 43, 41 and 22 kDa. Conclusions: Our data show that active MMP-20 proteinase is readily detectable in extracts of mature human teeth. This assignment was confirmed by immunoprecipitation and cleavage of amelogenin. Active MMP-20 could play a degradative role in tooth damage associated with caries and oral cancer radiotherapy.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
  • S.B. LOKAPPA, K.B. CHANDRABABU, J. MORADIAN-OLDAK
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    ABSTRACT: Amelogenin, an intrinsically disordered protein, is the major component in the dental enamel extracellular matrix (ECM). ECM also constitutes other phosphorylated, glycosylated and sulfated proteins, proteinases and lipids. Due to this heterogeneity, complex protein-protein, protein-mineral and protein-lipid interactions can be envisaged during amelogenesis. Lipids constitute 0.2% of the developing enamel matrix. As amelogenin is synthesized by the ameloblast cells and secreted via matrix secretory vesicles, the study of its structure in the presence of cell membrane or membrane mimicking models can give more insight into its function during amelogenesis. Objectives: To investigate interactions of amelogenin with liposomes as artificial models for ameloblast cell membrane and matrix vesicles, to analyze the structural changes involved in amelogenin upon its binding to liposomes, and address the transformation between its disordered and ordered conformation. Methods: We used recombinant porcine full-length amelogenin (rP172) and a mixture of commercially available lipids and phospholipids. Binding between amelogenin and liposomes was analyzed by fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. Conformational changes in amelogenin were analyzed by circular dichroism. Results: Fluorescence studies revealed that monomeric rP172 is peripherally bound to the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes but associated strongly with the anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) liposomes, as well as with the ameloblast cell membrane mimicking liposomes (ACML). Further, circular dichroism studies revealed that rP172 coils to form alpha helix following its interaction with the anionic POPG and the ACML liposomes. Fluorescence and CD studies indicated weak interactions of rP172 nanospheres with POPG and ACML liposomes. TEM studies suggests that amelogenin interactions are highly dependent on the lipid composition. Conclusions: Amelogenin molecules interact weakly with neutral liposomes, but strongly with negatively charged liposomes and the ameloblast mimicking liposomes, changing their conformation. These findings have important implications for understanding the interactions of amelogenin with ameloblasts and matrix secretory vesicles.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
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    ABSTRACT: Calcite crystals were grown in the presence of full-length amelogenin and during its proteolysis by recombinant human matrix metalloproteinase 20 (rhMMP-20). Recombinant porcine amelogenin (rP172) altered the shape of calcite crystals by inhibiting the growth of steps on the {104} faces and became occluded inside the crystals. Upon co-addition of rhMMP-20, the majority of the protein was digested resulting in a truncated amelogenin lacking the C-terminal segment. In rP172-rhMMP-20 samples, the occlusion of amelogenin into the calcite crystals was drastically decreased. Truncated amelogenin (rP147) and the 25-residue C-terminal domain produced crystals with regular shape and less occluded organic material. Removal of the C-terminal diminished the affinity of amelogenin to the crystals and therefore prevented occlusion. We hypothesize that HAP and calcite interact with amelogenin in a similar manner. In the case of each material, full-length amelogenin binds most strongly, truncated amelogenin binds weakly and the C-terminus alone has the weakest interaction. Regarding enamel crystal growth, the prevention of occlusion into maturing enamel crystals might be a major benefit resulting from the selective cleavage of amelogenin at the C-terminus by MMP-20. Our data have important implications for understanding the hypomineralized enamel phenotype in cases of amelogenesis imperfecta resulting from MMP-20 mutations and will contribute to the design of enamel inspired biomaterials.
    Crystal Growth & Design 10/2012; 12(10):4897-4905. DOI:10.1021/cg300754a · 4.56 Impact Factor
  • S.B. LOKAPPA, K. BALAKRISHNA, J. MORADIAN-OLDAK
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    ABSTRACT: Dental enamel extracellular matrix (ECM) constitutes phosphorylated and glycosylated proteins, proteinases, and traces of lipids. Due to this heterogeneity, complex protein-protein, protein-mineral and protein-lipid interactions can be envisaged during amelogenesis. The major (>90%) ECM protein is the proline-rich amelogenin, which is classified as an intrinsically disordered protein having the potential to bind to many targets. Lipids constitute 0.2% of the developing enamel matrix. Objective: To investigate interactions of amelogenin with liposomes as artificial models for ameloblast cell membrane, and analyze amelogenin conformational changes upon their binding. Methods: We used recombinant porcine full-length amelogenin rP172 and a mixture of commercially available lipids and phospholipids. Binding between amelogenin and liposomes was analyzed by fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. Conformational changes in amelogenin were analyzed by circular dichroism. Results: Fluorescence studies revealed that rP172 strongly interacted with the anionic liposome POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol), as well as with the liposomes formed by a mixture of different lipids and phospholipids to mimic ameloblast cell membrane. Further, circular dichroism studies revealed that rP172 coils to form alpha helix following its interaction with the anionic POPG and the mixture of ameloblast membrane mimicking liposomes. Conclusions: Amelogenin changes its conformation upon binding to phospholipids. Our comprehensive studies of amelogenin interactions with membrane models can provide detailed insight into mechanisms of amelogenin-cell interactions during amelogenesis, as well as into mechanisms of amelogenin signaling function.
