Uniaxial Hydroxyapatite Growth on a Self-Assembled Protein Scaffold.

Biomineralization is a crucial process whereby organisms produce mineralized tissues such as teeth for mastication, bones for support, and shells for protection. Mineralized tissues are composed of hierarchically organized hydroxyapatite crystals, with a limited capacity to regenerate when demineralized or damaged past a critical size. Thus, the development of protein-based materials that act as artificial scaffolds to guide hydroxyapatite growth is an attractive goal both for the design of ordered nanomaterials and for tissue regeneration. In particular, amelogenin, which is the main protein that scaffolds the hierarchical organization of hydroxyapatite crystals in enamel, amelogenin recombinamers, and amelogenin-derived peptide scaffolds have all been investigated for in vitro mineral growth. Here, we describe uniaxial hydroxyapatite growth on a nanoengineered amelogenin scaffold in combination with amelotin, a mineral promoting protein present during enamel formation. This bio-inspired approach for hydroxyapatite growth may inform the molecular mechanism of hydroxyapatite formation in vitro as well as possible mechanisms at play during mineralized tissue formation.

Enamel-like tissue regeneration by using biomimetic enamel matrix proteins.

Enamel regeneration currently -is limited by our inability to duplicate artificially its complicated and well-aligned hydroxyapatite structure. The initial formation of enamel occurs in enamel organs where the ameloblasts secret enamel extracellular matrix formed a unique gel-like microenvironment. The enamel extracellular matrix is mainly composed by amelogenin and non-amelogenin. In this study, an innovative strategy was proposed to regenerate enamel-like tissue by constructing a microenvironment using biomimetic enamel matrix proteins (biomimetic EMPs) composed of modified leucine-rich amelogenin peptide (mLRAP) and non-amelogenin analog (NAA). Impressively, the regenerated enamel in this biomimetic EMPs on etched enamel surface produced prismatic structures, and showed similar mechanical properties to natural enamel. The results of X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) showed that regenerated crystal was hydroxyapatite. Molecular dynamics simulation analysis showed the binding energy between mLRAP and NAA were electrostatic forces and Van der Walls. These results introduced a promising strategy to induce crystal growth of enamel-like hydroxyapatite for biomimetic reproduction of materials with complicated hierarchical microstructures.

Intrinsically disordered protein domain of human ameloblastin in synthetic fusion with calmodulin increases calmodulin stability and modulates its function.

Constantly increasing attention to bioengineered proteins has led to the rapid development of new functional targets. Here we present the biophysical and functional characteristics of the newly designed CaM/AMBN-Ct fusion protein. The two-domain artificial target consists of calmodulin (CaM) and ameloblastin C-terminus (AMBN-Ct). CaM as a well-characterized calcium ions (Ca2+) binding protein offers plenty of options in terms of Ca2+ detection in biomedicine and biotechnologies. Highly negatively charged AMBN-Ct belongs to intrinsically disordered proteins (IDPs). CaM/AMBN-Ct was designed to open new ways of communication synergies between the domains with potential functional improvement. The character and function of CaM/AMBN-Ct were explored by biophysical and molecular modelling methods. Experimental studies have revealed increased stability and preserved CaM/AMBN-Ct function. The results of molecular dynamic simulations (MDs) outlined different interface patterns between the domains with potential allosteric communication within the fusion.

Characterization of AMBN I and II Isoforms and Study of Their Ca2+-Binding Properties.

Ameloblastin (Ambn) as an intrinsically disordered protein (IDP) stands for an important role in the formation of enamel-the hardest biomineralized tissue commonly formed in vertebrates. The human ameloblastin (AMBN) is expressed in two isoforms: full-length isoform I (AMBN ISO I) and isoform II (AMBN ISO II), which is about 15 amino acid residues shorter than AMBN ISO I. The significant feature of AMBN-its oligomerization ability-is enabled due to a specific sequence encoded by exon 5 present at the N-terminal part in both known isoforms. In this study, we characterized AMBN ISO I and AMBN ISO II by biochemical and biophysical methods to determine their common features and differences. We confirmed that both AMBN ISO I and AMBN ISO II form oligomers in in vitro conditions. Due to an important role of AMBN in biomineralization, we further addressed the calcium (Ca2+)-binding properties of AMBN ISO I and ISO II. The binding properties of AMBN to Ca2+ may explain the role of AMBN in biomineralization and more generally in Ca2+ homeostasis processes.