    IADR General Session 2012; 06/2012
  • Janet Moradian‐Oldak, Yuwei Fan
    Nanotechnologies for the Life Sciences, 02/2012; , ISBN: 9783527610419
  • Biophysical Journal 01/2012; 102(3):259-. DOI:10.1016/j.bpj.2011.11.1427 · 3.83 Impact Factor
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    Janet Moradian-Oldak
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    ABSTRACT: Enamel is a hard nanocomposite bioceramic with significant resilience that protects the mammalian tooth from external physical and chemical damages. The remarkable mechanical properties of enamel are associated with its hierarchical structural organization and its thorough connection with underlying dentin. This dynamic mineralizing system offers scientists a wealth of information that allows the study of basic principels of organic matrix-mediated biomineralization and can potentially be utilized in the fields of material science and engineering for development and design of biomimetic materials. This chapter will provide a brief overview of enamel hierarchical structure and properties and the process and stages of amelogenesis. Particular emphasis is given to current knowledge of extracellular matrix protein and proteinases, and the structural chemistry of the matrix components and their putative functions. The chapter will conclude by discussing the potential of enamel for regrowth.
    Frontiers in Bioscience 01/2012; 17:1996-2023. DOI:10.2741/4034 · 4.25 Impact Factor
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    Xiudong Yang, Daming Fan, Shibi Mattew, Janet Moradian-Oldak
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    ABSTRACT: The structures and interactions among macromolecules in the enamel extracellular matrix play vital roles in regulating hydroxyapatite crystal nucleation, growth, and maturation. We used dynamic light scattering (DLS), circular dichroism (CD), fluorescence spectroscopy, and transmission electron microscopy (TEM) to investigate the association of amelogenin and the 32-kDa enamelin, at physiological pH 7.4, in phosphate-buffered saline (PBS). The self-assembly behavior of amelogenin (rP148) was altered following addition of the 32-kDa enamelin. Dynamic light scattering revealed a trend for a decrease in aggregate size in the solution following the addition of enamelin to amelogenin. A blue-shift and intensity increase of the ellipticity minima of rP148 in the CD spectra upon the addition of the 32-kDa enamelin, suggest a direct interaction between the two proteins. In the fluorescence spectra, the maximum emission of rP148 was red-shifted from 335 to 341 nm with a marked intensity increase in the presence of enamelin as a result of complexation of the two proteins. In agreement with DLS data, TEM imaging showed that the 32-kDa enamelin dispersed the amelogenin aggregates into oligomeric particles and stabilized them. Our study provides novel insights into understanding the possible cooperation between enamelin and amelogenin in macromolecular co-assembly and in controlling enamel mineral formation.
    European Journal Of Oral Sciences 12/2011; 119 Suppl 1:351-6. DOI:10.1111/j.1600-0722.2011.00916.x · 1.73 Impact Factor
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    ABSTRACT: Amelogenin self-assembles to form an extracellular protein matrix, which serves as a template for the continuously growing enamel apatite crystals. To gain further insight into the molecular mechanism of amelogenin nanosphere formation, we manipulated the interactions between amelogenin monomers by altering pH, temperature, and protein concentration to create isolated metastable amelogenin oligomers. Recombinant porcine amelogenins (rP172 and rP148) and three different mutants containing only a single tryptophan (Trp161, Trp45, and Trp25) were used. Dynamic light scattering and fluorescence studies demonstrated that oligomers were metastable and in constant equilibrium with monomers. Stable oligomers with an average hydrodynamic radius (RH) of 7.5 nm were observed at pH 5.5 between 4 and 10 mg·ml−1. We did not find any evidence of a significant increase in folding upon self-association of the monomers into oligomers, indicating that they are disordered. Fluorescence experiments with single tryptophan amelogenins revealed that upon oligomerization the C terminus of amelogenin (around residue Trp161) is exposed at the surface of the oligomers, whereas the N-terminal region around Trp25 and Trp45 is involved in protein-protein interaction. The truncated rP148 formed similar but smaller oligomers, suggesting that the C terminus is not critical for amelogenin oligomerization. We propose a model for nanosphere formation via oligomers, and we predict that nanospheres will break up to form oligomers in mildly acidic environments via histidine protonation. We further suggest that oligomeric structures might be functional components during maturation of enamel apatite.
    Journal of Biological Chemistry 10/2011; 286(40):34643-34653. · 4.60 Impact Factor

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