Controls of nature: Secondary, tertiary, and quaternary structure of the enamel protein amelogenin in solution and on hydroxyapatite.

Amelogenin, a protein critical to enamel formation, is presented as a model for understanding how the structure of biomineralization proteins orchestrate biomineral formation. Amelogenin is the predominant biomineralization protein in the early stages of enamel formation and contributes to the controlled formation of hydroxyapatite (HAP) enamel crystals. The resulting enamel mineral is one of the hardest tissues in the human body and one of the hardest biominerals in nature. Structural studies have been hindered by the lack of techniques to evaluate surface adsorbed proteins and by amelogenin's disposition to self-assemble. Recent advancements in solution and solid state nuclear magnetic resonance (NMR) spectroscopy, atomic force microscopy (AFM), and recombinant isotope labeling strategies are now enabling detailed structural studies. These recent studies, coupled with insights from techniques such as CD and IR spectroscopy and computational methodologies, are contributing to important advancements in our structural understanding of amelogenesis. In this review we focus on recent advances in solution and solid state NMR spectroscopy and in situ AFM that reveal new insights into the secondary, tertiary, and quaternary structure of amelogenin by itself and in contact with HAP. These studies have increased our understanding of the interface between amelogenin and HAP and how amelogenin controls enamel formation.

Protein nanoribbons template enamel mineralization.

As the hardest tissue formed by vertebrates, enamel represents nature's engineering masterpiece with complex organizations of fibrous apatite crystals at the nanometer scale. Supramolecular assemblies of enamel matrix proteins (EMPs) play a key role as the structural scaffolds for regulating mineral morphology during enamel development. However, to achieve maximum tissue hardness, most organic content in enamel is digested and removed at the maturation stage, and thus knowledge of a structural protein template that could guide enamel mineralization is limited at this date. Herein, by examining a gene-modified mouse that lacked enzymatic degradation of EMPs, we demonstrate the presence of protein nanoribbons as the structural scaffolds in developing enamel matrix. Using in vitro mineralization assays we showed that both recombinant and enamel-tissue-based amelogenin nanoribbons are capable of guiding fibrous apatite nanocrystal formation. In accordance with our understanding of the natural process of enamel formation, templated crystal growth was achieved by interaction of amelogenin scaffolds with acidic macromolecules that facilitate the formation of an amorphous calcium phosphate precursor which gradually transforms into oriented apatite fibers along the protein nanoribbons. Furthermore, this study elucidated that matrix metalloproteinase-20 is a critical regulator of the enamel mineralization as only a recombinant analog of a MMP20-cleavage product of amelogenin was capable of guiding apatite mineralization. This study highlights that supramolecular assembly of the scaffold protein, its enzymatic processing, and its ability to interact with acidic carrier proteins are critical steps for proper enamel development.

Tuftelin-derived peptide facilitates remineralization of initial enamel caries in vitro.

With the gradual discovery of functional domains in natural proteins, several biologically inspired peptides have been designed for use as biomaterials for hard tissue regeneration and repair. In this study, we designed a tuftelin-derived peptide (TDP) and tested its effects on hydroxyapatite crystallization and remineralization of initial enamel carious lesions in vitro. Using circular dichroism spectroscopy, we found that TDP contained 36.1% ß-sheets and ß-turns, which could be influenced by calcium ions. We verified the ability of TDP to crystallize hydroxyapatite using transmission electron microscopy and its ability to bind to the enamel surface and hydroxyapatite using confocal laser scanning microscopy and Langmuir adsorption isotherms (K = 881.56, N = 1.41 × 10-5 ). Artificial enamel lesions were generated on human enamel blocks and subjected to a 12-day pH cycling model and were treated with 25 µM TDP, 1 g/L sodium fluoride (NaF), or deionized water. We analyzed the results of remineralization by surface microhardness testing, polarized light microscopy, and transverse microradiography. The TDP group showed significantly higher surface microhardness recovery (49.21 ± 1.66%), shallower lesions (34.89 ± 4.05 µm), and less mineral loss (871.33 ± 81.49 vol%·µm) after pH cycling than the deionized water group (p < .05). There were no significant differences between the TDP and NaF groups. Our experiment indicated that TDP could regulate hydroxyapatite crystallization and promote remineralization of enamel caries in vitro.

Hydroxyapatite Formation Coexists with Amyloid-like Self-Assembly of Human Amelogenin.

Tooth enamel is formed in an extracellular environment. Amelogenin, the major component in the protein matrix of tooth enamel during the developing stage, could assemble into high molecular weight structures, regulating enamel formation. However, the molecular structure of amelogenin protein assembly at the functional state is still elusive. In this work, we found that amelogenin is able to induce calcium phosphate minerals into hydroxyapatite (HAP) structure in vitro at pH 6.0. Assessed using X-ray diffraction (XRD) and 31P solid-state NMR (SSNMR) evidence, the formed HAP mimics natural enamel closely. The structure of amelogenin protein assembly coexisting with the HAP was also studied using atomic force microscopy (AFM), transmission electron microscopy (TEM) and XRD, indicating the ß-amyloid structure of the protein. SSNMR was proven to be an important tool in detecting both the rigid and dynamic components of the protein assembly in the sample, and the core sequence 18EVLTPLKWYQSI29 was identified as the major segment contributing to the ß-sheet secondary structure. Our research suggests an amyloid structure may be an important factor in controlling HAP formation at the right pH conditions with the help of other structural components in the protein assembly.

Similar inductive effects of enamel and dentin matrix derivatives on osteoblast-like cell response over SLA titanium surface.

OBJECTIVES: The aim of this in vitro study was to investigate the behavior of osteoblasts on titanium discs under different concentrations of enamel matrix derivatives (EMD) and dentin matrix derivative (DMD). MATERIALS AND METHODS: MC3T3-E1 osteoblast-like cells were cultivated on coated titanium SLA discs with EMD or DMD at 100 µg/ml, 1 mg/ml, 10 mg/ml and 30 mg/ml or left uncoated. Cell viability, proliferation, adhesion and migration were assessed respectively with MTT, BrdU, DAPI and scratch wound healing assays. Messenger ribonucleic acid of different genes related to osteoblastic differentiation was quantified by means of real-time quantitative PCR. Data were analyzed using student t-test for adhesion and migration assay and ANOVA for proliferation assay (p < 0.05). RESULTS: BrdU incorporation was found in proliferative osteoblasts for both test solutions at all concentrations. Osteoblast migrated and covered approximately 70% of the wound area observed at time zero when exposed to EMD and DMD to all concentrations. The increase of gene expression was dependent on the concentration enhancement of EMD and DMD. Higher concentrations showed proliferation augmentation if compared to lower concentrations. CONCLUSIONS: Roughness surface of Ti SLA can limit cell adhesion independent of the presence EMD or DMD. DMD enhances cell migration of osteoblasts on SLA titanium implants in a concentration-dependent manner.

Reptile enamel matrix proteins: Selection, divergence, and functional constraint.

The three major enamel matrix proteins (EMPs): amelogenin (AMEL), ameloblastin (AMBN), and enamelin (ENAM), are intrinsically linked to tooth development in tetrapods. However, reptiles and mammals exhibit significant differences in dental patterning and development, potentially affecting how EMPs evolve in each group. In most reptiles, teeth are replaced continuously throughout life, while mammals have reduced replacement to only one or two generations. Reptiles also form structurally simple, aprismatic enamel while mammalian enamel is composed of highly organized hydroxyapatite prisms. These differences, combined with reported low sequence homology in reptiles, led us to predict that reptiles may experience lower selection pressure on their EMPs as compared with mammals. However, we found that like mammals, reptile EMPs are under moderate purifying selection, with some differences evident between AMEL, AMBN, and ENAM. We also demonstrate that sequence homology in reptile EMPs is closely associated with divergence times, with more recently diverged lineages exhibiting high homology, along with strong phylogenetic signal. Lastly, despite sequence divergence, none of the reptile species in our study exhibited mutations consistent with diseases that cause degeneration of enamel (e.g. amelogenesis imperfecta). Despite short tooth retention time and simplicity in enamel structure, reptile EMPs still exhibit purifying selection required to form durable enamel.

Characterization of inter-crystallite peptides in human enamel rods reveals contribution by the Y allele of amelogenin.

Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1-6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation.

Peptide coatings enhance keratinocyte attachment towards improving the peri-implant mucosal seal.

There is a critical need for preventing peri-implantitis as its prevalence has increased and dental implants lack features to prevent it. Research strategies to prevent peri-implantitis have focused on modifying dental implants to incorporate different antimicrobial agents. An alternative strategy consists of barring the expansion of the biofilm subgingivally by forming a long-lasting permucosal seal between the soft tissue and the implant surface. Here, we innovatively biofunctionalized titanium with bioinspired peptide coatings to strengthen biological interactions between epithelial cells and the titanium surface. We selected laminin 332- and ameloblastin-derived peptides (Lam, Ambn). Laminin 332 participates in the formation of hemidesmosomes by keratinocytes and promotes epithelial attachment around teeth; and ameloblastin, an enamel derived protein, is involved in tissue regeneration events following disruption of the periodontium. Lam, Ambn or combinations of both peptides were covalently immobilized on titanium discs. Successful immobilization of the peptides was confirmed by contact angle goniometry, X-ray photoelectron spectroscopy and fluorescent labelling of the peptides. Additionally, we confirmed the mechanical and thermochemical stability of the peptides on Ti substrates. Proliferation and hemidesmosome formation of human keratinocytes (TERT-2/OKF-6) were assessed by immunofluorescence labelling. The peptide-coated surfaces increased cell proliferation for up to 48 h in culture compared to control surfaces. Most importantly, formation of hemidesmosomes by keratinocytes was significantly increased on surfaces coated with Ambn + Lam peptides compared to control (p < 0.01) and monopeptide coatings (p < 0.005). Together, these results support the Ambn + Lam multipeptide coating as a promising candidate for inducing a permucosal seal around dental implants.

The enamel protein ODAM promotes mineralization in a collagen matrix.

Purpose/aim of the study: Odontogenic ameloblast-associated protein (ODAM) is predominantly expressed during the maturation stage of enamel formation and interacts strongly with amelotin (AMTN). AMTN is involved in enamel mineralization, but the effect of ODAM on mineralization has not been investigated. This study determined whether ODAM was able to induce hydroxyapatite (HA) mineralization in modified simulated body fluid (SBF) and in a collagen matrix in vitro. MATERIALS AND METHODS: To monitor the kinetics of calcium phosphate mineralization, recombinant human (rh) ODAM protein in SBF buffer was incubated at 37°C and a light-scattering assay was conducted at intervals. To investigate the nucleation of ODAM in collagen matrix, the ODAM-impregnated collagen hydrogel was incubated in SBF buffer for 24 hours. Bovine serum albumin (BSA) was used as negative control. Mineral deposits were visualized using electron microscopy. RESULTS: The presence of rh-ODAM protein in SBF resulted in higher light-scattering values after 18-24 hours. Calcium phosphate precipitates were observed on the surface of the ODAM-treated, but not BSA-treated collagen hydrogel after 24 hours in SBF. TEM and SAED analyses showed that these crystals consisted of needle-like HA. CONCLUSION: Similar to AMTN, ODAM is able to promote HA nucleation in a dose-dependent manner in SBF, and even outside of its biological context in vitro.

Changes in the Proteomic Profile of Acquired Enamel Pellicles as a Function of Their Time of Formation and Hydrochloric Acid Exposure.

OBJECTIVE: Changes in the protein profile of acquired enamel pellicles (AEP) formed in vivo over different time periods were evaluated after the application of hydrochloric acid (HCl). METHODS: Nine subjects were submitted to dental prophylaxis with pumice. After 3 or 120 min, the teeth were isolated with cotton rolls and 50 µL of 0.1 M HCl (pH 1.0), 0.01 M HCl (pH 2.0), or deionized water were applied on the buccal surface of the teeth for 10 s. The AEP was then collected using an electrode filter paper presoaked in 3% citric acid. After protein extraction, the samples were submitted to reverse-phase liquid chromatography coupled to mass spectrometry (nano LC-ESI-MS/MS). Label-free quantification was performed (Protein Lynx Global Service software). RESULTS: A total of 180 proteins were successfully identified in the AEP samples. The number of identified proteins increased with the time of pellicle formation. Only 4 proteins were present in all the groups (isoforms of IgA, serum albumin, and statherin). The greatest number of proteins identified uniquely in one of the groups was obtained for the groups treated with HCl after 2 h of pellicle formation (approx. 50 proteins). CONCLUSION: Proteins resistant to removal by HCl, such as serum albumin and statherin, were identified even in the short-term AEP. In addition, 120-min pellicles present many proteins that are resistant to removal by HCl. This suggests an increase in protection against intrinsic acids with the time of pellicle formation, which should be evaluated in future studies.

Proteomic Mapping of Dental Enamel Matrix from Inbred Mouse Strains: Unraveling Potential New Players in Enamel.

Enamel formation is a complex 2-step process by which proteins are secreted to form an extracellular matrix, followed by massive protein degradation and subsequent mineralization. Excessive systemic exposure to fluoride can disrupt this process and lead to a condition known as dental fluorosis. The genetic background influences the responses of mineralized tissues to fluoride, such as dental fluorosis, observed in A/J and 129P3/J mice. The aim of the present study was to map the protein profile of enamel matrix from A/J and 129P3/J strains. Enamel matrix samples were obtained from A/J and 129P3/J mice and analyzed by 2-dimensional electrophoresis and liquid chromatography coupled with mass spectrometry. A total of 120 proteins were identified, and 7 of them were classified as putative uncharacterized proteins and analyzed in silico for structural and functional characterization. An interesting finding was the possibility of the uncharacterized sequence Q8BIS2 being an enzyme involved in the degradation of matrix proteins. Thus, the results provide a comprehensive view of the structure and function for putative uncharacterized proteins found in the enamel matrix that could help to elucidate the mechanisms involved in enamel biomineralization and genetic susceptibility to dental fluorosis.

Duplicated or Hybridized Peptide Functional Domains Promote Oral Homeostasis.

Proteins that have existed for millions of years frequently contain repeats of functional domains within their primary structure, thereby improving their functional capacity. In the evolutionary young statherin protein contained within the in vivo-acquired enamel pellicle (AEP), we identified a single functional domain (DR9) located within the protein N-terminal portion that exhibits a higher affinity for hydroxyapatite and more efficient protection against enamel demineralization compared to other native statherin peptides. Thus, we tested the hypothesis that multiplication of functional domains of naturally occurring pellicle peptides amplifies protection against enamel demineralization. In addition, a specific amino acid sequence from histatin 3 (RR-14) was introduced to the hybrid peptides for further testing. Enamel specimens were sectioned to 150-µm thickness and randomly grouped as follows: DR9, DR9-DR9, DR9-RR14, statherin, histatin 1, or distilled water (control). After submersion for 2 h at 37°C, the specimens were placed in 2 mL demineralization solution for 12 d at 37°C. Upon sample removal, the remaining solution was subjected to colorimetric assays to determine the amount of calcium and phosphate released from each specimen. DR9-DR9 amplified protection against enamel demineralization when compared to single DR9 or statherin. Notably, the hybrid peptide DR9-RR14 demonstrated relatively strong protection when the antimicrobial property of these peptides was tested against Candida albicans and Streptococcus mutans. DR9-RR14 was able to maintain 50% of the antifungal activity compared with RR14 for C. albicans and similar values of S. mutans killing activity. This study has pioneered the functional exploration of the natural peptide constituents of the AEP and their evolution-inspired engineered peptides. The knowledge obtained here may provide a basis for the development of stable (proteinase-resistant) synthetic peptides for therapeutic use against dental caries, dental erosion, and/or oral candidiasis.

Scaffold requirements for periodontal regeneration with enamel matrix derivative proteins.

Periodontitis affects the attachment of natural teeth, and infection or inflammation associated with periodontitis may affect peri-implant tissues. Enamel matrix derivative (EMD) proteins provide stimulation for self-regeneration of the damaged tissue when applied to wide intrabony defects as part of a mixture with bone graft material. As a first step of the process enhancing cell proliferation and ligament formation, we demonstrated that EMD protein precipitation depends strongly on the physical and chemical characteristics of the bone grafts used in the mixture. To guarantee optimum protein-stimulated self-regulation, the pH of the initial EMD formulation must therefore be adjusted between 3.9 and 4.2 in order to compensate the change in pH induced by the bone graft. Moreover, the interaction between the two components resulted in precipitates of different shape and size differently covering the grafts. This outcome might potentially have clinical implications on cell attachment and periodontal ligament extension, which deserve further in vitro and in vivo tests.

Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding.

Mutations in FAM20A cause tooth enamel defects known as Amelogenesis Imperfecta (AI) and renal calcification. We previously showed that Fam20A is a secretory pathway pseudokinase and allosterically activates the physiological casein kinase Fam20C to phosphorylate secreted proteins important for biomineralization (Cui et al., 2015). Here we report the nucleotide-free and ATP-bound structures of Fam20A. Fam20A exhibits a distinct disulfide bond pattern mediated by a unique insertion region. Loss of this insertion due to abnormal mRNA splicing interferes with the structure and function of Fam20A, resulting in AI. Fam20A binds ATP in the absence of divalent cations, and strikingly, ATP is bound in an inverted orientation compared to other kinases. Fam20A forms a dimer in the crystal, and residues in the dimer interface are critical for Fam20C activation. Together, these results provide structural insights into the function of Fam20A and shed light on the mechanism by which Fam20A mutations cause disease.

In vitro re-hardening of artificial enamel caries lesions using enamel matrix proteins or self-assembling peptides.

OBJECTIVES: To assess the re-hardening potential of enamel matrix derivatives (EMD) and self-assembling peptides in vitro, hypothesizing that these materials may increase the mineralization of artificial carious lesions and improve hardness profiles. MATERIAL AND METHODS: Forty-eight enamel samples were prepared from extracted bovine lower central incisors. After embedding and polishing, nail varnish was applied, leaving a defined test area. One third of this area was covered with a flowable composite (non-demineralized control). The remaining area was demineralized in an acidic buffer solution for 18 d to simulate a carious lesion. Half the demineralized area was then covered with composite (demineralized control), while the last third was left open for three test and one control treatments: (A) Application of enamel-matrix proteins (EMD - lyophilized protein fractions dissolved in acetic acid, Straumann), (B) self-assembling peptides (SAP, Curodont), or (C) amine fluoride solution (Am-F, GABA) for 5 min each. Untreated samples (D) served as control. After treatment, samples were immersed in artificial saliva for four weeks (remineralization phase) and microhardness (Knoop) depth profiles (25-300 µm) were obtained at sections. Two-way ANOVA was calculated to determine differences between the areas (re-hardening or softening). RESULTS: Decalcification resulted in significant softening of the subsurface enamel in all groups (A-D). A significant re-hardening up to 125 µm was observed in the EMD and SAP groups. CONCLUSIONS: This study showed that EMD and SAP were able to improve the hardness profiles when applied to deep demineralized artificial lesions. However, further research is needed to verify and improve this observed effect.

In vitro re-hardening of artificial enamel caries lesions using enamel matrix proteins or self-assembling peptides

ABSTRACT Objectives To assess the re-hardening potential of enamel matrix derivatives (EMD) and self-assembling peptides in vitro, hypothesizing that these materials may increase the mineralization of artificial carious lesions and improve hardness profiles. Material and Methods Forty-eight enamel samples were prepared from extracted bovine lower central incisors. After embedding and polishing, nail varnish was applied, leaving a defined test area. One third of this area was covered with a flowable composite (non-demineralized control). The remaining area was demineralized in an acidic buffer solution for 18 d to simulate a carious lesion. Half the demineralized area was then covered with composite (demineralized control), while the last third was left open for three test and one control treatments: (A) Application of enamel-matrix proteins (EMD - lyophilized protein fractions dissolved in acetic acid, Straumann), (B) self-assembling peptides (SAP, Curodont), or (C) amine fluoride solution (Am-F, GABA) for 5 min each. Untreated samples (D) served as control. After treatment, samples were immersed in artificial saliva for four weeks (remineralization phase) and microhardness (Knoop) depth profiles (25-300 µm) were obtained at sections. Two-way ANOVA was calculated to determine differences between the areas (re-hardening or softening). Results Decalcification resulted in significant softening of the subsurface enamel in all groups (A-D). A significant re-hardening up to 125 µm was observed in the EMD and SAP groups. Conclusions This study showed that EMD and SAP were able to improve the hardness profiles when applied to deep demineralized artificial lesions. However, further research is needed to verify and improve this observed effect.